baptistearbezandhélènelibouban(universityofangers, · 112 editorial •...

Morphologie (2017) 101, 111—112

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EDITORIAL

Beta-tricalcium phosphate and bonesurgery: EditorialLe bêta-tricalcium phosphate en chirurgie osseuse : éditorial

Bone is a dynamic connective tissue that is continuouslyremodel throughout life to remain well-adapted to hor-monal and mechanical changes. In addition, bone has ahigh regenerative capacity in the young but, as the cellularfunctions decline upon ageing, the capacity to heal spon-taneously is reduced. Bone regeneration appears reducedin the elderly and in orthopedic, oral and maxillofacial andneurosurgery when large bone defects have not the capac-ity to heal spontaneously. This includes fracture non-union,trauma, infection, revision arthroplasty, tumor resection,avascular necrosis and osteoporosis. . . In these cases, it isnecessary to provide a template allowing bone cells to buildnew bone (‘‘one can only build on ruins’’). The preferredtreatment is the graft of autologous bone but the supplyof this material is necessarily limited and associated withan increased morbidity [1]. This can be overcome by usingallogenic bone but this implies a bone bank with a potentialrisk of transmission of bacterial, viral, or prion infections[2] and the risk of immune adverse reactions [3]. Many bio-materials intended to replace the need for autologous orallogeneic bone have been proposed either of natural orsynthetic origin. Synthetic bioactive ceramics are promisingmaterials if they are osteoconductive (i.e. osteoblasts fromthe margin of the grafted area utilize the biomaterial as ascaffold upon which to spread and build newly regeneratedbone) and resorbed simultaneously by bone resorbing cells.Among them, hydroxyapatite and �-TCP (beta tri-calciumphosphate) are interesting but resorption of the former isusually very long. �-TCP appears as very interesting bio-material as it is easily resorbable and can be prepared invarious forms: porous blocks, granules. . . with a high poros-ity. This factor appears extremely important as trabecularbone is a naturally porous material with a very particu-lar microarchitecture [4]. The pores within trabecular boneare interconnected and allow the distribution of vesselsthroughout the whole cancellous space. Industry can nowprovide blocks of �-TCP with a high porosity (Fig. 1A) sim-ilar to that of trabecular bone (Fig. 1B). There is now a

Figure 1 A. Scanning electron microscopy of a commercialhighly porous �-TCP block with an interconnected porosity(Kasios HPTM, France). B. Microcomputed tomography of a blockof human trabecular bone. The microarchitectures of these twostructures appear very similar.

general consensus that a biomaterial scaffold suitable forbone grafting must have a high porosity (in the range of40—60%), large pores (100—300 �m) and an interconnectedporosity providing a high permeability for fluid transport andvascular invasion [5].

In this special issue of Morphologie, we provide an updateof the basic and clinical use of �-TCP in humans. We haveasked several of the participants to prepare an article ontheir own research:

• Hassan CHAAIR et al. (Hassan II University, Mohammedia,Morocco), will present a paper on the synthesis and char-acterization of �-TCP from a chemical point of view;

• Sergey Dorozhkin, (Moscow, Russia) has written tworeviews: one on the classification of the different typesof calcium orthophosphates (�-TCP belong to this class ofcompounds) and a second one on the history of their usein medical practice;

http://dx.doi.org/10.1016/j.morpho.2017.09.0011286-0115/© 2017 Elsevier Masson SAS. All rights reserved.

dx.doi.org/10.1016/j.morpho.2017.09.001

http://www.sciencedirect.com/science/journal/12860115

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112 Editorial

• Baptiste Arbez and Hélène Libouban (University of Angers,France) explored the in vivo behavior of macrophages andosteoblasts which were cultured on �-TCP;

• Bernard Guillaume (CFI, Paris) presents significantimprovements obtained with porous �-TCP in oral andmaxillofacial surgery;

• Takaaki Tanaka et al. (NHO Utsunomiya National Hospital,Tochigi, Japan), is working with porous �-TCP in orthope-dic surgery for more than twenty years. He presents someastonishing results obtained in bone regeneration;

• Philippe Hernigou et al. (H. Mondor Hospital, Créteil,France) analyzes the outcomes of patients who received�-TCP as a bone substitute in orthopedic surgery.

We hope that a special issue on this biomaterial in ourjournal will interest all the morphologists and will open theirmind to fruitful reflections.

Disclosure of interest

The author has not supplied their declaration of competinginterest.

References

[1] Younger EM, Chapman MW. Morbidity at bone graft donor sites.J Orthop Trauma 1989;3:192—5.

[2] Delloye C, Cornu O, Druez V, Barbier O. Bone allografts. BoneJoint J 2007;89:574—80.

[3] de Lacerda PE, Pelegrine AA, Teixeira ML, Montalli VAM,Rodrigues H, Napimoga MH. Homologous transplantation withfresh frozen bone for dental implant placement can induceHLA sensitization: a preliminary study. Cell Tissue Bank2016;17:465—72.

[4] Chappard D, Baslé MF, Legrand E, Audran M. Trabecu-lar bone microarchitecture: a review. Morphologie 2008;92:162—70.

[5] Stevens MM. Biomaterials for bone tissue engineering. MaterialsToday 2008;11:18—25.

D. ChappardGEROM (groupe études remodelage osseux et

biomatériaux), LHEA, IRIS, IBS, LabCom NextBone,université d’Angers, CHU d’Angers, 49933 Angers cedex,

FranceE-mail address: [email protected]

mailto:[email protected]

Morphologie (2017) 101, 113—119


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GENERAL REVIEW

Filling bone defects with �-TCP inmaxillofacial surgery: A reviewComblement osseux par �-TCP en chirurgie maxillofaciale :revue des indications

B. Guillaume a,b,∗

a Collège Francais d’Implantologie (CFI), 6, rue de Rome, 75005 Paris, Franceb Groupe Études Remodelage Osseux et bioMatériaux (GEROM), Institut de Biologie en Santé (IRIS-IBS),LUNAM Université, CHU d’Angers, 49933 Angers cedex, France

Available online 29 May 2017

KEYWORDSTricalcium phosphate;�-TCP;Bone graft;Sinus lift;Biomaterial

Summary Reconstruction of bone defects prior to implant placement now involves syntheticsubstitutes such as �-TCP because of its ability to promote bone remodeling. Its capacity tobe progressively substituted by the patient’s bone allows to regenerate a dense bone volume.In addition, its availability in large quantities, avoiding the morbidity observed with harvest-ing autogenous bone, widens the operative indications. In this paper, the main indicationsof �-TCP in maxillofacial surgery (dentistry, parodontology and dental implant surgery) arereviewed. They include periodontal bone disease, bone disjunction, pre-implant surgery (sinusfloor elevation and lateralization of the inferior alveolar nerve).© 2017 Elsevier Masson SAS. All rights reserved.

MOTS CLÉS�-TCP ;Biomatériau ;Greffe osseuse ;Sinus lift ;Tri calcium phosphate

Résumé La reconstruction des déficits osseux avant la pose d’implant fait appel désormais àdes matériaux de substitution synthétique comme le �-TCP de par ses capacités à favoriser leremodelage osseux. Il est progressivement substitué par l’os du patient permettant de régénérerun volume osseux dense. Enfin, sa disponibilité en grande quantité, évitant la morbidité liéeà un site de prélèvement d’autogreffe, élargit les indications opératoires. Dans ce travail,les principales indications d’utilisation du �-TCP en chirurgie maxillofaciale (chirurgie den-taire, parodontologie et implantation dentaire) sont revues. Elles comprennent la maladieosseuse parodontale, la disjonction osseuse, la chirurgie pré-implantaire (élévation de sinuset latéralisation du nerf alvéolaire inférieur).© 2017 Elsevier Masson SAS. Tous droits reserves.

∗ 6, rue de Rome, 75005 Paris, France.E-mail address: [email protected]

Introduction

In dental and maxillofacial surgery, the repair of bonedefects aims at recreating a bony site that is suitable for







114 B. Guillaume

Figure 1 Scanning electron microscopic image of beta-tricalcium phosphate (�-TCP). A. A scaffold suitable for bonegrafting with an interconnected macroporosity. B. High magni-fication of the surface of a wall of this scaffold showing thetypical surface of sintered grains with microporosity (arrows).

morphological, prosthetic or implant-prosthetic rehabilita-tion [1,2]. Causes of bone deficiency are numerous: genetic,post-traumatic, secondary to tooth removal, infectious oriatrogenic. The volume of bone to reconstruct varies accord-ing to the anatomical situation. The characteristics of the

Figure 2 Filling a bone socket and reconstruction of the corti-cal side with beta-tricalcium phosphate (�-TCP) granules aftera tooth extraction. Insert: CT analysis of the reconstructed boneseveral months after the graft, note the high bone density.

graft depend on the volumes to be filled (alveolar area) or tobe restored (vertical or horizontal ridge insufficiency, bonecyst or sinus lifting). The volume and form of the defectinfluence the choice of the grafting material. A consider-able number of possibilities are available: e.g., harvestingautologous bone particles to fill a post-extraction socket [3],thickening of a thin ridge caused by hypodontia [4], bone dis-junction [5], sinus lifting [6] or lateralization of the inferioralveolar nerve [7]. Surgical techniques also use various sur-gical protocols with a local or a general anesthesia; it shouldbe noticed that local anesthesia is being practiced more andmore frequently. Although autologous bone grafts and allo-grafts have been a recognized surgical modality for severaldecades, the use of synthetic biomaterials has continued todevelop as substitute products, especially in the context ofpre-implant surgery.

Definitions of graft-related criteria

A bone substitute must be biocompatible and fill severalcriteria: bio-inertia is defined as the absence of physico-chemical reaction of the product in direct contact withbone. Bioactivity is the capacity to develop reactions favor-ing osseointegration of the product and the adaptation of the

Figure 3 A. Disjunction on a premolar zone. B. Beta-tricalcium phosphate (�-TCP) granules (1000—2000 �m) withhigh porosity are inserted in the gap created between the twocortices.

Bone filling with �-TCP 115

receiving tissue. Osteoinduction is defined as the ability toinduce bone formation in an extra-skeletal area. Osteocon-duction is the ability of the recipient bone cells to colonizethe graft. An evolution in the choice of materials to fill bonedefects occurred in the past few years. Autologous graft isconsidered as the ‘‘gold standard’’ in terms of osteogenic-ity but its main disadvantage is that obtaining a sufficientvolume of bone is sometimes a problem (except when largebone samples are harvested but with a significant increasein morbidity) [8,9]. So, due to the justified reluctance to useextra-oral bone grafts and the medicolegal constraints, theinterest for synthetic bone substitutes is increasingly grow-ing. Most surgical teams seek to restore sufficient amountsof high quality bone allowing implant placement by usingbiomaterials to restore a part or the whole bone. Beta-tricalcium phosphate (�-TCP) is now one of the most usedsynthetic materials for bone reconstruction in orthopedicand maxillofacial surgery and now in pre-implant surgery.In implantology, the resorbable or non-resorbable natureof the grafted material appears of the upmost importance.Some materials are resorbable but there are considerabledifferences in term of disappearance upon time, some ofthem can persist indefinitely. The use of a biomaterial in amaxillary or mandibular site after a tooth extraction mustallow implant placement in a second surgical time. In addi-tion, synthetic biomaterials preserve the macroarchitecture

of the implanted areas and they can be produced in largequantities by industry.

Characteristics of �-TCP

�-TCP - Ca3(POH4)2 - belongs to the family of tricalciumphosphates in the beta phase. Being considerably much moreresorbable than hydroxyapatite, this biomaterial is highlybiocompatible when implanted in bone; it is resorbablewithin 6 to 9 months as shown in animal and human his-tological studies [10]. One of the main characteristics ofthe biomaterial is to be commercially available as scaf-folds with a macroporosity of 100 to 600 �m that ensuresosteoconduction and can reach 85% of the total mass of thegrafted material (Fig. 1A). �-TCP has also a microporosity(< 100 �m) due to sintering of elementary grains, which facil-itates the exchange of extracellular fluxes of Ca2+ and PO4

3−

ions (Fig. 1B). This ensures an optimal bone remodelingwith increased osteoblastic apposition and the appositionof lamellar bone. Its biocompatibility, its bioresorption andosteoconductive properties from the recipient bone makeit a reliable material particularly adapted to fill alveolarsockets of variable sizes. In addition, reconstruction of amaxillary sinus in a pre-implantation step can also be easilyobtained. �-TCP can also be prepared in the form of plates

Figure 4 Sinus lift elevation. A. The sinus membrane is gently pushed away. B. The space available for grafting is then exposed.C. Beta-tricalcium phosphate (�-TCP) granules (1000—2000 �m) with a high porosity are used to fill this area. D. Implants have beenplaced in the grafted area 9 months later, note the increase density of the grafted zone.

116 B. Guillaume

to fill the sinus ceiling. Such plates also allow to densify theunderlying graft with granules and can be used to protect thesinus mucosa (Schneider’s membrane) in case of perforation.

Clinical indications of bone filling biomaterials

Periodontics and pre-implant surgery

Periodontal bone diseaseIt has been proposed for more than 15 years to fill peri-odontal pockets with biomaterial granules. Loss of theperi-radicular bone tissue induces mobility of the dental rootand can cause loss of the tooth. Under local anesthesia, theprocedure consists in detaching the full thickness of the gin-giva to scrap the inflammatory tissues and cementum alongthe root and to fill the defect. �-TCP is available in variousgranule sizes varying from 550—1000 �m to 1000—2000 �m.

Figure 5 Histological analysis of a bone biopsy harvestedin a patient six months after grafting with beta-tricalciumphosphate (�-TCP) granules. A. Mineralized bone is apposeddirectly onto the biomaterial surfaces. (Goldner’s trichrome,calcified bone is in green osteoid tissue in red). B. Remodel-ing of implanted �-TCP granules by osteoclasts. Histoenzymaticidentification of these cells by their tartrate resistant acidphosphatase (TRAcP) content; osteoclasts are in brown, newly-apposed bone is counterstained in blue and �-TCP remainsunstained.

The characteristics of �-TCP scaffolds prepared with a highporosity by industry are to promote reconstruction withlamellar bone. In case of apicoectomy and elimination ofthe cystic tissues at the apex of the tooth, a bone loss mayoccur with a variable volume ranging from a few mm3 to 1or 2 cm3. The cystic tissue is removed by a careful curettageand 1000—2000 �m granules of high porosity �-TCP are usedto fill the cavity.

Pre-implant surgeryIncreasing the volume of the dental arch will allow theplacement of dental implants. In implant surgery, it is oftenmandatory to respect the healing time necessary for thegrafted area to be remodeled before placing an implant ina bone environment that is sufficient in volume and willremain stable over time. Post-extraction sockets, as peri-odontal pockets, can benefit from the use of �-TCP granulesto support bone reconstruction (Fig. 2).

Bone disjunctionWhether at the maxilla or the mandible, insufficient thick-ness of the ridge imposes to recreate the bone thickness.Apposition graft is an alternative technique but requires asecond site to harvest the autogenous bone; sometimes thisis not possible or rejected by the patient. Bone disjunctionrepresents in fact a greenstick fracture [11]. With a bonechisel or with a fissure burr, vertical sections are done at thedesired height (Fig. 3A). Similarly, a cleavage plane is madein the whole thickness of the ridge. Finally, the cortical boneis gradually moved away while maintaining its apical inser-tion. The gap (4 to 5 mm in thickness) is then filled with�-TCP 1000—2000 �m granules, this prevents the vestibularcortical bone from returning in place (Fig. 3B). This allowsbone remodeling and osseointegration of the biomaterial byosteoconduction in 8 to 9 months.

Sinus floor elevation

Placement of an implant in the posterior zone of the upperjaw is often challenging because bone loss is observed inmost patients with edentulation and thickness of the sinusfloor may be reduced up to 5 to 6 mm. It is evident thatsuch a reduced vertical bone height limits implant place-ment since an 8 to 10 mm of bone height is required to allowimplant placement. Thus, elevation of the maxillary sinusfloor is a recently described solution to restore a suitable 5to 10 mm height of available bone for implant placement.Sinus lifting was first reported by Boyne and James in the1960s [12]. Sinus floor elevation for dental implant place-ment is obtained by detachment of the sinus mucosa usinga lateral maxillary window approach (lateral anthrotomy, inmost cases) or by the crestal route, followed by the filling bya grafting material [13—15]. A crestal incision of the max-illa is made on the alveolar ridge with a vertical dischargeincision in the canine region and opposite to the posteriortuberosity. The gingival flap is then reclined to ensure goodvisibility of the anterior surface of the sinus and allow therealization of a bone window whose upper limit is 15 mmfrom the crest. The bone window is done with a diamondround bur under irrigation (Fig. 4A). The central trapdoorof cortical bone is preserved to maintain a bone layer for


the mucosa during the other surgical steps. The sinus mem-brane is then gently raised from the bone floor to delimit asufficient volume. At this stage, one should carefully checkthe presence of any perforation of the membrane becausethey can lead to complications at the time of placementof the biomaterial (Fig. 4B). A suture may be done with a6/0 thread or a �-TCP plate (Sinus-upTM, Kasios, France) toensure a complete closure of the sinus. The cavity is thenfilled with a biomaterial that will latter provide the bony bedsuitable for implant placement. Autogenous bone chips havelong been considered as the gold standard due to their pre-sumptive maintenance of cellular viability [12]. The choiceof a biomaterial must fulfill several qualities: it should bebiocompatible in the grafted site and should not inducean inflammatory reaction of the sinus mucosa, which couldbe deleterious to the whole surgical procedure. It must beintegrated within the grafted areas by combining its resorp-tion and its substitution by new bone. Resorption shouldbe progressive to allow bone remodeling with appositionof lamellar bone. Non-resorbable hydroxyapatite should beavoided [16]. Macroporosity of the grafted biomaterial playsa key role in bone osteoconduction because it determinesthe invasion of vascular and cellular sprouts [17,18]. Lastly,the grafting material must be provided in sufficient volumeto allow an easy adaptation in these complex surgical areasto fill volumes up 3 to 6 cm3. Over the years, several typesof materials including allografts and xenografts of various

origins have been used either alone or mixed with morcel-lized autogenic bone [19]. �-tricalcium phosphate was thefirst synthetic bone substitute to be proposed for sinus floorelevation [14]. The use of 1000—2000 �m granules of highporosity �-TCP HP is recommended nowadays [20]. Anthralfilling begins by placing granules on the posterior wall thenon the latero-anterior and anteroposterior walls withoutexcessive compression of the biomaterial (Fig. 4C). Theanterior wall is then filled and a resorbable SurgicelTM mem-brane (Ethicon, Johnson and Johnson) to avoid migrationof granules and sutured with Vicryl 5/0TM thread (Ethicon,Johnson and Johnson). A computed tomographic analysis isusually done 8 months later. There is a marked densificationof the grafted area filled by the biomaterial and no edemaof the sinus membrane can be evidenced (Fig. 4D).

The grafted area appears to be more radio-opaque thanthe surround bone tissue (due to the greater calcium contentof �-TCP). The density will progressively decrease as thebiomaterial is replaced by bone [21]. Histological analysisconfirmed the direct bonding of mineralized bone matrixto �-TCP granules together with the biomaterial resorption(Fig. 5A) [10,22,23]. Depending on the area, the bone has amatrix of lamellar or non-lamellar texture. The detection ofa key enzyme present in the osteoclasts (tartrate resistantacid phosphatase [TRAcP]) evidences these cells at the sur-face of the newly formed bone and at the surface of �-TCPgranules (Fig. 5B).

Figure 6 Lower alveolar nerve (IAN) lateralization. A. X-ray of a patient with an included canine whose ablation will create alarge bone defect. B. A significant bone defect is obtained and the emergence of the IAN is evidenced. C. Beta-tricalcium phosphate(�-TCP) granules (1000—2000 �m) with a high porosity are used to fill this area. D. X-ray of the patient after lateralization of theIAN and placement of four implants is now possible.

118 B. Guillaume

Lateralization of the inferior alveolar nerve (IAN)

Vertical atrophy of alveolar ridge is encountered at themandible as a sequela of dental extractions (Fig. 6A) or inpatients with a long history of wearing a removable pros-thesis. An onlay graft allows bone reconstruction in thevertical direction but requires the use autologous cortico-trabecular bone harvested at the calvarium, iliac crest,tibia or mandibular symphysis, retromolar area, mandibularramus [24]. The use of allogenic or xenogenic bone par-ticles has also been suggested. In all cases, the graft ismaintained to the alveolar ridge by osteosynthesis using tita-nium screws. The IAN remains one of the main anatomicallimitation to the placement of implants in the poste-rior mandibular zone. Another alternative is to move theIAN laterally from its canal by nerve lateralization andrepositioning to provide a bone area suitable for implantplacement [25]. The displacement of the IAN can be pro-posed in several situations: in case of compression of thenerve sheath following a trauma, when a removable prosthe-sis exerts compressive strains on the IAN or when an implantapex directly compresses the nerve [7,11,26]. Lateraliza-tion of the IAN radically changes the nature of the prostheticconcept where only the anterior region is usually concerned.Displacement of the nerve allows the placement of implantsin the posterior zone with a removable prosthesis with an O-ring system or a posterior bar. In fixed dental prosthesis, theposterior implants increase the holding of an overdentureor a fixed bridge by providing supporting pillars. In case of aremovable prosthesis, attachment of the prosthesis is con-siderably improved when an O-ring system or a posterior baris used.

The surgical technique is based on a vestibular osteotomyof the external cortical bone in front of the mental fora-men, and extended by fashioning of a bone flap towards therear [27—29]. The cortico-trabecular bone block is removedto gain access to the nerve and to gently tease it out ofthe mandible (Fig. 6B). The bone block is both removedin toto or morcellized and kept until the end of the inter-vention. Replacement of the bony flap allows an underlyingbone growth and prevents the nerve from returning to themandibular canal. Nevertheless, displacement of the IANcreates a bone void. It can be filled with �-TCP particles(Fig. 6C) held in place with a SurgicelTM membrane. Compactblocks of �-TCP can also be implanted. After an 8-monthbone healing period, the mandibular bone has been recon-stituted and offers a suitable site for implant placement(Fig. 6D). High porosity �-TCP granules plays an essentialrole in the local remodeling of bone volume and lead to theformation of a quality site.

Conclusion

Bone remodeling is an essential step in the healing processof a grafted area. The contribution of synthetic biomaterialsis now confirmed and guarantees an optimal reconstruc-tion that allows implant placement in a second step. Theavailability of �-TCP in the form of granules or compactslabs offers new therapeutic perspectives in maxillofacialand pre-implant surgery.


The author declares that he has no competing interest.

References

[1] Fennis J, Stoelinga P, Jansen J. Mandibular reconstruction: ahistological and histomorphometric study on the use of autog-enous scaffolds, particulate cortico-cancellous bone graftsand platelet rich plasma in goats. Int J Oral Maxillofac Surg2004;33:48—55.

[2] Guillaume B. Dental implants: a review. Morphologie2016;100:189—98.

[3] Holmquist P, Dasmah A, Sennerby L, Hallman M. A new tech-nique for reconstruction of the atrophied narrow alveolar crestin the maxilla using morselized impacted bone allograft andlater placement of dental implants. Clin Implant Dent RelatRes 2008;10:86—92.

[4] Matalova E, Fleischmannova J, Sharpe P, Tucker A. Tooth agen-esis: from molecular genetics to molecular dentistry. J DentRes 2008;87:617—23.

[5] Cortese A, Savastano G, Amato M, Cantone A, Boschetti C, Clau-dio PP. New palatal distraction device by both bone-borne andtooth-borne force application in a paramedian bone anchoragesite: surgical and occlusal considerations on clinical cases. JCraniofac Surg 2014;25:589—95.

[6] Browaeys H, Bouvry P, De Bruyn H. A literature review on bio-materials in sinus augmentation procedures. Clin Implant DentRelat Res 2007;9:166—77.

[7] Guillaume B. Latéralisation du nerf alvéolaire inférieurà visée pré-implantaire. Rev Stomatol Chir Maxillofac2012;113:327—34.

[8] Misch CM. Comparison of intraoral donor sites for onlay graftingprior to implant placement. Int J Oral Maxillofac Implants 1997,173-195;12.

[9] Nyström E, Lundgren S, Gunne J, Nilson H. Interpositional bonegrafting and Le Fort I osteotomy for reconstruction of theatrophic edentulous maxilla: a two-stage technique. Int J OralMaxillofac Surg 1997;26:423—7.

[10] Chappard D, Guillaume B, Mallet R, Pascaretti-Grizon F, BasléMF, Libouban H. Sinus lift augmentation and beta-TCP: amicroCT and histologic analysis on human bone biopsies. Micron2010;41:321—6.

[11] Guillaume B. Accroissement osseux pré-implantaire par dis-jonction osseuse. Inform Dent 2004;86:1640—8.

[12] Boyne PJ, James RA. Grafting of the maxillary sinus floor withautogenous marrow and bone. J Oral Surg 1980;38:613.

[13] Woo I, Le B. Maxillary sinus floor elevation: review of anatomyand two techniques. Implant Dent 2004;13:28—32.

[14] Tatum Jr H. Maxillary and sinus implant reconstructions. DentClin North Am 1986;30:207—29.

[15] Zerbo IR, Zijderveld SA, De Boer A, Bronckers AL, De Lange G,Ten Bruggenkate CM, et al. Histomorphometry of human sinusfloor augmentation using a porous �-tricalcium phosphate: aprospective study. Clin Oral Implants Res 2004;15:724—32.

[16] Smiler DG. Sinus lift procedure using porous hydroxyapatite: apreliminary clinical report. J Oral Implantol 1987;13:239—53.

[17] Ndiaye M, Terranova L, Mallet R, Mabilleau G, Chappard D.Three-dimensional arrangement of beta-tricalcium phosphategranules evaluated by microcomputed tomography and fractalanalysis. Acta Biomater 2015;11:404—11.

[18] Habibovic P, Yuan H, van der Valk CM, Meijer G, van Blit-terswijk CA, de Groot K. 3D microenvironment as essentialelement for osteoinduction by biomaterials. Biomaterials2005;26:3565—75.


[19] Malorana C, Sigurtà D, Mirandola A, Garlini G, Santoro F. Sinuselevation with alloplasts or xenogenic materials and implants:an up to 4-year clinical and radiologic follow-up. Int J OralMaxillofac Implants 2006, 426-432;21.

[20] Zerbo IR, Bronckers AL, De Lange GL, Burger EH, Van BeekGJ. Histology of human alveolar bone regeneration with aporous tricalcium phosphate. Clin Oral Implants Res 2001;12:379—84.

[21] Marchand-Libouban H, Guillaume B, Bellaiche N, Chappard D.Texture analysis of computed tomographic images in osteo-porotic patients with sinus lift bone graft reconstruction. ClinOral Investig 2013;17:1267—72.

[22] Guillaume B, Libouban H, Baslé MF, Chappard D. Comblementsinusien par �-TCP avant pose d’implants chez l’homme : étudeclinique et histologique. Titane 2010;7:201—10.

[23] Trombelli L, Franceschetti G, Stacchi C, Minenna L, Riccardi O,Di Raimondo R, et al. Minimally invasive transcrestal sinus floorelevation with deproteinized bovine bone or �-tricalcium phos-phate: a multicenter, double-blind, randomized, controlledclinical trial. J Clin Periodontol 2014;41:311—9.

[24] Schwartz-Arad D, Levin L, Sigal L. Surgical success of intra-oral autogenous block onlay bone grafting for alveolar ridgeaugmentation. Implant Dent 2005;14:131—8.

[25] Chrcanovic BR, Custódio ALN. Inferior alveolar nerve lateraltransposition. Oral Maxillofac Surg 2009;13:213—9.

[26] Khajehahmadi S, Rahpeyma A, Bidar M, Jafarzadeh H. Vital-ity of intact teeth anterior to the mental foramen afterinferior alveolar nerve repositioning: nerve transposition-ing versus nerve lateralization. Int J Oral Maxillofac Surg2013;42:1073—8.

[27] Metzger MC, Bormann K, Schoen R, Gellrich N, SchmelzeisenR. Inferior alveolar nerve transposition—–an in vitro compar-ison between piezosurgery and conventional bur use. J OralImplantol 2006;32:19—25.

[28] Jensen O, Nock D. Inferior alveolar nerve repositioning in con-junction with placement of osseointegrated implants: a casereport. Oral Surg Oral Med Oral Pathol 1987;63:263—8.

[29] Chossegros C, Cheynet F, Aldegheri A, Blanc J. Complete lat-eralization of the inferior alveolar nerve. A preliminary study,apropos of a case. Rev Stomatol Chir Maxillofac 1994;96:171—4.

Morphologie (2017) 101, 120—124


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ORIGINAL ARTICLE

Synthesis of �-tricalcium phosphateSynthèse du phosphate tricalcique-�

H. Chaair ∗, H. Labjar, O. Britel

Engineering laboratory processes and environment, faculty of Sciences and Technology, University HassanII-Casablanca , B.P. 146, 20650 Mohammedia, Morocco

Available online 21 September 2017

KEYWORDSBiomaterials;Bone substitutes;Apatite;Tricalciumphosphate;�-TCP

Summary Ceramics play a key role in several biomedical applications. One of them is bonegrafting, which is used for treating bone defects caused by injuries or osteoporosis. Calcium-phosphate based ceramic are preferred as bone graft biomaterials in hard tissue surgery becausetheir chemical composition is close to the composition of human bone. They also have amarked bioresorbability and bioactivity. In this work, we have developed methods for synthe-sis of �-tricalcium phosphate apatite (�-TCP). These products were characterized by differenttechniques such as X-ray diffraction, infrared spectroscopy, scanning electron microscopy andchemical analysis.© 2017 Elsevier Masson SAS. All rights reserved.

Résumé Les céramiques sont des biomatériaux jouant un rôle clé dans plusieurs applica-tions biomédicales. L’un d’eux est la possibilité de les utiliser comme matériaux de greffeosseuse dans le traitement des défauts osseux causés par une blessure ou une ostéoporose. Lescéramiques à base de phosphate de calcium sont préférées comme greffons osseux en chirurgiedes tissus durs en raison de leur composition chimique puisqu’elles sont proches de la compo-sition de l’os humain, bio-résorbables et ont une forte bio-activité. Dans ce travail, nous avonsdéveloppé différentes méthodes de synthèse de l’apatite phosphate tricalcique � (�-TCP). Cesproduits ont été caractérisés par différentes techniques telles que la diffraction des rayons X,la spectroscopie infrarouge, la microscopie électronique à balayage et l’analyse chimique.© 2017 Elsevier Masson SAS. Tous droits reserves.

Introduction

The physicochemical characteristics of calcium phosphatesare responsible for their biological activity [1]. Hydroxy-apatite chemically is a calcium phosphate close to the

∗ Corresponding author.E-mail address: [email protected] (H. Chaair).

crystalline phase of the bone matrix. However, because ofits molar Ca/P ratio of 1.67, the apatite is very slightly sol-uble in biological media and hence its degradation rate isvery low in vivo [2—5]. Tricalcium phosphate (Ca3(PO4)2) ischaracterized by an atomic ratio Ca/P of 1.5, exists in fourdifferent forms:

• the � form is stable between 1120 and 1470 oC and ismetastable at room temperature;







Synthesis of �-tricalcium phosphate 121

Figure 1 Elementary mesh of Ca3(PO4) (according to refer-ence [8]).

• the � form is stable above 1470 oC;• the � form i �-TCP s stable below 1120 oC;• the � form obtained under high pressure.

The � form cannot be directly synthesized by precipi-tation in aqueous environment. It is produced by heatingamorphous calcium phosphate (ACP) between 800 and1000 oC [6,7]. �-TCP crystallizes in the trigonal system(hexagonal mark) (space group R3c) [8]; the crystallographyparameters are a = b = 10.439 A; and c = 37.375 A. It is alsopossible to synthesize �-TCP by heating an intimate mixtureof solid as CaHPO4 and CaCO3 at 1000 oC for one hour. In crys-tallographic studies, the content of the asymmetric unit,taking into account the multiplicity of different Wyckoffpositions occupied by atoms, leads to a stoichiometry consis-tent with the formulation Ca3(PO4)2. The phosphate anionsoccupy coordinated sites of four generated by the arrange-ment of four oxygen anions first neighbours. The averagedistance equal to 1.535 A, is conform to the expected dis-tances for the anion (PO4) (Fig. 1). In contrast, �-TCP whichhas a Ca/P ratio of 1.5 is much more soluble than hydroxyap-atite and is more subjected to degradation. �-TCP producesceramic granules usable as bone substitutes [8,9]. Theseceramics are bioactive and allow interactions between cellsand body fluids, thus allowing a good bone integration. Sim-ilarly, their biocompatibility is well known from decades.They do not induce immunological or tissue toxic reactionsnor foreign body reaction. However, they have no osteoin-ductive properties because there is no bone formation when�-TCP particles are implanted in muscle: in particular theyinduce inflammation and encapsulation. However, �-TCPgranules are osteoconductive because they induce a directcontact at the periphery and inside the internal macroporesof the biomaterial.

�-TCP is an anhydrous tricalcium phosphate. It isobtained by calcination at 900 oC of apatitic tricalcium phos-phate or amorphous tricalcium phosphate [6,10]. The latterare prepared by different routes:

• in aqueous environment by neutralization;• in an aqueous environment by hydrolysis of brushite

(DCPD) [11—13] or in alcoholic medium by double decom-position [8,13];

• by the sol-gel method [19,21].

Materials and methods for synthesis of TCPand �-TCP

Method of synthesis of phosphate tricalcium (TCP)from Ca(NO3)2 and (NH4)2HPO4 or doubledecomposition

Tricalcium phosphate apatitic was prepared by precipita-tion by fast double decomposition from solution A (calciumnitrate) and solution B (ammonia orthophosphate ions)[18,20]. The molar ratio Ca/P in solution was fixed at 1.5:

• solution A: 46.73 g of calcium nitrate Ca(NO3)2, 4H2Owere dissolved in 550 mL of distilled decarbonated water(0.36 M) added with 40 mL of pure ammonia (d = 0.92);

• solution B: 25.74 g of ammonium dihydrogen phosphate(NH4)2HPO4 were dissolved in 1300 mL of decarbonateddistilled water (0.15 M). To this solution 40 mL of pureammonia (d = 0.92) were added.

The precipitation was carried at a temperature of37 ± 0.1 oC by rapidly pouring, with stirring, solution A ina reactor of two-litre containing the solution B. The precipi-tate was separated from the mother liquors by filtration on aBuchner’s funnel, washed several times with distilled watercontaining ammonia. It was then dried at 80 oC overnight.The product no dried obtained under these conditions isamorphous and can be described by the following formula:

Ca9(PO4)6, nH2OThe beta tricalcium phosphate (�-TCP) was prepared by

calcination at 900 oC of the synthesized precipitate. Thisis a transition phase during the precipitation of deficientapatites. The amorphous tricalcium phosphate was charac-terized by X-ray diffraction by a broad halo correspondingto an amorphous product. Apatitic tricalcium phosphate isthe crystalline form at low temperature of amorphous tri-calcium phosphate. It presents a chemical formula differentsince during crystallization, a hydrolysis internal of PO4

3−

group occurs simultaneously [10]. This latter form an apatitestructure of compound of the following formula:

Ca9 (HPO4) (PO4)5 (OH)

Method of synthesis of phosphate tricalcium (TCP)per neutralisation from CaCO3 and H3PO4

We also propose to prepare tricalcium phosphate apatitefrom low-cost reagents available such as calcium carbonate,CaCO3 as a calcium source and phosphoric acid H3PO4 as aphosphate source using the method established by Heughe-baert [8,14].

Solution A was prepared from the attack of 66 g of cal-cium carbonate CaCO3 by 91.4 mL of nitric acid HNO3 (d = 1.4and P = 65%) according to the following reaction:

CaCO3 + 2HNO3 → Ca(NO3)2 + H2O + CO2

After complete dissolution of the solid and cooling of thesolution was made up to 550 mL with distilled water and then40 mL decarbonated pure ammonia (d = 0.92).

122 H. Chaair et al.

Table 1 XRD Analysis. Inter-reticular spacing and intensityof the principal diffraction lines for ß-TCP.

D theoretical(oA) I/I0 HkL

5.21 20 1104.06 16 0243.45 25 10103.21 55 2143.01 16 3002.88 100 02102.76 20 1282.61 65 220

Table 2 FTIR Analysis. Position of the bands and theirintensities for ß-TCP.

Wave number (cm−1) Intensity

3420 Low1125 Strong1051 Very strong985 Very strong945 Strong609 Strong604 Shoulder557 Strong503 Shoulder

Solution B was obtained by neutralization of 30 mL oforthophosphoric acid H3PO4 (d = 1.68 and P = 85%) with pureammonia (d = 0.92) according to the following reaction:

H3PO4 + 2NH4OH → (NH4)2 + H2O2PO4

After cooling the solution, the mixture was completedto 1300 mL with distilled and decarbonated water. On theother hand, one proceeds to the precipitation of the solidby rapidly introducing the solution B in a reactor of capacity2 litres already containing the solution A at a temperatureof 60 oC. The pH of the reaction medium was kept constantat a value of 7 by the addition of pure ammonia (d = 0.92)by means of a metering pump controlled by a pH stat con-nected to a measuring electrode of the pH. After 4 hours ofstirring at the synthesis temperature, the product formedwas Buchner filtered, washed, dried at 80 oC and calcined at900 oC in air for one hour.

Method of synthesis of phosphate tricalcium (TCP)by sol gel

Calcium nitrate tetrahydrate (Ca(NO)2 4H2O, Merck) andphosphorus pentoxide (P, Merck) were selected as Ca andP precursors, respectively. A design amount of Ca(NO3), 4H2O and P2O5 was dissolved in absolute ethanol to form0.01 mol and 0.003 mol solution, respectively. The solutionswere mixed as initial mixed precursor solution and werecontinuously stirred for 30 minutes at ambient temperatureuntil a white transparent gel was obtained. The gel was driedat 80 oC in an oven for 24 hours and then further calcined at

Figure 2 XRD of ß-TCP sintered at 900 oC.

Figure 3 FTIR analysis of ß-TCP sintered at 900 oC.

600, 700, 800, 900 and 1000 oC at a heating rate of 5 oC/minwith six hours holding time. The samples then characterizedusing XRD, SEM-EDS and FTIR techniques [15—17].

Results and discussions

The calcination temperature plays an important role inthe formation of ß-TCP. The precipitated products werecharacterized by X-ray diffraction, infrared absorptionspectroscopy and scanning electron microscopy (SEM) andchemical analysis. �-TCP is generally used to produceceramics that can be use a bone substitute. It is obtained byheating at 900 oC, The equation proposed for synthesis is:

Ca9 (HPO4) (PO4)5(OH)3900◦C−→� Ca3(PO4)2 + H2O

�-TCP has a rhombohedral structure (space group R3c)[10] and the following lattice parameters: a = 10.429 A;c = 37.380 A.

XRD Analysis

The XRD diffractogram of powder obtained at calcinationtemperature of 900 oC is shown in Fig. 2. X-ray diffractionspectra were obtained using a diffractometer XRD 6000 typeof meter (Shimadzu, Japan) using a radiation emitted from acopper anticathode (� = 1.54060 A). The inter-reticular spac-ing’s and intensities of the principal diffraction lines aregiven in Table 1.

Synthesis of �-tricalcium phosphate 123

Figure 4 SEM micrograph of ß-TCP sintered at 900 oC.

FTIR Analysis

The infrared spectrometry analysis was done with a Shi-madzu IR spectrometer type solution 1.30 wavenumber FTIRbetween 4000 and 600 cm−1 (Fig. 3). Table 2 presents theposition of the bands and their intensities.

Scanning electron microscopy (SEM)

Fig. 4 shows the SEM micrographs of the powder producedat a calcination temperature of 900 oC. It is clearly observedthat the size of the grains of the powder is bigger with theincrease of the calcination temperature from 900 oC. Thepurity of the powder obtained at a 900 oC calcination tem-perature was also confirm by EDS analysis. The elementalanalysis shows the existence of elements such as O, P andCa of calcium phosphates as shows in Fig. 5. This EDS datashows similar result of pure ß-TCP which was produced at thetemperature of 900 oC as well as determined by XRD analysis(Fig. 4 and Fig. 5) [21].

Conclusion

The methods used in the present study allowed us toobtain the pure apatite tricalcium phosphate with a ratioCa/P = 1.5. The synthesized powder was transformed intopure �-TCP after calcination at 900 oC. These products werecharacterized by different techniques such as X-ray diffrac-tion, infrared spectroscopy, SEM and chemical analysis. Thesintering of powders has strengthened the �-TCP withouttransformation of the � phase of tricalcium phosphate.

Figure 5 Composition of powder determined by SEM-EDS for a calcination temperature of 900 oC (according to reference [21]).

124 H. Chaair et al.


The authors declare that they have no competing interest.

References

[1] Barralet JE, Best SM, Bonfield W. Effect of sintering parameterson the density and microstructure of carbonate hydroxyapa-tite. J Mater Sci Mater Med 2000;11:719—24.

[2] Doi Y, Shibutani T, Moriwaki Y, Kajimoto T, Iwayama Y. Sin-tered carbonated apatites as bioresorbable bone substitutes. JBiomed Mater Res 1998;39:603—10.

[3] Chaair H, Mansouri I, Heughebaert M, Nadir S. Statisticalanalysis of calcium phosphates preparation. Phosphorus SulfurSilicon Rel Elem 2001;173:163—74.

[4] Nelson DGA. The influence of carbonate on the Atomicstructure and reactivity of hydroxyapatite. J Dent Res1981;60:1621—9.

[5] Ghosh R, Sarkar R, Paul S, Pal SK. Biocompatibility and drillingperformance of beta-tricalcium phosphate: yttrium phosphatebioceramic composite. Ceram Int 2016;42:8263—73.

[6] Macarovici D. Les réactions de synthèse du phosphate ter-tiaire de calcium par voie thermique. Rev Roum Chim1966;11:725—31.

[7] Chaair H, Mansouri I, Heughebaert M, Nadir N. Statisticalanalysis of calcium phosphates preparation. Phosphorus SulfurSilicon Rel Elem 2001;173:163—74.

[8] Britel O. Modélisation et optimisation par la méthodologiedes plans d’expériences de la synthèse: de l’hydroxyapatitephosphocalcique, du phosphate tricalcique apatitique, duphosphate de calcium apatitique carbonate. In: PhD Thesis.Rabat; 2017.

[9] Heughebaert JC. Contribution à l’étude de l’évolutiondes orthophosphates de calcium précipités amorphes enorthophophates apatitiques. PhD Thesis. France: INP, Toulouse;1977.

[10] Dickens B, Schroeder LW, Brown WE. Crystallographic studiesof the role of Mg as a Stabilizing impurity in beta-Ca3(PO4)2,I. The crystal structure of pure beta-Ca3(PO4)2. J Solid StateChem 1974;10:232—48.

[11] Schmitt M. Contribution à l’élaboration de nouveaux matéri-aux biphasés en phosphates de calcium. PhD Thesis. France:Nantes; 2000.

[12] Ducheyne P, Radin S, King L. The effect of calcium phosphateceramic composition and structure on in vitro behavior. I. Dis-solution. J Biomed Mater Res 1993;27:25—34.

[13] Ereiba KMT, Mostafa AG, Gamal GA, Said AH. In vitro study ofiron doped hydroxyapatite. J Biophys Chem 2013;4:122—30.

[14] Heughebeart JC, Montel G. Conversion of Amorphous trical-cium phosphate into apatitic tricalcium phosphate. CalcifTissue Int 1982;34:103—8.

[15] Montel G. Sur la formation de certaines apatites par hydrol-yse du phosphate bicalcique hydraté. Bull Soc Chim F1953;7:506—11.

[16] Wallaeys R. Contribution à l’étude des apatites phosphocal-ciques. Ann Chim 1952;7:808—48.

[17] Gallur A, Descamps M, Richart O, Thierry B, Anselme K, LuJX, et al. Le naphtalène: agent porogène pour l’élaboration desubstituts osseux en hydroxyapatite et phosphate tricalciquebêta. Ann Biomater 1997;6:127—35.

[18] Marraha M, Heughebeart JC, Bonel G. Nouvelles méthodes depréparation de matériaux à base de phosphate de calcium àusages biologiques. Innov Tech Biol Med 1984;5:360—8.

[19] Sanosh KP, Chu MC, Balakrishnan A, Kim TN, Cho SJ. Sol gelsynthesis of pure nanosized � tricalcium phosphate crystallinepowders. Curr Appl Phys 2010;10:68—71.

[20] Chaair H. Optimisation de la synthèse en continu desphosphates de calcium. PhD Thesis. France: INP Toulouse;1993.

[21] Muhamad MA, Mohd Ridzuan M, Arifin Ahmad Z. Syn-thesis and characterization of ß-tricalcium phosphateceramic via sol-gel method. J Nucl Rel Technol 2009;6:199—205.

Morphologie (2017) 101, 125—142


ScienceDirect


GENERAL REVIEW

Calcium orthophosphates (CaPO4):Occurrence and propertiesLes orthophosphates de calcium (CaPO4) : occurrence etpropriétés

S.V. Dorozhkin

1-155, Kudrinskaja sq., Moscow 123242, Russia


KEYWORDSCalciumorthophosphates;Hydroxyapatite;Fluorapatite;Applications

Summary The present overview is intended to point the readers’ attention to the importantsubject of calcium orthophosphates (CaPO4). This type of materials is of the special signifi-cance for the human beings because they represent the inorganic part of major normal (bones,teeth and antlers) and pathological (those appearing due to various diseases) calcified tissuesof mammals. For example, atherosclerosis results in blood vessel blockage caused by a solidcomposite of cholesterol with CaPO4, while dental caries (tooth decay) and osteoporosis (a lowbone mass with microarchitectural changes) mean a partial decalcification of teeth and bones,respectively, that results in replacement of a less soluble and harder biological apatite by moresoluble and softer calcium hydrogenorthophosphates. Due to the compositional similarities tothe calcified tissues of mammals, CaPO4 are widely used as biomaterials for bone grafting pur-poses. In addition, CaPO4 have many other applications. Thus, there is a great significance ofCaPO4 for the humankind and, in this paper, an overview on the current knowledge on thissubject is provided.© 2017 Elsevier Masson SAS. All rights reserved.

Résumé Cette revue vise à attirer l’attention des lecteurs sur l’important sujet desorthophosphates de calcium (CaPO4). Ce type de matériaux revêt une importance spé-ciale pour l’Homme car ils représentent, chez les mammifères, la partie inorganique de lamajorité des tissus calcifiés normaux (os, dents et bois) et pathologiques (ceux qui appa-raissent lors de diverses maladies). Par exemple, l’athérosclérose entraîne une obturationdes vaisseaux sanguins causée par un complexe solide de cholestérol et de CaPO4 tan-dis que les caries dentaires et l’ostéoporose (caractérisée par une faible masse osseuseavec des changements microarchitecturaux) traduit une perte partielle de la matrice desdents et de l’os qui aboutit au remplacement d’une apatite biologique moins soluble et

E-mail address: [email protected]







126 S.V. Dorozhkin

plus difficile par des hydrogéno-orthophosphates de calcium plus solubles et moins résistants.En raison de la composition similaire avec les tissus calcifiés des mammifères, le CaPO4 estlargement utilisé comme biomatériau pour la réalisation de greffes osseuses. De plus, leCaPO4 possède de nombreuses autres applications. Ainsi, il existe un grand intérêt des phos-phates de calcium pour l’humanité et, dans cette revue, nous faisons le point des connaissancessur le sujet.© 2017 Elsevier Masson SAS. Tous droits reserves.

Introduction

Due to the abundance in nature (as phosphate ores) andpresence in living organisms (as bones, teeth, deer antlersand the majority of various pathological calcifications), cal-cium phosphates are the inorganic compounds of a specialinterest for human being. They were discovered in 1769and have been investigated since then [1,2]. According tothe databases of scientific literature (Web of science, Sco-pus, Medline, etc.), the total amount of currently availablepublications on the subject exceeds 40,000 with the annualincrease for, at least, 2000 papers. This is a clear confirma-tion of the importance.

Briefly, by definition, all known calcium phosphatesconsist of three major chemical elements: calcium (oxi-dation state +2), phosphorus (oxidation state +5) andoxygen (reduction state —2), as a part of the phosphateanions. These three chemical elements are present in abun-dance on the surface of our planet: oxygen is the mostwidespread chemical element of the earth’s surface (∼47mass %), calcium occupies the fifth place (∼3.3—3.4 mass%) and phosphorus (∼0.08—0.12 mass %) is among the firsttwenty of the chemical elements most widespread on ourplanet. These concentrations correspond to a calculatedtheoretical normative value of ∼0.3 vol.% calcium phos-phates in the upper continental and oceanic crusts. Inaddition, the chemical composition of many calcium phos-phates includes hydrogen, as an acidic orthophosphate anion(for example, HPO4

2− or H2PO4−), hydroxide (for example,

Ca10(PO4)6(OH)2) and/or incorporated water (for exam-ple, CaHPO4·2H2O). Regarding their chemical composition,diverse combinations of CaO and P2O5 oxides (both in thepresence of water and without it) provide a large variety ofcalcium phosphates, which are differentiated by the type ofthe phosphate anion. Namely, ortho- (PO4

3−), meta- (PO3−),

pyro- (P2O74−) and poly- ((PO3)n

n−) phosphates are known.Furthermore, in the case of multi-charged anions (validfor orthophosphates and pyrophosphates), calcium phos-phates are also differentiated by the number of hydrogenions substituted by calcium ones. The examples comprisemono- (Ca(H2PO4)2), di- (CaHPO4), tri- (Ca3(PO4)2) and tetra-(Ca2P2O7) calcium phosphates [3]. However, to narrow thesubject, calcium orthophosphates (abbreviated as CaPO4)will be considered and discussed only. Their names, standardabbreviations, chemical formulae and solubility values arelisted in Table 1 [4,5]. Since all of them belong to CaPO4,strictly speaking, all abbreviations in Table 1 are incorrect;however, they have been extensively used in literature fordecades and, to avoid confusion, there is no need to modifythem.

In general, the atomic arrangement of all CaPO4 is builtup around a network of orthophosphate (PO4) groups, whichstabilize the entire structure. Therefore, the majority ofCaPO4 are sparingly soluble in water (Table 1); however, allof them are easily soluble in acids but insoluble in alka-line solutions. In addition, all chemically pure CaPO4 arecolorless transparent crystals of moderate hardness but,as powders, they are of white color. Nevertheless, natu-ral minerals of CaPO4 are always colored due the presenceof impurities and dopants, such as ions of Fe, Mn andrare earth elements [6]. Biologically formed CaPO4 are themajor component of all mammalian calcified tissues, whilethe geologically formed ones are the major raw materialto produce phosphorus-containing agricultural fertilizers,chemicals and detergents.

Geological and biological occurrences

Geologically, natural CaPO4 are found in different regionsmostly as deposits of apatites, mainly as ion-substituted FA(igneous rocks), and phosphorites (sedimentary rocks) [7].In addition, natural ion-substituted CDHA was also found [8]but it is a very rare mineral. Some types of sedimentary rockscan be formed by weathering of igneous rocks into smallerparticles. Other types of sedimentary rocks can be com-posed of minerals precipitated from the dissolution productsof igneous rocks or minerals produced by biomineralization(Fig. 1). Thus, due to a sedimentary origin, both a generalappearance and a chemical composition of natural phos-phorites vary a lot [9]. It is a common practice to considerfrancolite (or carbonate-hydroxyfluorapatite regarded as itssynonym) as the basic phosphorite mineral [7,10]. Accord-ing to Henry [11], the name francolite was given by Mr.Brooke and Mr. Nuttall to a mineral from Wheal Franco, Tavi-stock, Devon, some years prior to 1850. A cryptocrystalline(almost amorphous) variety of francolite (partly of a bio-logical origin) is called collophane (synonyms: collophanit,collophanita, collophanite, grodnolite, kollophan), named in1870 by Karl Ludwig Fridolin von Sandberger from the Greekroots �о�� (= glue) and �� (to appear) referringto the appearance of the mineral [12]. Francolite is foundin natural phosphorites predominantly as fossil bones andphosphatized microbial pseudomorphs: phosphatic crusts ofchasmolithic biofilms (or microstromatolites) and globularclusters with intra-particular porosities [13,14]. The trans-formation process of bones into fossil bones is well describedelsewhere [15]. Natural phosphorites (therefore, francol-ite and collophane as well) occur in various forms, suchas nodules, crystals or masses. Occasionally, other types

Calcium orthophosphates (CaPO4): Occurrence and properties 127

Table 1 Existing calcium orthophosphates and their major properties [4].

Ca/P molar ratio Compound Formula Solubility at25 ◦C, -log(Ks)

Solubility at25 ◦C, g/L

pH stabilityrange in aqueoussolutions at 25 ◦C

0.5 Monocalcium phosphatemonohydrate (MCPM)

Ca(H2PO4)2·H2O 1.14 ∼18 0.0—2.0

0.5 Monocalcium phosphateanhydrous (MCPA or MCP)

Ca(H2PO4)2 1.14 ∼17 c

1.0 Dicalcium phosphatedihydrate (DCPD), mineralbrushite

CaHPO4·2H2O 6.59 ∼0.088 2.0—6.0

1.0 Dicalcium phosphateanhydrous (DCPA or DCP),mineral monetite

CaHPO4 6.90 ∼0.048 c

1.33 Octacalcium phosphate(OCP)

Ca8(HPO4)2

(PO4)4·5H2O96.6 ∼0.0081 5.5—7.0

1.5 �-Tricalcium phosphate(�-TCP)

�-Ca3(PO4)2 25.5 ∼0.0025 a


�-Ca3(PO4)2 28.9 ∼0.0005 a

1.2—2.2 Amorphous calciumphosphates (ACP)

CaxHy(PO4)z

·nH2O,n = 3—4.5;15—20% H2O

b b ∼5—12d

1.5—1.67 Calcium-deficienthydroxyapatite (CDHA orCa-def HA)e

Ca10−x(HPO4)x(PO4)6−x(OH)2−x

(0 < x < 1)

∼85 ∼0.0094 6.5—9.5

1.67 Hydroxyapatite (HA, HAp orOHAp)

Ca10(PO4)6(OH)2 116.8 ∼0.0003 9.5—12

1.67 Fluorapatite (FA or FAp) Ca10(PO4)6F2 120.0 ∼0.0002 7—121.67 Oxyapatite (OA, OAp or

OXA)f, mineral voelckeriteCa10(PO4)6O ∼69 ∼0.087 a

2.0 Tetracalcium phosphate(TTCP or TetCP), mineralhilgenstockite

Ca4(PO4)2O 38—44 ∼0.0007 a

a These compounds cannot be precipitated from aqueous solutions.b Cannot be measured precisely.c Stable at temperatures above 100 ◦C.d Always metastable.e Occasionally, it is called ‘‘precipitated HA (PHA)’’.f Existence of OA remains questionable.

Figure 1 A simplified schematic of the phosphorus cycle from apatitic igneous rock to phosphorite sedimentary rock throughchemical or physical weathering. Life forms accumulate soluble phosphorus species and can produce apatite through biomineral-ization.

128 S.V. Dorozhkin

Table 2 Comparative composition and structural parameters of inorganic phases of adult human calcified tissues. Due to theconsiderable variation found in biological samples, typical values are given in these cases.

Enamel Dentin Cementum Bone HA

Composition, wt%Calciuma 36.5 35.1 ∼35 34.8 39.6Phosphorus (as P)a 17.7 16.9 ∼16 15.2 18.5Ca/P (molar ratio)a 1.63 1.61 ∼1.65 1.71 1.67Sodiuma 0.5 0.6 c 0.9 —Magnesiuma 0.44 1.23 0.5—0.9 0.72 —Potassiuma 0.08 0.05 c 0.03 —Carbonate (as CO3

2−)b 3.5 5.6 c 7.4 —Fluoridea 0.01 0.06 Up to 0.9 0.03 —Chloridea 0.30 0.01 c 0.13 —Pyrophosphate (as P2O7

4−)b 0.022 0.10 c 0.07 —Total inorganicb 97 70 60 65 100Total organicb 1.5 20 25 25 —Waterb 1.5 10 15 10 —

Crystallographic properties: Lattice parameters (± 0.003 Å)a-axis, Å 9.441 9.421 c 9.41 9.430c-axis, Å 6.880 6.887 c 6.89 6.891Crystallinity index (HA = 100) 70—75 33—37 ∼30 33—37 100Typical crystal sizes (nm) 100 �m ×50 × 50 35 × 25 × 4 c 50 × 25 × 4 200—600Ignition products (800 ◦C) �-TCP + HA �-TCP + HA �-TCP + HA �-TCP + HA HAElastic modulus (GPa) 80 23.8 ± 3.7 15.0 ± 3.6 0.34—13.8 10Tensile strength (MPa) 10 100 c 150 100

a Ashed samples.b Unashed samples.c Numerical values were not found in the literature but they should be similar to those for dentin.

of natural CaPO4 are found as minerals, for example clino-hydroxylapatite, staffelite (synonyms: staffelit, staffelita)belonging to carbonate-rich fluorapatites (chemical for-mula: Ca5[(F,O)(PO4,CO3)3]) and DCPD. Furthermore, CaPO4

were found in meteoric stones [16]. The world deposits ofnatural CaPO4 are estimated to exceed 150 billion tons; fromwhich approximately 85% belong to phosphorites and theremaining ∼15% belong to apatites [7].

As minor constituents (< ∼5%), natural CaPO4 (bothapatites and phosphorites) occur in many geological envi-ronments [17]. Concentrations sufficient for economic use(> 15%) are also available. Namely, the largest world depositsof natural apatites are located in Russia (the Khibiny andKovdor massifs, Kola peninsula), Brazil and Zambia, whilethe largest world deposits of natural phosphorites arelocated in Morocco, Russia, Kazakhstan, USA (Florida, Ten-nessee), China and Australia [7]. In addition, they are foundat seabed and ocean floor [18]. The majority of naturalCaPO4 occur as small polycrystalline structures (spheruliticclusters). Larger crystals are rare. They usually have thecrystal structure of apatites (hexagonal system, space groupP63/m). Giant crystals including ‘‘a solid but irregular massof green crystalline apatite, 15 feet long and 9 feet wide’’[19] and a single euhedral crystal from the Aetna mine mea-suring 2.1 × 1.2 m with an estimated weight of 6 tons [20]were found. None of them is a pure compound; they alwayscontain dopants of other elements.

Apatites represent a special supergroup of minerals witha generic chemical formula IXM12

VIIM23(IVTO4)3X with 2 dis-tinct metal-cation sites (M1 and M2), a tetrahedral-cation

site (T), an anion column along four edges of the unit celland 2 formula units per unit cell (Z = 2), in which M = Ca2+,Pb2+, Ba2+, Sr2+, Mn2+, Mg2+, Fe2+, Na+, K+, Ce3+, La3+, Y3+,Bi3+; T = P5+, As5+, V5+, Si4+, S6+, B3+; X = F−, OH−, Cl−. Chem-ically, apatites can be orthophosphates, orthoarsenates,orthovanadates, orthosilicates and sulfates; however, CaPO4

apatites (FA, HA and CDHA) appear to be the most commonmembers of the apatite supergroup. Since this supergroupcontains over 40 mineral species, it is not surprising thatmore than half the elements occurring as long-lived isotopeson Earth can be incorporated into the apatite structure inalmost any valence state [21]. In Ref. [21], the interestedreaders can find the Periodic Chart, in which the elementsfound in the apatite supergroup minerals in amounts rangingfrom ppm to tens of weight percent are marked. Namely,substituents such as the first row transition elements andthe lanthanides (they act as activators and chromophores)impart colors and can lead to luminescence. Furthermore,crystal imperfections, such as site vacancies, vacancies withtrapped electrons and point defect clusters, can likewiseinfluence both color and luminescence. The substitutionsin apatites are usually in trace concentrations; however,for some dopants (e.g., F− and OH−) large concentrationsand even complete solid solutions exist. To make thingseven more complicated, some ions in the crystal structuremay be missing, leaving the crystallographic defects, whichleads to formation of non-stoichiometric compounds, suchas CDHA. Due to their affinity for chromophoric substituentsand propensity for other defects, natural apatites are foundin just about all colors of the rainbow. Ease of atomic


Figure 2 Phase diagram of the system CaO—P2O5 (C = CaO, P = P2O5) at elevated temperatures. Here: C7P5 means 7CaO·5P2O5;other abbreviations should be written out in the same manner. Reprinted from Ref. [36] with permission.

substitution for apatite leaves this mineral open to a widearray of compositions [21]. In medicine, this property mightbe used as an antidote for heavy metal intoxication [22].Furthermore, organic compounds have been found in naturalapatites [23].

Manufacturing of elementary phosphorus (white andred), phosphoric acids, various P-containing chemicals,agricultural fertilizers (namely, superphosphate, ammo-nium orthophosphates) and detergents (principally sodiumtripolyphosphate) are the major industrial applications ofnatural CaPO4. The annual consumption of a phosphate rockhas approached ∼150 million tons and about 95 percent ofthis production is utilized in the fertilizer industry. In addi-tion, natural CaPO4 appear to be the important chemicals inenvironmental remediation of contaminated groundwater.In this process, the groundwater reacts with CaPO4 in thereactive barrier and metal contaminants are precipitated asinsoluble and non-bioavailable orthophosphate phases. Sim-ilarly, they can be used in permeable reactive barriers toisolate radionuclides from groundwater and as a waste formin planned nuclear waste repositories.

In biological systems, many organisms, ranging frombacteria and isolated cells to invertebrates and vertebrates,synthesize CaPO4. Formation of solid CaPO4 in primitiveorganisms is believed to enable the storage and regulationof essential elements such as calcium, phosphorus and, pos-sibly, magnesium. The morphology of precipitates in theseorganisms (small intracellular nodules of ACP often locatedin mitochondria) complies with the necessities for rapidmobilization and intracellular control of the concentrationof these elements [24]. In vertebrates CaPO4 occur as theprincipal inorganic constituent of normal (bones, teeth,fish enameloid, deer antlers and some species of shells)and pathological (dental and urinary calculus and stones,

atherosclerotic lesions, etc.) calcifications [25]. In addition,they are found in ganoid fish scales (in alligator gar andSenegal bichir), turtle shells, as well as in armadillo andalligator osteoderms [26]. In minute quantities CaPO4 existin the brain (brain sand), without significantly affecting itsfunction [27]. Therefore, the expression ‘‘having sand inthe head’’ is not without a reason. Except for small por-tions of the inner ear, all hard tissues of the human body areformed of CaPO4. Structurally, they occur mainly in the formof poorly crystalline, non-stoichiometric, Na−, K−, Mg− andcarbonate-containing CDHA. It is often called ‘‘biologicalapatite’’ (which might be abbreviated as BAp), bioapatite ordahllite. The latter term was introduced in 1888 by Bröggerand Bäckström [28] after the Swedish mineralogist brothersTellef and Johan Martin Dahll. In addition, in 2016, the firstreport on the CaPO4 presence in some parts of higher plantswas published [29], which may become the starting point forthe development of biomimetic CaPO4 biocomposites basedon a cellulose matrix.

The main constituents of human bones are CaPO4

(∼60—70 wt.%), collagen (∼20—30 wt.%) and water (up to10 wt.%) [27]. The detailed information on the chemicalcomposition of the most important human normal calci-fied tissues is comprised in Table 2. One should note thatthe values mentioned in Table 2 are approximate; the mainconstituents can vary by a percent or more. Due to the afore-mentioned effect of lattice flexibility, bones act as boththe mineral reservoir of the body and the storage for toxicelements, thus fulfilling two of its essential physiologicalroles.

Finally, one should mention, that, in a dissolved state,CaPO4 are found in many biological liquids, such as bloodserum [30], urine [31], sweat [32], milk [33], etc. [34] and,therefore, in dairy products [35].

130 S.V. Dorozhkin

Figure 3 pH variation of ionic concentrations in triproticequilibrium for orthophosphoric acid solutions. Reprinted fromRef. [38] with permission.

The members of CaPO4 family

In the ternary aqueous system Ca(OH)2—H3PO4—H2O(or CaO—P2O5—H2O) there are twelve known non-ion-substituted CaPO4 with the Ca/P molar ratio rangingbetween 0.5 and 2.0 (Table 1). An anhydrous phase diagramCaO—P2O5 at temperatures within 200—2200 ◦C is shown inFig. 2 [36]. Table 3 comprises crystallographic data of theexisting CaPO4 [3]. The most important parameters of CaPO4

are the ionic Ca/P ratio, basicity/acidity and solubility. Allthese parameters strongly correlate with the solution pH.The lower the Ca/P molar ratio is, the more acidic andwater-soluble the CaPO4 is [3]. Therefore, the Ca/P ratiocan be used as a fingerprint of the CaPO4 phases. One cansee that the solubility ranges from high values for acidiccompounds, such as MCPM, to very low values for basic com-pounds, such as apatites, which allow CaPO4 to be dissolved,transported from one place to another and precipitated,when necessary. In general, solubility of CaPO4 phases isproportional to their resorbability. Crystallization, dissolu-tion and phase transformation processes of different CaPO4

under various experimental conditions have been reviewed[37]. Since all types of CaPO4 appear to be chemically quiteclose compounds, the discrimination among them requiresan application of various techniques. Regarding their appli-cations, some of them might be used in food industry and,according to the European classification of food additives,CaPO4 of food grade quality are known as E341 additive.

Due to the triprotic equilibrium that exists withinorthophosphate-containing solutions, variations in pH alterthe relative concentrations of the four types of anionicspecies of orthophosphoric acid (Fig. 3) [38] and thus boththe chemical composition (Fig. 4) [39] and the amountof the CaPO4 that are formed by a direct precipitation.The solubility isotherms of different CaPO4 are shown inFig. 5 [3,40,41]. However, in 2009, the classic solubilitydata of CaPO4 were mentioned to be inappropriate [42].According to the authors of the latter study, all previoussolubility calculations were based on simplifications, whichwere only crudely approximate. The problem lies in incon-gruent dissolution, leading to phase transformations andlack of the detailed solution equilibriums. Using an absolute

Figure 4 Various types of CaPO4 obtained by neutralizing oforthophosphoric acid by calcium hydroxide. The Ca/P valuesof the known types of CaPO4 (Table 1) are reported in the fig-ure. The solubility of CaPO4 in water decreases drastically fromleft to right, HA being the most insoluble and stable phase.Reprinted from Ref. [39] with permission.

solid-titration approach, the true solubility isotherm ofHA was found to lie substantially lower than previouslyreported. In addition, contrary to a wide belief, DCPDappeared not to be the most stable phase below pH ∼4.2,where CDHA was less soluble [42].

A brief description of all known CaPO4 (Table 1) is givenbelow.

MCPM

Monocalcium phosphate monohydrate (Ca(H2PO4)2·H2O; theIUPAC name is calcium dihydrogen orthophosphate mono-hydrate) is both the most acidic and water-soluble CaPO4.Although acidic CaPO4 in general were known by 1795 as‘‘superphosphate of lime’’, their differentiation started in1800s. Namely, by 1807, researchers first prepared a calciumphosphate, which could be attributed to MCPM [1,2].

MCPM crystallizes from aqueous solutions containing dis-solved ions of H2PO4

− and Ca2+ at the Ca/P ratio ∼0.5 andsolution pH below ∼2.0. Besides, MCPM might be precipi-tated from aqueous solutions containing organic solvents.At temperatures above ∼100 ◦C, MCPM releases a moleculeof water and transforms into MCPA but at temperatures> ∼500 ◦C MCPA further transforms into Ca(PO3)2.

Due to high acidity and solubility, MCPM is never found inbiological calcifications. Moreover, pure MCPM is not biocom-patible with bones. However, in medicine MCPM is used as acomponent of several self-setting CaPO4 formulations [43].In addition, MCPM is used as a nutrient, acidulant and min-eral supplement for food, feed and some beverages. Coupledwith NaHCO3, MCPM is used as a leavening agent for both drybaking powders and bakery dough. MCPM might be added tosalt-curing preserves, pickled and marinated foods. In addi-tion, MCPM might be added to tooth pastes and chewinggums [44]. Besides, MCPM might be added to ceramics andglasses, while agriculture is the main consumer of a tech-nical grade MCPM, where it is used as a fertilizer, triplesuperphosphate [45].


Table 3 Crystallographic data of calcium orthophosphates [3].

Compound Space group Unit cell parameters Za Density, gcm−3

MCPM Triclinic P1 a = 5.6261(5), b = 11.889(2),c = 6.4731(8) A,

= 98.633(6)◦, = 118.262(6)◦, = 83.344(6)◦

2 2.23

MCPA Triclinic P1 a = 7.5577(5), b = 8.2531(6),c = 5.5504(3) A,

= 109.87(1)◦, = 93.68(1)◦, = 109.15(1)◦

2 2.58

DCPD MonoclinicIa

a = 5.812(2), b = 15.180(3),c = 6.239(2) A, ˇ = 116.42(3)◦

4 2.32

DCPA Triclinic P1 a = 6.910(1), b = 6.627(2),c = 6.998(2) A,

= 96.34(2)◦, = 103.82(2)◦, = 88.33(2)◦

4 2.89

OCP Triclinic P1 a = 19.692(4), b = 9.523(2),c = 6.835(2) A, ˛ = 90.15(2)◦,

= 92.54(2)◦, = 108.65(1)◦

1 2.61

�-TCP MonoclinicP21/a

a = 12.887(2), b = 27.280(4),c = 15.219(2) A, = 126.20(1)◦

24 2.86

�-TCP RhombohedralR3cH

a = b = 10.4183(5), c = 37.3464(23) A, = 120◦

21b 3.08

HA MonoclinicP21/borhexagonalP63/m

a = 9.84214(8), b = 2a, c = 6.8814(7) A, = 120◦ (monoclinic)a = b = 9.4302(5), c = 6.8911(2) A, = 120◦ (hexagonal)

42 3.16

FA HexagonalP63/m

a = b = 9.367, c = 6.884 A, = 120◦ 2 3.20

OA HexagonalP6

a = b = 9.432, c = 6.881 A, = 90.3◦, = 90.0◦, = 119.9◦

1 ∼3.2

TTCP MonoclinicP21

a = 7.023(1), b = 11.986(4),c = 9.473(2) A, ˇ = 90.90(1)◦

4 3.05

a Number of formula units per unit cell.b Per the hexagonal unit cell.

MCPA (or MCP)

Monocalcium phosphate anhydrous (Ca(H2PO4)2; the IUPACname is calcium dihydrogen orthophosphate anhydrous) isthe anhydrous form of MCPM. Although MCPM has beenknown since 1807, MCPA was differentiated as ‘‘tetra-hydrogen calcium phosphate, H4Ca(PO4)2’’ by 1879 [1,2].It crystallizes under the same conditions as MCPM but attemperatures above ∼100 ◦C (e.g., from concentrated hotmother liquors during fertilizer production). In addition,MCPA can be prepared from MCPM by dehydration. Further-more, it can be also prepared at ambient temperatures bycrystallization in water-restricted or non-aqueous systems.Like MCPM, MCPA never appears in calcified tissues and is notbiocompatible due to its acidity. There is no current applica-tion of MCPA in medicine. Due to the similarity with MCPM, inmany cases, MCPA might be used instead of MCPM; however,highly hydroscopic properties of MCPA reduce its commercialapplications.

DCPD

Dicalcium phosphate dihydrate (CaHPO4·2H2O; the IUPACname is calcium hydrogen orthophosphate dihydrate; themineral brushite) has been known since, at least, 1804.As a mineral, brushite was first discovered in phosphaticguano from Avis Island (Caribbean) in 1865 and named tohonor an American mineralogist Prof. George Jarvis Brush(1831—1912), Yale University, New Haven, Connecticut, USA[1,2].

DCPD can be easily crystallized from aqueous solutionscontaining dissolved ions of HPO4

2− and Ca2+ at the Ca/Pratio ∼1 and solution pH within ∼2.0 < pH < ∼6.5. Otherpreparation techniques such as neutralization of H3PO4

and/or MCPM solutions by CaO, CaCO3 or more basic CaPO4

(�- or �-TCP, CDHA, HA, TTCP) are also known. Interestingly,that precipitation of DCPD by mixing a Ca(OH)2 suspensionand a H3PO4 solution in the equimolar quantities was foundto occur in five stages, being HA the first precipitated phase.

132 S.V. Dorozhkin

Figure 5 Top: a 3D version of the classical solubility phase dia-grams for the ternary system Ca(OH)2—H3PO4—H2O. Reprintedfrom Ref. [40] with permission. Middle and bottom: sol-ubility phase diagrams in two-dimensional graphs, showingtwo logarithms of the concentrations of (a) calcium and (b)orthophosphate ions as a function of the pH in solutionssaturated with various salts. Reprinted from Ref. [41] with per-mission.

Besides, DCPD can be prepared in gels. DCPD transforms intoDCPA at temperatures above ∼80 ◦C and this transformationis accompanied by ∼11% decrease in volume and structuralchanges [45]. The value for �rG0 for DCPD → DCPA transfor-mation is —1.032 kJ/mol [45]. Briefly, DCPD crystals consistof CaPO4 chains arranged parallel to each other, while lat-tice water molecules are interlayered between them [46].Many additional data on DCPD including a good drawing ofits atomic structure might be found in Ref. [47].

DCPD is of biological importance because it is oftenfound in pathological calcifications (dental calculi, crys-talluria, chondrocalcinosis and urinary stones) and somecarious lesions [25]. It was proposed as an intermediatein both bone mineralization and dissolution of enamel inacids (dental erosion). In medicine, DCPD is used in self-setting CaPO4 formulations [43] and as an intermediate fortooth remineralization. DCPD is added to toothpaste bothfor caries protection (in this case, it is often coupled withF-containing compounds such as NaF and/or Na2PO3F) and asa gentle polishing agent [44]. Other applications include aflame retardant, a slow release fertilizer, using in glass pro-duction, as well as calcium supplement in food, feed andcereals. In food industry, it serves as a texturizer, bakeryimprover and water retention additive. In diary industry,DCPD is used as a mineral supplement.

DCPA (or DCP)

Dicalcium phosphate anhydrous (CaHPO4; the IUPAC nameis calcium hydrogen orthophosphate anhydrate; the mineralmonetite) is the anhydrous form of DCPD. Although DCPD hasbeen known since, at least, 1804, DCPA was differentiated as‘‘mono-hydrogen CaPO4, HCaPO4’’ by 1879 [1,2]. As a min-eral, monetite was first described in 1882 in rock-phosphatedeposits from the Moneta (now Monito) Island (archipelagoof Puerto Rico), which contains a notable occurrence [48].

Due to the absence of water inclusions, DCPA is less solu-ble than DCPD (Table 1). Like DCPD, DCPA can be crystallizedfrom aqueous solutions containing Ca/P ratio ∼1 at solutionpH within ∼2.0 < pH < ∼6.5 but at temperatures > ∼90 ◦C. Inaddition, DCPA can be prepared by dehydration of DCPD.Furthermore, it can be also prepared at ambient tempera-tures in water-restricted or non-aqueous systems, such asgels, ethanol, as well as in the oil-in-water and water-in-oilsystems. According to Table 3, DCPA is crystallized in tri-clinic P1 space group; however, in 2016, a new polymorphof DCPA crystallized in orthorhombic Ccm21 space group wasprepared [49]. DCPA is physically stable and was found toresist hydration even when dispersed in water for over 7months in the temperature range of 4—50 ◦C [50]. A calcium-deficient DCPA was also prepared. It might be sintered at∼300 ◦C. Unlike DCPD, DCPA occurs in neither normal norpathological calcifications. It is used in self-setting CaPO4

formulations [43]. Besides, DCPA might be implanted as bio-ceramics [51]. Other applications include using as a polishingagent, a source of calcium and phosphate in nutritional sup-plements (e.g., in prepared breakfast cereals, enriched flourand noodle products), a tabletting aid [52] and a toothpastecomponent [44]. In addition, it is used as a dough condi-tioner in food industry. DCPA of a technical grade of puritycan be used as a fertilizer.

OCP

Octacalcium phosphate (Ca8(HPO4)2(PO4)4·5H2O; theIUPAC name is tetracalcium hydrogen orthophosphatediorthophosphate pentahydrate, another name is octa-calcium bis(hydrogenphosphate) tetrakis(phosphate)pentahydrate) is often found as an unstable transient inter-mediate during the precipitation of the thermodynamically


more stable CaPO4 (e.g., CDHA) in aqueous solutions. Tothe best of my findings, OCP has been known since, at least,1843, when Percy published a paper, in which he describedformation of ‘‘a new hydrated phosphate of lime’’ witha chemical formula 2CaO + PO5 + 6HO, in which ‘‘1 equiv.water being basic and 5 constitutional’’. However, a CaPO4

with the OCP composition was first described by Berzeliusin 1836 [1,2].

The preparation techniques of OCP are available in lit-erature. Briefly, to prepare OCP, Ca- and PO4-containingchemicals must be mixed to get the supersaturated aque-ous solutions with the Ca/P ratio equal to 1.33 at slightlyacidic to neutral pH (pH from ∼5.0 to ∼7.0). Typically, OCPcrystals are smaller if compared to DCPD ones, extremelyplaty and almost invariably twinned. However, OCP mightbe non-stoichiometric and be either Ca-deficient (down toCa/P = 1.26) or include excessive calcium (up to Ca/P = 1.48)in the structure [53]. It has been proposed that the struc-ture of a non-stoichiometric OCP contains an excess ofhydrogen, resulting in a non-stoichiometric chemical for-mula Ca16H4+x(PO4)12(OH)x·(10—x)H2O, which resembles thestructure of HA even more closely than previously antici-pated [54]. Furthermore, a partially hydrolyzed form of OCPwith Ca/P molar ratio of 1.37 might be prepared [53]. Atsolution pH = 7.2 and temperature 60 ◦C, the full hydrolysisof OCP into CDHA occurs within ∼6 hours.

The triclinic structure of OCP crystals displays threesub-layer structures along the a-axis, namely, a HA-likelayer structure (Apa: Ca/PO4 ratio 2.00, Ca:PO4 = 4:2), atransition structure (Tra: Ca/PO4 ratio 1.33, Ca:PO4 = 4:3)and a HPO4—OH layered structure (Lay: Ca/PO4 ratio 0.00,Ca:PO4 = 0:2). Thus, OCP crystals exhibit a layered struc-ture with Apa—Tra—Lay—Tra—Apa stacking along the a-axis[55]. A combination Tra—Lay—Tra is also called a hydratedsub-layer because all water molecules are located within it.In addition, it has the atomic arrangements of calcium andorthophosphate ions similar to those in DCPD [3,53]. Causedby the sub-layer structure, the a-axis parameter in OCP crys-tals appears to be much larger than the b-axis and c-axisones (Table 3). Due to both the large a-axis parameter and alow symmetry, a strong reflection at a low angle (2 = ∼4.7◦)is observed in the typical X-ray diffraction patterns of OCP.This reflection is commonly used as a signature of the OCPpresence [3,53,55]. A similarity in crystal structure betweenOCP and HA is the reason that the epitaxial growth of thesephases is observed. It is generally assumed that, in solu-tions, the hydrated sub-layer of the (100) face is the layermost likely exposed to solution. The water content of OCPcrystals is ∼20% that of DCPD and this is partly responsiblefor its lower solubility.

OCP is of a great biological importance because it isone of the stable components of human dental and urinarycalculi [56,57]. OCP was first proposed by W.E. Brown toparticipate as the initial phase in enamel mineral forma-tion and bone formation through subsequent precipitationand stepwise hydrolysis of OCP [58]. It plays an importantrole in formation of apatitic biominerals in vivo [59]. A‘‘central OCP inclusion’’ (also known as ‘‘central dark line’’)is seen by transmission electron microscopy in many biolog-ical apatites and in synthetically precipitated CDHA [60].Although OCP has not been observed in vascular calcifica-tions, it has been strongly suggested as a precursor phase

to biological apatite found in natural and prosthetic heartvalves [61,62]. In surgery, OCP is used for implantation intobone defects [63—66]. For the comprehensive informationon OCP, the readers are referred to other reviews [53,56,66].

�-TCP

�-tricalcium phosphate (�-Ca3(PO4)2; the IUPAC nameis tricalcium diorthophosphate beta, other names arecalcium orthophosphate tribasic beta or tricalciumbis(orthophosphate) beta) is one of the polymorphs of TCP.Although CaPO4 with the composition close to that of TCP,CDHA and HA were known in 1770s, �- and �-polymorphs ofTCP were differentiated only by 1932 [1,2].

�-TCP cannot be precipitated from aqueous solutions.It is a high temperature phase, which can be prepared attemperatures above ∼800 ◦C by thermal decomposition ofCDHA or by solid-state interaction of acidic CaPO4, e.g.,DCPA, with a base, e.g., CaO. In all cases, the chemi-cals must be mixed in the proportions to get the Ca/Pratio equal to 1.50. However, in water-free mediums, �-TCP can be precipitated at relatively low temperatures(∼150 ◦C) in ethylene glycol or even at room temperature inmethanol. Apart from the chemical preparation routes, ion-substituted �-TCP can be prepared by calcining of bones:such type of CaPO4 is occasionally called ‘‘bone ash’’. Attemperatures above ∼1125 ◦C, �-TCP is transformed into ahigh-temperature phase �-TCP. Being the stable phase atroom temperature, �-TCP is less soluble in water than �-TCP (Table 1). Both ion-substituted and organically modifiedforms of �-TCP can be synthesized, as well. In addition,Ca-deficient hydrogen substituted �-TCP with the chemi-cal formula Ca21−x(HPO4)2x(PO4)14−2x, where x = 0.80 ± 0.04(Ca/P = 1.443 ± 0.003), was synthesized. Upon sintering thiscompound was decomposed into a blend of the stoichiomet-ric �-TCP and �-calcium pyrophosphate. Furthermore, anability of �-TCP to store an electrical charge by electricalpolarization was studied and this material was found to havea suitable composition and structure for both ion conductionand charge storage [67].

Pure �-TCP never occurs in biological calcifications. Onlya Mg-substituted form (�-TCMP—�-tricalcium magnesiumphosphate, which is often called magnesium whitlockite(chemical formula Ca18Mg2(HPO4)2(PO4)12) to honor Mr. Her-bert Percy Whitlock (1868—1948), an American mineralogist,the curator of the American Museum of Natural History, NewYork City, New York, USA) is found [68]. Since �-TCMP isless soluble than �-TCP, it is formed instead of �-TCP indental calculi and urinary stones, dentinal caries, salivarystones, arthritic cartilage, as well as in some soft-tissuedeposits [25,69—71]. However, it has not been observed inenamel, dentin or bone. In medicine, �-TCP is used in theself-setting CaPO4 formulations [43] and other types of bonegrafts [72—75]. Dental applications of �-TCP are also known.For example, �-TCP is added to some brands of toothpaste asa gentle polishing agent [44]. Multivitamin complexes withCaPO4 are widely available in the market and �-TCP is usedas the calcium phosphate there. In addition, �-TCP servesas a texturizer, bakery improver and anti-clumping agent fordry powdered food (flour, milk powder, dried cream, cocoapowder). Besides, �-TCP is added as a dietary or mineral

134 S.V. Dorozhkin

supplement to food and feed. Occasionally, �-TCP can beused as inert filler in pelleted drugs. Other applications com-prise porcelains, pottery, enamel, using as a component formordants and ackey, as well as a polymer stabilizer. �-TCPof a technical grade (as either calcined natural phosphoritesor bone dust) is used as a slow release fertilizer for acidicsoils.

To conclude, one should briefly mention on an existenceof -TCP polymorph, naturally known as tuite, which wasnamed after Prof. Guangzhi Tu (born in 1920), a foundingdirector of the Guangzhou Institute of Geochemistry, Chi-nese Academy of Sciences, Guangzhou, China. Tuite appearsto be a high-pressure polymorph of whitlockite with anempirical formula Ca2.51Na0.28Mg0.27Fe2+

0.02(PO4)2.02. Pure -TCP polymorph can be synthesized from �-TCP at pressuresabove ∼4 GPa and temperatures above ∼1000 ◦C [76].

�-TCP

�-tricalcium phosphate (�-Ca3(PO4)2; the IUPAC nameis tricalcium diorthophosphate alpha, other names arecalcium orthophosphate tribasic alpha or tricalciumbis(orthophosphate) alpha) is another polymorph of TCP,which was differentiated by 1932 [1,2]. �-TCP is also ahigh-temperature phase; therefore, it cannot be precipi-tated from aqueous solutions either. Thus, �-TCP is usuallyprepared by the same techniques as �-TCP (see the pre-vious section) but, since the �-TCP → �-TCP transitiontemperature is ∼1125 ◦C, calcining is performed at tem-peratures above ∼1200 ◦C. Consequently, �-TCP is oftenconsidered as a high-temperature polymorph of �-TCP. How-ever, data are available that �-TCP might be prepared atlower temperatures. Namely, at the turn of the millennium,the previously forgotten data that the presence of silicatesstabilized �-TCP at temperatures of 800—1000 ◦C were redis-covered again. Such type of �-TCP is called ‘‘silica stabilized�-TCP’’. Furthermore, sometimes, �-TCP might be preparedat even lower temperatures (∼700 ◦C) by a thermal decom-position of low-temperature ACPs.

Although �-TCP and �-TCP have exactly the same chemi-cal composition, they differ by the crystal structure (Table 3)and solubility (Table 1). In the absence of humidity, bothpolymorphs of TCP are stable at room temperatures; how-ever, according to a density functional study, stability of�-TCP crystal lattice exceeds that of �-TCP. Therefore, ofthem, �-TCP is more reactive in aqueous systems, has ahigher specific energy and in aqueous solutions it can behydrolyzed to CDHA. Milling was found to increase the �-TCPreactivity even more [77]. Although, �-TCP never occurs inbiological calcifications, in medicine, it is used as a compo-nent of self-setting CaPO4 formulations [43]. On the otherhand, the chemically pure �-TCP has received not muchinterest in the biomedical field [72]. The disadvantage forusing �-TCP is its quick resorption rate (faster than forma-tion of a new bone), which limits its application in this area.However, the silicon stabilized �-TCP (more precisely as abiphasic composite with HA) has been commercialized as astarting material to produce bioresorbable porous ceramicscaffolds to be used as artificial bone grafts. Upon implan-tation, �-TCP tends to convert to CDHA, which drasticallyreduces further degradation rate. The structure of �-TCP

is well described in literature. Similar to �-TCP, �-TCP ofa technical grade might be used slow release fertilizer foracidic soils.

To conclude, one should briefly mention on an existenceof �-TCP polymorph, which was discovered in 1959. How-ever, this TCP polymorph lacks of any practical interestbecause it only exists at temperatures between ∼1450 ◦Cand its melting point (∼1756 ◦C). It reverts to �-TCPpolymorph by cooling below the transition temperature.Additional details on �-TCP are available in the topicalreview [78].

ACP

Amorphous calcium phosphates (ACPs) represent a specialclass of CaPO4 salts, having variable chemical but ratheridentical glass-like physical properties, in which there areneither translational nor orientational long-range orders ofthe atomic positions. Since ACPs do not have the definitechemical composition, the IUPAC nomenclature is not appli-cable to describe them. To the best of my findings, ACPwas first prepared in 1845 [1,2]. Nevertheless, until recently[79], ACP has often been considered as an individual CaPO4

compound with a variable chemical composition, while, inreality, ACP is just an amorphous state of other CaPO4.Therefore, in principle, all compounds mentioned in Table 1might be somehow fabricated in an amorphous state but,currently, only few of them (e.g., an amorphous TCP) areknown [79]. Thus, strictly speaking, ACP should be excludedfrom Table 1.

Depending on the production temperatures, all types ofACP are divided into two major groups: low-temperatureACPs (prepared in solutions, usually aqueous ones) and high-temperature ACPs [79]. Low-temperature ACPs (describedby the chemical formula CaxHy(PO4)z·nH2O, n = 3—4.5;15—20% H2O) are often encountered as a transient precursorphase during precipitation of other CaPO4 in aqueous sys-tems. Usually, an ACP is the first phase precipitated fromsupersaturated solutions (the higher supersaturation, thebetter) prepared by rapid mixing of solutions containing ionsof calcium and orthophosphate [3,79]. Such ACP precipitatesusually look like spherical particles with diameters in therange 200 to 1200 A without a definite structure. Generally,the ACP particles are smaller if prepared under conditions ofhigh supersaturation and/or high pH, while for a given pH,higher temperatures give larger particles. The freshly pre-cipitated ACPs contain 10—20% by weight of tightly boundwater, which is removed by vacuum drying at elevated tem-perature [80]. The amorphization degree of ACPs increaseswith the concentration increasing of Ca- and PO4-containingsolutions, as well as at a high solution pH and a low crystal-lization temperature. A continuous gentle agitation of asprecipitated ACPs in the mother solution, especially at ele-vated temperatures, results in a slow recrystallization andformation of better crystalline CaPO4, such as CDHA [3]. Inaddition, other production techniques of ACPs are known[79].

The lifetime of ACPs in aqueous solutions was reportedto be a function of the presence of additive molecules andions, pH, ionic strength and temperature. In addition, con-finement was found to increase their lifetime. Thus, ACPs


may persist for appreciable periods and retain the amor-phous state under some specific experimental conditions.The chemical composition of ACPs strongly depends on thesolution pH and the concentrations of mixing solutions. Forexample, ACPs with Ca/P ratios in the range of 1.18 (precip-itated at solution pH = 6.6) to 1.53 (precipitated at solutionpH = 11.7) [3] and even to 2.5 [25] were described. In deedand not in name, these data mean that various types ofCaPO4 were prepared in an amorphous state [79]. It shouldbe noted that unsubstituted ACPs are unstable in aqueoussolutions and even when stored dry they tend to transforminto more crystalline CaPO4, such as poorly crystalline CDHA.The presence of poly(ethylene glycol), ions of pyrophos-phate, carbonate and/or magnesium in solutions during thecrystallization promotes formation of ACPs and slows downtheir further transformation, while the presence of fluoridehas the opposite effect [3]. In general, low-temperaturesACPs heated to ∼550 ◦C (so that all volatiles have alreadyescaped) remain amorphous, but further heating above∼650 ◦C causes their transformation into crystalline CaPO4,such as �- or �-TCP, HA, mixtures thereof, depending on theCa/P ratio of the ACP heated.

High-temperature ACPs can be prepared using highenergy processing at elevated temperatures [79]. Thismethod is based on a rapid quenching of melted CaPO4

occurring, e.g., during plasma-spraying of HA. A plasma jet,possessing very high temperatures (∼5000—∼20,000 ◦C),partly decomposes HA. That results in formation of a com-plicated mixture of products, some of which would beACPs. Obviously, all types of high-temperature ACPs aredefinitively anhydrous contrary to the precipitated ACPs.Unfortunately, no adequate chemical formula is availableto describe the high-temperature ACPs.

Since all types of amorphous compounds are character-ized by a lack of long-range order, it is problematic to discussthe structure of ACPs (they are X-ray amorphous). Concern-ing a short-range order (SRO) in ACPs, it exists, just due tothe nature of chemical bonds. Unfortunately, in many cases,the SRO in ACPs is uncertain either, because it dependson many variables, such as Ca/P ratio, preparation condi-tions, storage, admixtures, etc. Infrared spectra of ACPsshow broad featureless phosphate absorption bands. Elec-tron microscopy of freshly precipitated ACPs usually showsfeatureless nearly spherical particles with diameters in therange of 20 to 200 nm. However, there is a questionable opin-ion that ACPs might have an apatitic structure but with acrystal size so small, that they are X-ray amorphous. Thisis supported by X-ray absorption spectroscopic data (EXAFS)on biogenic and synthetic samples. On the other hand, itwas proposed that the basic structural unit of the precip-itated ACPs is a 9.5 A diameter, roughly spherical clusterof ions with the composition of Ca9(PO4)6 (Fig. 6) [3,81].These clusters were found experimentally as first nuclei dur-ing the crystallization of CDHA and a model was developedto describe the crystallization of HA as a stepwise assemblyof these units [82] (see section 3.10 HA (or HAp, or OHAp)below). Biologically, ion-substituted ACPs (always contain-ing ions of Na, Mg, carbonate and pyrophosphate) are foundin soft-tissue pathological calcifications (e.g., heart valvecalcifications of uremic patients) [25].

In medicine, ACPs are used in self-setting CaPO4 formula-tions [43]. Bioactive composites of ACPs with polymers have

Figure 6 A model of ACP structure. Reprinted from Ref. [81]with permission.

properties suitable for use in dentistry [44,79] and surgery[79]. Due to a reasonable solubility and physiological pH ofaqueous solutions, ACPs appeared to be consumable by somemicroorganisms and, due to this reason, it might be addedas a mineral supplement to culture media. Non-biomedicalapplications of ACPs comprise their using as a componentfor mordants and ackey. In food industry, ACPs are used forsyrup clearing. Occasionally, they might be used as inertfiller in pelleted drugs. In addition, ACPs are used in glassand pottery production and as a raw material for productionof some organic phosphates. To get further details on ACPs,the readers are referred to the special reviews [79,81,83].

CDHA (or Ca-def HA, or CDHAp)

Calcium-deficient hydroxyapatite (Ca10−x(HPO4)x(PO4)6−x

(OH)2−x (0 < x < 1)) became known since the earliest exper-iments on establishing the chemical composition of bonesperformed in 1770s. However, the first appropriate term‘‘subphosphate of lime’’ appeared by 1819 [1,2]. Otherchemical formulae such as Ca10−x(HPO4)2x(PO4)6−2x(OH)2

(0 < x < 2), Ca10−x−y(HPO4)x(PO4)6−x(OH)2−x−2y (0 < x < 2 andy < x/2), Ca10−x(HPO4)x(PO4)6−x(OH)2−x(H2O)x (0 < x < 1),Ca9−x(HPO4)1+2x(PO4)5−2x(OH), etc. were also proposedto describe its variable composition [3]. As seen fromthese formulae, Ca-deficiency is always coupled with bothOH-deficiency and protonation of some PO4 groups withsimultaneous formation of the ionic vacancies in the crystalstructure [84]. In addition, CDHA often contains tightlybound water molecules, which might occupy some ofthese ionic vacancies. For example, there is an approachdescribing a lack of the hydroxide vacancies in CDHA: toperform the necessary charge compensation of the missingCa2+ ions, a portion of OH− anions is substituted by neutralwater molecules [85]. This water is removed by vacuumdrying at elevated temperature. Concerning possible vacan-cies of orthophosphate ions, nothing is known about theirpresence in CDHA. It is just considered that a portion ofPO4

3− ions is either protonated (as HPO42−) or substituted

by other ions (e.g., CO32−). Since CDHA does not have any

definite chemical composition, the IUPAC nomenclature isnot applicable to describe it.

CDHA can be easily prepared by simultaneous addition ofCa- and PO4-containing solutions in the proportions to getCa/P ratio within 1.50—1.67 into boiling water followed by

136 S.V. Dorozhkin

boiling the suspension for several hours (an ageing stage).That is why, in literature it might be called as ‘‘precipitatedHA (PHA)’’. Besides, it might be prepared by hydrolysis of�-TCP. Other preparation techniques of CDHA are known aswell. During ageing, initially precipitated ACPs are restruc-tured and transformed into CDHA. Therefore, there aremany similarities in the structure, properties and applica-tion between the precipitated in alkaline solutions (pH > 8)ACPs and CDHA. Some data indicated on a presence of inter-mediate phases during further hydrolysis of CDHA to a morestable HA-like phase [86]. In general, CDHA crystals arepoorly crystalline and of submicron dimensions. They have avery large specific surface area, typically 25—100 m2/g. Onheating above ∼700 ◦C, CDHA with Ca/P = 1.5 converts to �-TCP and that with 1.5 < Ca/P < 1.67 converts into a biphasiccomposite of HA and �-TCP (see section 3.14. Biphasic,triphasic and multiphasic CaPO4 formulations below) [87]. Asolid-state transformation mechanism of CDHA into HA + �-TCP biocomposite was proposed [88,89].

The variability in Ca/P molar ratio of CDHA has beenexplained through different models: surface adsorption, lat-tice substitution and intercrystalline mixtures of HA andOCP. Due to a lack of stoichiometry, CDHA is usually doped byother ions [24]. The doping extent depends on the counter-ions of the chemicals used for CDHA preparation. Directdeterminations of the CDHA structures are still missing andthe unit cell parameters remain uncertain. However, unlikethat in ACPs (see section 3.8. ACP above), a long-range orderexists in CDHA. Namely, the following lattice parameterswere reported for CDHA with Ca/P = 1.5: a = 9.4418(20) A andc = 6.8745(17) Å.

Systematic studies of defect constellations in CDHA areavailable in literature [85]. As a first approximation, CDHAmay be considered as HA with some ions missing (ionicvacancies). The more amount of Ca is deficient, the moredisorder, imperfections and vacancies are in the CDHAstructure. Furthermore, a direct correlation between theCa-deficiency and the mechanical properties of the crys-tals was found: calcium deficiency lead to an 80% reductionin the hardness and elastic modulus and at least a 75%reduction in toughness in plate-shaped HA crystals. Theo-retical investigations of the defect formation mechanismrelevant to non-stoichiometry in CDHA are available else-where [90].

Undoped CDHA (i.e., that containing ions of Ca2+, PO43−,

HPO42− and OH− only) does not exist in biological systems.

However, the ion-substituted CDHA: Na+, K+, Mg2+, Sr2+ forCa2+; CO3

2− for PO43− or HPO4

2−; F−, Cl−, CO32− for OH−, plus

some water forms biological apatite — the main inorganicpart of animal and human normal and pathological calcifi-cations [24]. Therefore, CDHA is a very promising compoundfor industrial manufacturing of artificial bone substitutes,including drug delivery applications [91]. Non-biomedicalapplications of CDHA are similar to those of ACP and HA.

HA (or HAp, or OHAp)

Hydroxyapatite (Ca5(PO4)3(OH), but is usually written asCa10(PO4)6(OH)2 to denote that the crystal unit cell com-prises two molecules; the IUPAC name is pentacalciumhydroxide tris(orthophosphate)) is the second most stable

and least soluble CaPO4 after FA. Apatites were recognizedas calcium phosphates by, at least, 1789 [1,2]. Here, it isworth noting that hydroxylapatite would be a more accurateabbreviation expansion of HA (perhaps, hydroxideapatitewould be even better because it relates to calcium hydrox-ide) while by both the medical and material communitiesHA is usually expanded as hydroxyapatite.

Chemically pure HA crystallizes in the monoclinic spacegroup P21/b. However, at temperatures above ∼250 ◦C,there is a monoclinic to hexagonal phase transition in HA(space group P63/m) [3]. The structural difference betweenthe two modifications represents the ordered, head-to-tailarrangement of OH groups located in the center of everyother Ca2 triangle in the monoclinic low-temperature sym-metry and the disordered arrangement of OH groups, wherethe head-to-tail and tail-to-head arrangements alternatethroughout the channel in the hexagonal high-temperaturesymmetry. This induces strains that might be compensatedby substitutions and/or ion vacancies. Some impurities, likepartial substitution of hydroxide by fluoride or chloride,stabilize the hexagonal structure of HA at ambient tem-perature. Due to this reason, hexagonal HA is seldom thestoichiometric phase and very rare single crystals of naturalHA always exhibit the hexagonal space group. The detaileddescription of the HA structure was first reported in 1964and its interpretation in terms of aggregation of Ca9(PO4)6

clusters, the so-called Posner’s clusters, has been widelyused since publication of the article by Posner and Betts[80].

Due to the exceptional importance of HA for the humanbeings, its properties have been thoroughly investigated bymany research groups and further studies are kept going.Many techniques might be utilized for HA preparation; theycan be divided into solid-state reactions and wet methods[92], which include precipitation, hydrothermal synthesisand hydrolysis of other CaPO4. However, in all cases, Ca- andPO4-containing chemicals must be mixed to get the Ca/Pratio strictly equal to 1.67. Nevertheless, even under theideal stoichiometric conditions, the precipitates are gener-ally non-stoichiometric, suggesting intermediate formationof precursor phases, such as ACP and CDHA. Usually, unsin-tered HA is poorly crystalline and often non-stoichiometric,resembling the aforementioned CDHA. However, well crys-talline HA can be prepared from aqueous solutions atrelatively high (10—11) pH and elevated (> 90 ◦C) temper-atures. HA with the Ca/P ratio > 1.67 (Ca-rich HA) might beprepared as well [93]. The detailed information on HA syn-thesis is available elsewhere [94]. In addition, there are goodreviews on HA solubility, crystal growth and intermediatephases of HA crystallization [95], as well as on HA dissolution[96].

Pure HA never occurs in biological systems. However,due to the chemical similarities to bone and teeth mineral(Table 2), HA is widely used as coatings on orthopedic (e.g.,hip joint prosthesis) and dental implants [44,97]. Due to agreat similarity to biological apatite, over a long time HA hasbeen used in liquid chromatography of nucleic acids, pro-teins and other biological compounds and for drug deliverypurposes. Furthermore, HA-containing formulations werefound to possess the haemostatic properties [98]. Also, HAis added to some brands of toothpaste as a gentle polishingagent instead of calcium carbonate [44].


Figure 7 A biomimetically grown aggregate of FA that wascrystallized in a gelatin matrix. Its shape can be explained andsimulated by a fractal growth mechanism. Scale bar: 10 �m.Reprinted from Ref. [99] with permission.

FA (or FAp)

Fluorapatite (Ca5(PO4)3F, but is usually written asCa10(PO4)6F2 to denote that the crystal unit cell com-prises two molecules; the IUPAC name is pentacalciumfluoride tris(orthophosphate)) is the only ion-substitutedCaPO4, considered in this review. Since the presence of2.5% of fluorides in natural apatites was established by 1798[1,2], this date might be accepted as the earliest hearingof FA.

FA is the hardest (5 according to the Mohs’ scale ofmineral hardness), most stable and least soluble compoundamong all CaPO4 (Table 1). In addition, it is the most ther-mally stable CaPO4 with the melting point at ∼1650 ◦C.Perhaps, such ‘‘extreme’’ properties of FA are related tothe specific position of F− ions in the center of Ca(2) trian-gles of the crystal structure [3]. Preparation techniques ofthe chemically pure FA are similar to the aforementionedones for HA but the synthesis must be performed in pres-ence of the necessary amount of F− ions (usually, NaF orNH4F is added). Under some special crystallization condi-tions (e.g., in presence of gelatin or citric acid), FA mightform unusual dumbbell-like fractal morphology that finallyare closed to spheres (Fig. 7) [99]. In addition, FA is the onlyCaPO4, which melts without decomposition; therefore, big(up to 30 cm long and, for shorter lengths, up to 1.9 cm wide)single FA crystals can be grown from FA melts. Similar to thatfor HA (see CDHA), an existence of CaF2-deficient FA was alsodetected but for the crystals grown from the FA melt only. Ahierarchical structure for FA was proposed [100]. The crystalstructure of FA for the first time was studied in 1930 [1,2]and is well described elsewhere [3]. In addition, there arereviews on FA solubility [95] and the dissolution mechanism[96].

FA easily forms solid solutions with HA with any desiredF/OH molar ratio. Such compounds are called fluorhy-droxyapatites (FHA) or hydroxyfluorapatites (HFA) anddescribed with a chemical formula Ca10(PO4)6(OH)2−xFx,where 0 < x < 2. If the F/OH ratio is either uncertain or notimportant, the chemical formula of FHA and HFA is often

written as Ca10(PO4)6(F,OH)2. The lattice parameters, crys-tal structure, solubility and other properties of FHA and HFAlay in between of those for the chemically pure FA and HA.Namely, the substitution of F for OH results in a contractionin the a-axis with no significant change in the c-axis dimen-sions and greater resolution of the IR absorption spectra.

Similar to pure HA, pure FA never occurs in biologi-cal systems. Obviously, a lack of the necessary amount oftoxic fluorides (the acute toxic dose of fluoride is ∼5 mg/kgof body weight) in living organisms is the main reason ofthis fact (pure FA contains 3.7% mass. F). Enameloid ofshark teeth [101] and some exoskeletons of mollusks [102]seem to be the only exclusions because they contain sub-stantial amounts of fluoride, with is presented there asion-substituted, non-stoichiometric FHA or HFA. Among allnormal calcified tissues of humans, the highest concentra-tion of fluorides is found in dentin and cementum, whilethe lowest—in dental enamel (Table 2). Nevertheless, oneshould stress that the amount of fluorides on the very sur-face of dental enamel might be substantially increasedby using fluoride-containing toothpastes and mouthwashes[103]. However, in no case, the total amount of fluorides isenough to form pure FA.

Contrary to the initial expectations, chemically pure FA isnot used for grafting purposes. Presumably, this is due to thelowest solubility, good chemical stability of FA and toxicityof high amounts of fluorides. However, attempts to test FA-containing formulations, ion-substituted FA, FHA and porousFA bioceramics are kept performing. The effect of fluoridecontents in FHA on both osteoblast behavior [104,105] andleukemia cells proliferation [106] has been described.

OA (or OAp, or OXA)

Oxyapatite (Ca10(PO4)6O; the IUPAC name is decacalciumoxide hexakis (phosphate), mineral voelckerite) is the leaststable and, therefore, the least known CaPO4, which,probably, does not exist at all. Nevertheless, a name‘‘voelckerite’’ was introduced in 1912 by A.F. Rogers(1887—1957) for a hypothetical mineral with the chemi-cal composition of 3Ca3(PO4)2 + CaO, to honor an Englishagricultural chemist John Christopher Augustus Voelcker(1822—1884), who, in 1883, first showed an apparent halo-gen deficiency in some natural apatites [1,2]. Therefore,1883 might be accepted as the earliest hearing on OA.

To the best of my findings, phase pure OA has never beenobtained at room temperatures; therefore, its propertiesare not well established. Furthermore, still there are seri-ous doubts that pure OA can exist. Since hydroxyl ions in HAappear to be the most mobile ones and upon exposure to hightemperatures are the first to leave the lattice, a mixture (ora solid solution?) of OA and HA (so-called ‘‘oxy-HA’’, chemi-cal formula: Ca10(PO4)6(OH)2−2xOxVx, where V represents anOH− vacancy) can be prepared by a partial dehydroxyla-tion of HA at temperatures exceeding ∼900 ◦C (e.g., duringplasma-spray of HA) strictly in the absence of water vapor[107,108]. It also can be crystallized in glass-ceramics. OAis very unstable and has no stability field in aqueous con-ditions. Namely, data are available, that oxy-HA containingless than 25% HA (i.e., almost OA) during further dehydrationdecomposes to a mixture of �-TCP and TTCP. In addition,

138 S.V. Dorozhkin

OA is very reactive and transforms to HA in contact withwater vapor (HA reconstitution) [107]. The largest imped-iment to active research in OA is the non-availability ofa user-friendly approach to measure the concentration ofhydroxyl ions.

Therefore, computer-modeling techniques have beenemployed to qualitatively and quantitatively investigate thedehydration of HA to OA [109]. OA has the hexagonal spacegroup symmetry P6 (174) of cesanite type, while the spacegroup symmetry for partially dehydrated HA was found tochange from hexagonal P63/m to triclinic P1 when more thanca. 35% of the structurally bound water had been removed[108]. On the c-axis, pure OA should have a divalent ion O2−

coupled with a vacancy instead of two neighboring monova-lent OH− ions.

Due to the aforementioned problems with OA prepara-tion, it cannot be found in biological systems. In addition,no information on the biomedical applications of OA is avail-able either. Plasma-sprayed coatings of CaPO4, in which OAmight be present as an admixture phase, seem to be the onlyexception [107].

TTCP (or TetCP)

Tetracalcium phosphate or tetracalcium diorthophosphatemonoxide (Ca4(PO4)2O; the IUPAC name is tetracalciumoxide bis(orthophosphate); the mineral hilgenstockite) is themost basic CaPO4, however, its solubility in water is higherthan that of HA (Table 1). TTCP has been known since 1883,while the mineral hilgenstockite was named to honor a Ger-man metallurgist Gustav Hilgenstock (1844—1913), who firstdiscovered it in Thomas slag from blast furnaces [1,2]. Itsmajor industrial importance stems from the fact that it isformed by the reactions between orthophosphates and limein the manufacture of iron, and through these reactions,TTCP has a significant role in controlling the properties ofthe metal.

TTCP cannot be precipitated from aqueous solutions. Itcan be prepared only under the anhydrous conditions bysolid-state reactions at temperatures above ∼1300 ◦C, e.g.,by heating homogenized equimolar quantities of DCPA andCaCO3 in dry air, or in a flow of dry nitrogen [3]. Thesereactions should be carried out in a dry atmosphere, invacuum or with rapid cooling (to prevent uptake of waterand formation of HA). Easily DCPA might be replaced byammonium orthophosphates, while calcium carbonate mightbe replaced by calcium acetate; however, in all cases,Ca/P ratio must be equal to 2.00. Furthermore, TTCP oftenappears as an unwanted by-product in plasma-sprayed HAcoatings, where it is formed as a result of the thermaldecomposition of HA to a mixture of high-temperaturephases of �-TCP, TTCP and CaO [110]. Nevertheless, TTCPcan be produced at 900 ◦C by calcining of a precipitated ACPwith Ca/P = 2 in vacuum; the process is suggested to occurvia a intermediate formation of a mixture of OA + CaO.

TTCP is metastable: in both wet environment andaqueous solutions it slowly hydrolyses to HA and calciumhydroxide [3]. Consequently, TTCP is never found in bio-logical calcifications. In medicine, TTCP is widely usedfor preparation of various self-setting CaPO4 formulations[43,110]; however, to the best of my knowledge, there is

no commercial bone-substituting product consisting solelyof TTCP. For the comprehensive information on TTCP, thereaders are referred to a special review [110].

To finalize the description of individual CaPO4, oneshould mention on an interesting opinion, that all typesof CaPO4 listed in Table 1 might be classified into threemajor structural types [56]. They comprise: (i) the apatitetype, Ca10(PO4)6X2, which includes HA, FA, OA, CDHA, OCPand TTCP; (ii) the glaserite type, named after the min-eral glaserite, K3Na(SO4)2, which includes all polymorphsof TCP and, perhaps, ACP; (iii) the CaPO4 sheet-containingcompounds, which include DCPD, DCPA, MCPM and MCPA.According to the authors, a closer examination of the struc-tures revealed that all available CaPO4 could be includedinto distorted glaserite type structures, but with varyingdegrees of distortion [56].

Biphasic, triphasic and multiphasic CaPO4formulations

CaPO4 might form biphasic, triphasic and multiphasic(polyphasic) formulations, in which the individual compo-nents cannot be separated from each other. Presumably, theindividual phases of such compositions are homogeneously‘‘mixed’’ at a far submicron level (< 0.1 �m) and stronglyintegrated with each other. Nevertheless, the presence ofall individual phases is easily seen by X-ray diffraction tech-nique [87].

The usual way to prepare multiphasic formulations con-sists of sintering non-stoichiometric compounds, such as ACPand CDHA, at temperatures above ∼700 ◦C. Furthermore,a thermal decomposition of the stoichiometric CaPO4 attemperatures above ∼1300 ◦C can be used as well; how-ever, this approach often results in formation of complicatedmixtures of various products including admixtures of CaO,calcium pyrophosphates, etc. [87]. Namely, transformationof HA into polyphasic CaPO4 by annealing in a vacuum occursas this: the outer part of HA is transformed into �-TCPand TTCP, while the �-TCP phase of the surface furthertransforms into CaO. Besides, in the boundary phase, HAis transformed into TTCP.

Historically, Nery and Lynch with co-workers first usedthe term biphasic calcium phosphate (BCP) in 1986 todescribe a bioceramic, that consisted of a mixture of HAand �-TCP [111]. Based on the results of X-ray diffractionanalysis, these authors found that the ‘‘tricalcium phos-phate’’ preparation material used in their early publication[112] was in fact a mixture of ∼20% HA and ∼80% �-TCP.Currently, only biphasic and triphasic CaPO4 formulationsare known; perhaps, more complicated formulations will bemanufactured in future. Furthermore, nowadays, multipha-sic (polyphasic) compositions consisting of high-temperaturephases of CaPO4, such as �-TCP, �-TCP, HA and, perhaps,high-temperature ACP, OA and TTCP, are known only. Noprecise information on multiphasic compositions, contain-ing MCPM, MCPA, DCPD, DCPA, low-temperature ACP, OCPand CDHA has been found in literature [87]. Perhaps, suchformulations will be produced in future.

All BCP formulations might be subdivided into two majorgroups: those consisting of CaPO4 with either the same (e.g.,�-TCP and �-TCP) or different (e.g., �-TCP and HA) molar


Ca/P ratios. Among all known BCP formulations, BCP con-sisting of HA and �-TCP is both the most known and the bestinvestigated [87]. In 1986, LeGeros in USA and Daculsi inFrance initiated the basic studies on preparation of this typeof BCP and its in vitro properties. This material is solubleand gradually dissolves in the body, seeding new bone for-mation as it releases calcium and orthophosphate ions intothe biological medium. Presently, commercial BCP productsof different or similar HA/�-TCP ratios are manufacturedin many parts of the world as bone graft or bone substitutematerials for orthopaedic and dental applications under var-ious trademarks and several manufacturers [87]. A similarcombination of �-TCP with HA forms BCP as well.

Recently the concept of BCP has been extended bypreparation and characterization of biphasic TCP (BTCP),consisting of �-TCP and �-TCP phases. It is usually preparedby heating ACP precursors, in which the �-TCP/�-TCP ratiocan be controlled by aging time and pH value during synthe-sis of the amorphous precursor [113]. Furthermore, triphasicformulations, consisting of HA, �-TCP and �-TCP or HA, �-TCP and TTCP have been prepared [87].

It is important to recognize, that the major biomedicalproperties (such as bioactivity, bioresorbability, osteo-conductivity and osteoinductivity) of the multiphasicformulations might be adjusted by changing the ratiosamong the phases. When compared to both �- and �-TCP,HA is a more stable phase under the physiological condi-tions, as it has a lower solubility (Table 1) and, thus, slowerresorption kinetics. Therefore, due to a higher biodegrad-ability of the �- or �-TCP component, the reactivity of BCPincreases with the TCP/HA ratio increasing. Thus, in vivobioresorbability of BCP can be adjusted through the phasecomposition. Similar conclusions are also valid for both thebiphasic TCP (in which �-TCP is a more soluble phase) andthe triphasic (HA, �-TCP and �-TCP) formulations. Furtherdetails on this subject might be found in a topical review[87].

Ion-substituted CaPO4

Last, one should very briefly mention on existence of car-bonated HA [114], chlorapatite [115], as well as on a greatnumber of CaPO4 with various ionic substitutions (CaPO4

with dopants) [21,24]. In principle, any ion in CaPO4 mightbe substituted by other ion(s). Usually, the ion-substitutedCaPO4 are of a non-stoichiometric nature with just a par-tial ionic substitution and there are too many of them to bedescribed here.

To finalize this brief topic on the ion-substituted CaPO4,it is important to note, that chemical elements not foundin natural bones can be intentionally incorporated intoCaPO4 biomaterials to get special properties. For example,addition of Ag+, Zn2+ and Cu2+ was used for imparting antimi-crobial effect, while radioactive isotopes of 90Y, 153Sm and186Re were incorporated into HA bioceramics and injectedinto knee joints to treat rheumatoid joint synovitis. Moreto the point, apatites were found to incorporate individ-ual molecules, such as water, oxygen and carbon dioxide[24].

To finalize the description of the known CaPO4,one should mention that in spite of the well defined

crystallographic data (Table 3) and, therefore, the welldefined shapes of the single crystals, various types of CaPO4

can be prepared with the controllable sizes from nano- tomacro-scale (up to centimeter size) and the diverse shapesincluding zero- (particles), one- (rods, fibers, wires andwhiskers), two- (sheets, disks, plates, belts, ribbons andflakes) and three-dimensional morphologies [94,116]. Thelatter might be of versatile morphologies and shapes (Fig. 7is an example), including porous and hollow structures.

Conclusions

To date, although CaPO4-based biomaterials and bioceram-ics have been extensively studied for over 50 years, theirability to trigger bone formation is still incomparable withother biomaterials. Nowadays, the biomaterials’ field isshifting towards biologically active systems to improve theirperformance and to expand their use. Therefore, tissueengineering is the strongest direction of current research,which, in the case of CaPO4, means fabrication of propersubstrates and/or scaffolds to carry cells, hormones and bio-chemical factors to be further used in surgery and medicine[117,118]. Presumably, a synthesis of various types of CaPO4-based biocomposites and hybrid biomaterials occupies thesecond important place [119]. The third important placeis occupied by investigations devoted to the synthesis andcharacterization of various nano-sized particles and nan-odimensional crystals of CaPO4 [120], ACP [79], as wellas CaPO4 with controlled particle geometry and shapes[116]. In general, the geometry of crystal phases can bevaried by controlling the precipitation conditions, such astemperature, solution pH, concentration of the reagents,hydrodynamics, presence of various admixtures, inhibitorsor promoters, ultrasonication, etc. All these approachesmight be useful in preparation of CaPO4 fibers, whiskers, hol-low microspheres, etc. [121]. In addition, a great attentionis paid to manufacturing of the self-setting CaPO4 formu-lations [43] and multiphase formulations [87] mimickingas closely as possible the mineral component of biologi-cal apatite. A work along the ecological ways of synthesisof CaPO4 might be of a great importance as well [122]. Adeeper study of the fascinating growth rate of deer antlersand the ability of some animals, such as newts, to regen-erate amputated limbs might provide new and unexpectedapproaches to the bone-healing concept, as well as this willbe important for further development of both biomimet-ics and biomineralization fields. Unfortunately, no currentlyavailable grafting biomaterials can substitute the bones’mechanical function, illustrating yet unmet medical needthat would entirely substitute and regenerate a damaged tis-sue or organ. In a close future, the foreseeable application ofCaPO4 will be as an integrated component of the third gen-eration of biomaterials, where they will support cells and/orother biologically active substances (peptides, growth fac-tors, hormones, drugs, etc.) to guide regeneration of hardtissues.


The author declares that he has no competing interest.

140 S.V. Dorozhkin

References

[1] Dorozhkin SV. Calcium orthophosphates and human beings. Ahistorical perspective from the 1770s until 1940. Biomatter2012;2:53—70.

[2] Dorozhkin SV. A detailed history of calcium orthophosphatesfrom 1770s till 1950. Mater Sci Eng C 2013;33:3085—110.

[3] Elliott JC. Structure and chemistry of the apatites and othercalcium orthophosphates. Studies in inorganic chemistry, 18.Amsterdam, Netherlands: Elsevier; 1994 [389 pp].

[4] Dorozhkin SV. Calcium orthophosphates: applications innature, biology, and medicine. Singapore: Pan Stanford; 2012[854 pp].

[5] Dorozhkin SV. Calcium orthophosphate-based bioceramics andbiocomposites. Weinheim, Germany: Wiley-VCH; 2016 [405pp].

[6] Ribeiro HB, Guedes KJ, Pinheiro MVB, Greulich-Weber S,Krambrock K. About the blue and green colours in naturalfluorapatite. Physica Status Solidi C 2005;2:720—3.

[7] Cook PJ, Shergold JH, Davidson DF, editors. Phosphatedeposits of the world: phosphate rock resources, 2.Cambridge, MA, USA: Cambridge University Press; 2005 [600pp].

[8] Mitchell L, Faust GT, Hendricks SB, Reynolds DS. Themineralogy and genesis of hydroxylapatite. Am Miner1943;28:356—71.

[9] Jarvis I. Phosphorite geochemistry: state-of-the-art andenvironmental concerns. Eclogae Geologicae Helvetiae1994;87:643—700.

[10] McClellan GH. Mineralogy of carbonate fluorapatites (Franco-lites). J Geol Soc 1980;137:675—81.

[11] Henry TH. On francolite, a supposed new mineral. Phil Mag1850;36:134—5.

[12] Rogers AF. Collophane, a much neglected mineral. Am J Sci1922;3:269—76.

[13] Hubert B, Álvaro JJ, Chen JY. Microbially mediated phospha-tization in the Neoproterozoic Doushantuo Lagerstätte, SouthChina. Bull Soc Geol Fr 2005;176:355—61.

[14] Xiao S, Yuan X, Knoll AH. Eumetazoan fossils in ter-minal Proterozoic phosphorites? Proc Natl Acad Sci USA2000;97:13684—9.

[15] Keenan SW. From bone to fossil: a review of the diagenesis ofbioapatite. Am Mineral 2016;101:1943—51.

[16] McCubbin FM, Shearer CK, Burger PV, Hauri EH, Wang J, ElardoSM, et al. Volatile abundances of coexisting merrillite andapatite in the martian meteorite shergotty: implications formerrillite in hydrous magmas. Am Miner 2014;99:1347—54.

[17] Webster JD, Piccoli PM. Magmatic apatite: a powerful, yetdeceptive, mineral. Elements 2015;11:177—82.

[18] Baturin GN. Phosphorites of the Sea of Japan. Oceanology2012;52:666—76.

[19] Hogarth DD. The discovery of apatite on the Lievre River,Quebec. Mineral Rec 1974;5:178—82.

[20] van Velthuizen J. Giant fluorapatite crystals: a question oflocality. Mineral Rec 1992;23:459—63.

[21] Hughes JM, Rakovan JF. Structurally robust, chemicallydiverse: apatite and apatite supergroup minerals. Elements2015;11:165—70.

[22] Sánchez-Salcedo S, Vila M, Izquierdo-Barba I, Cicuéndez M,Vallet-Regí M. Biopolymer-coated hydroxyapatite foams: anew antidote for heavy metal intoxication. J Mater Chem2010;20:6956—61.

[23] Gilinskaya LG, Zanin YN. Geochemistry of organic matter innatural apatites of phosphorites according to EPR spectra offree radicals. Geochem Int 2012;50:1007—25.

[24] Rey C, Combes C, Drouet C, Sfihi H. Chemical diversity ofapatites. Adv Sci Technol 2006;49:27—36.

[25] LeGeros RZ. Formation and transformation of calciumphosphates: relevance to vascular calcification. Z Kardiol2001;90(Suppl. 3):III116—25.

[26] Currey JD. Mechanical properties and adaptations of someless familiar bony tissues. J Mech Behav Biomed Mater2010;3:357—572.

[27] Bocchi G, Valdre G. Physical, chemical, and mineralogicalcharacterization of carbonate-hydroxyapatite concretions ofthe human pineal gland. J Inorg Biochem 1993;49:209—20.

[28] Brögger WC, Bäckström H. Über den Dahllit, ein neues Min-eral von Ödegärden, Bamle, Norwegen. Neues Jb. Miner GeolPalaont 1890:223—4.

[29] Ensikat HJ, Geisler T, Weigend M. A first report of hydroxy-lated apatite as structural biomineral in Loasaceae—plants’teeth against herbivores. Sci Rep 2016;6:26073.

[30] Floege J, Kim J, Ireland E, Chazot C, Drueke T, de FranciscoA, et al. Serum iPTH, calcium and phosphate, and the riskof mortality in a European haemodialysis population. NephrolDial Transpl 2011;26:1948—55.

[31] Suller MTE, Anthony VJ, Mathur S, Feneley RCL, Greenman J,Stickler DJ. Factors modulating the pH at which calcium andmagnesium phosphates precipitate from human urine. UrolRes 2005;33:254—60.

[32] Prompt CA, Quinton PM, Kleeman CR. High concentrations ofsweat calcium, magnesium and phosphate in chronic renalfailure. Nephron 1978;20:4—9.

[33] Holt C. Inorganic constituents of milk, III. The colloidal cal-cium phosphate of cow’s milk. J Dairy Res 1982;49:29—38.

[34] Lenton S, Nylander T, Teixeira SCM, Holt C. A review of thebiology of calcium phosphate sequestration with special ref-erence to milk. Dairy Sci Technol 2015;95:3—14.

[35] Gaucheron F. Calcium phosphates in dairy products. In:Heimann RB, editor. Calcium phosphates: structure, synthe-sis, properties, and applications. New York, NY, USA: NovaScience Publishers; 2012. p. 381—97.

[36] Kreidler ER, Hummel FA. Phase relationships in the systemSrO—P2O5 and the influence of water vapor on the formationof Sr4P2O9. Inorg Chem 1967;6:884—91.

[37] Wang L, Nancollas GH. Calcium orthophosphates: crystalliza-tion and dissolution. Chem Rev 2008;108:4628—69.

[38] Lynn AK, Bonfield W. A novel method for the simultaneous,titrant-free control of pH and calcium phosphate mass yield.Acc Chem Res 2005;38:202—7.

[39] León B, Jansen JJ. Thin calcium phosphate coatings for medi-cal implants. New York, USA: Springer; 2009 [326 pp].

[40] Chow LC. Next generation calcium phosphate-based bioma-terials. Dent Mater J 2009;28:1—10.

[41] Ishikawa K. Bone substitute fabrication based on dissolution-precipitation reactions. Materials 2010;3:1138—55.

[42] Pan HB, Darvell BW. Calcium phosphate solubility: the needfor re-evaluation. Cryst Growth Des 2009;9:639—45.

[43] Dorozhkin SV. Self-setting calcium orthophosphate formula-tions. J Funct Biomater 2013;4:209—311.

[44] Dorozhkin SV. Calcium orthophosphates (CaPO4) and den-tistry. Bioceram Dev Appl 2016;6:96 [28 pages].

[45] Landin M, Rowe RC, York P. Structural changes during thedehydration of dicalcium phosphate dihydrate. Eur J PharmacSci 1994;2:245—52.

[46] Curry NA, Jones DW. Crystal structure of brushite, calciumhydrogen orthophosphate dihydrate: a neutron-diffractioninvestigation. J Chem Soc A Inorg Phys Theoret Chem1971:3725—9.

[47] Qiu SR, Orme CA. Dynamics of biomineral formation at thenear-molecular level. Chem Rev 2008;108:4784—822.

[48] Shepard CU. On two new minerals, monetite and monite, witha notice of pyroclasite. Am J Sci 1882;23:400—5.


[49] Ouerfelli N, Zid MF. New polymorph of CaHPO4 (mon-etite): synthesis and crystal structure. J Struct Chem2016;57:628—31.

[50] Miyazaki T, Sivaprakasam K, Tantry J, Suryanarayanan R. Phys-ical characterization of dibasic calcium phosphate dihydrateand anhydrate. J Pharmac Sci 2009;98:905—16.

[51] Tamimi F, Torres J, Bassett D, Barralet J, Cabarcos EL. Resorp-tion of monetite granules in alveolar bone defects in humanpatients. Biomaterials 2010;31:2762—9.

[52] Takami K, Machimura H, Takado K, Inagaki M, Kawashima Y.Novel preparation of free flowing spherically granulated diba-sic calcium phosphate anhydrous for direct tabletting. ChemPharm Bull 1996;44:868—70.

[53] Suzuki O. Octacalcium phosphate: osteoconductivity andcrystal chemistry. Acta Biomater 2010;6:3379—87.

[54] Mathew M, Brown WE, Schroeder LW, Dickens B. Crys-tal structure of octacalcium bis(hydrogenphosphate)tetrakis(phosphate)pentahydrate, Ca8(HPO4)2(PO4)4·5H2O.J Crystallogr Spectrosc Res 1988;18:235—50.

[55] Sugiura Y, Onuma K, Yamazaki A. Enhancement of HPO4—OHlayered structure in octacalcium phosphate and its mor-phological evolution by acetic acid. J Ceram Soc Jpn2016;124:1178—84.

[56] Chow LC, Eanes ED, editors. Octacalcium phosphate. Mono-graphs in oral science, 18. Basel, Switzerland: Karger; 2001[167 pp].

[57] Kakei M, Sakae T, Yoshikawa M. Electron microscopy of octa-calcium phosphate in the dental calculus. J Electron Microsc2009;58:393—8.

[58] Brown WE. Crystal growth of bone mineral. Clin Orthop RelatRes 1966;44:205—20.

[59] Suzuki O. Biological role of synthetic octacalcium phos-phate in bone formation and mineralization. J Oral Biosci2010;52:6—14.

[60] Rodríguez-Hernández AG, Fernández ME, Carbajal-de-la-Torre G, García-García R, Reyes-Gasga J. Electron microscopyanalysis of the central dark line defect of the human toothenamel. Mater Res Soc Symp Proc 2005;839:157—62.

[61] Tomazic BB, Brown WE, Shoen FJ. Physicochemical propertiesof calcific deposits isolated from porcine bioprosthetic heartvalves removed from patients following 2-13 years function.J Biomed Mater Res 1994;28:35—47.

[62] Nancollas GH, Wu W. Biomineralization mechanisms: akinetics and interfacial energy approach. J Cryst Growth2000;211:137—42.

[63] Suzuki O, Kamakura S, Katagiri T, Nakamura M, Zhao B, HondaY, et al. Bone formation enhanced by implanted octacalciumphosphate involving conversion into Ca-deficient hydroxyap-atite. Biomaterials 2006;27:2671—81.

[64] Kikawa T, Kashimoto O, Imaizumi H, Kokubun S, SuzukiO. Intramembranous bone tissue response to biodegrad-able octacalcium phosphate implant. Acta Biomater2009;5:1756—66.

[65] Murakami Y, Honda Y, Anada T, Shimauchi H, Suzuki O.Comparative study on bone regeneration by synthetic octa-calcium phosphate with various granule sizes. Acta Biomater2010;6:1542—8.

[66] Suzuki O. Octacalcium phosphate (OCP)-based bone substi-tute materials. Jpn Dent Sci Rev 2013;49:58—71.

[67] Wang W, Itoh S, Yamamoto N, Okawa A, Nagai A, Yamashita K.Electrical polarization of �-tricalcium phosphate ceramics. JAm Ceram Soc 2010;93:2175—7.

[68] Lagier R, Baud CA. Magnesium whitlockite, a calcium phos-phate crystal of special interest in pathology. Pathol Res Pract2003;199:329—35.

[69] Reid JD, Andersen ME. Medial calcification (whitlockite) in theaorta. Atherosclerosis 1993;101:213—24.

[70] Scotchford CA, Ali SY. Magnesium whitlockite deposition inarticular cartilage: a study of 80 specimens from 70 patients.Ann Rheum Dis 1995;54:339—44.

[71] P’ng CH, Boadle R, Horton M, Bilous M, Bonar F. Magnesiumwhitlockite of the aorta. Pathology 2008;40:539—40.

[72] Kamitakahara M, Ohtsuki C, Miyazaki T. Review paper: behav-ior of ceramic biomaterials derived from tricalcium phosphatein physiological condition. J Biomater Appl 2008;23:197—212.

[73] Liu Y, Pei GX, Shan J, Ren GH. New porous beta-tricalciumphosphate as a scaffold for bone tissue engineering. J ClinRehabil Tiss Eng Res 2008;12:4563—7.

[74] Epstein NE. Beta tricalcium phosphate: observation of usein 100 posterolateral lumbar instrumented fusions. Spine J2009;9:630—8.

[75] Liu B, Lun DX. Current application of �-tricalcium phosphatecomposites in orthopaedics. Orthop Surg 2012;4:139—44.

[76] Zhai S, Akaogi M, Kojitani H, Xue W, Ito E. Thermodynamicinvestigation on �- and -Ca3(PO4)2 and the phase equilibria.Phys Earth Planet Inter 2014;228:144—9.

[77] Camiré CL, Gbureck U, Hirsiger W, Bohner M. Correlatingcrystallinity and reactivity in an �-tricalcium phosphate. Bio-materials 2005;26:2787—94.

[78] Carrodeguas RG, de Aza S. �-Tricalcium phosphate: synthe-sis, properties and biomedical applications. Acta Biomater2011;7:3536—46.

[79] Dorozhkin SV. Amorphous calcium orthophosphates: nature,chemistry and biomedical applications. Int J Mater Chem2012;2:19—46.

[80] Posner AS, Betts F. Synthetic amorphous calcium phosphateand its relation to bone mineral structure. Acc Chem Res1975;8:273—81.

[81] Boskey AL. Amorphous calcium phosphate: the contention ofbone. J Dent Res 1997;76:1433—6.

[82] Onuma K, Ito A. Cluster growth model for hydroxyapatite.Chem Mater 1998;10:3346—51.

[83] Combes C, Rey C. Amorphous calcium phosphates: syn-thesis, properties and uses in biomaterials. Acta Biomater2010;6:3362—78.

[84] Wilson RM, Elliott JC, Dowker SEP, Rodriguez-Lorenzo LM.Rietveld refinements and spectroscopic studies of the struc-ture of Ca-deficient apatite. Biomaterials 2005;26:1317—27.

[85] Zahn D, Hochrein O. On the composition and atomicarrangement of calcium-deficient hydroxyapatite: an ab-initio analysis. J Solid State Chem 2008;181:1712—6.

[86] Brès EF, Duhoo T, Leroy N, Lemaitre J. Evidence of a transientphase during the hydrolysis of calcium-deficient hydroxyapa-tite. Z Metallkd/Mater Res Adv Tech 2005;96:503—6.

[87] Dorozhkin SV. Multiphasic calcium orthophosphate (CaPO4)bioceramics and their biomedical applications. Ceram Int2016;42:6529—54.

[88] Dorozhkina EI, Dorozhkin SV. Mechanism of the solid-statetransformation of a calcium-deficient hydroxyapatite (CDHA)into biphasic calcium phosphate (BCP) at elevated tempera-tures. Chem Mater 2002;14:4267—72.

[89] Dorozhkin SV. Mechanism of solid-state conversion of non-stoichiometric hydroxyapatite to diphase calcium phosphate.Russ Chem Bull 2003;52:2369—75.

[90] Matsunaga K. Theoretical investigation of the defect forma-tion mechanism relevant to nonstoichiometry in hydroxyapa-tite. Phys Rev B 2008;77:104106 [14 pages].

[91] Bourgeois B, Laboux O, Obadia L, Gauthier O, Betti E,Aguado E, et al. Calcium-deficient apatite: a first in vivostudy concerning bone ingrowth. J Biomed Mater Res A2003;65A:402—8.

[92] Briak-Ben E, Abdeslam H, Ginebra MP, Vert M, BoudevilleP. Wet or dry mechanochemical synthesis of calcium

142 S.V. Dorozhkin

phosphates? Influence of the water content on DCPD—CaOreaction kinetics. Acta Biomater 2008;4:378—86.

[93] Bonel G, Heughebaert JC, Heughebaert M, Lacout JL,Lebugle A. Apatitic calcium orthophosphates and relatedcompounds for biomaterials preparation. Ann NY Acad Sci1988;523:115—30.

[94] Sadat-Shojai M, Khorasani MT, Dinpanah-Khoshdargi E,Jamshidi A. Synthesis methods for nanosized hydroxyapatitewith diverse structures. Acta Biomater 2013;9:7591—621.

[95] Rakovan J. Growth and surface properties of apatite. In:Hughes JM, Kohn M, Rakovan J, editors. Phosphates: geo-chemical, geobiological and materials importance. Series:reviews in mineralogy and geochemistry, 48. Washington,D.C., USA: Mineralogical Society of America; 2002. p. 51—86.

[96] Dorozhkin SV. Dissolution mechanism of calcium apatites inacids: a review of literature. World J Methodol 2012;2:1—17.

[97] Suchanek W, Yoshimura M. Processing and properties ofhydroxyapatite-based biomaterials for use as hard tissuereplacement implants. J Mater Res 1998;13:94—117.

[98] Hama C, Umeda T, Musha Y, Koda S, Itatani K. Prepa-ration of novel hemostatic material containing sphericalporous hydroxyapatite/alginate granules. J Ceram Soc Jpn2010;118:446—50.

[99] Busch S, Dolhaine H, Duchesne A, Heinz S, Hochrein O, LaeriF, et al. Biomimetic morphogenesis of fluorapatite-gelatincomposites: fractal growth, the question of intrinsic electricfields, core/shell assemblies, hollow spheres and reorganiza-tion of denatured collagen. Eur J Inorg Chem 1999:1643—53.

[100] Dorozhkin SV. A hierarchical structure for apatite crystals. JMater Sci Mater Med 2007;18:363—6.

[101] Enax J, Janus AM, Raabe D, Epple M, Fabritius HO. Ultrastruc-tural organization and micromechanical properties of sharktooth enameloid. Acta Biomater 2014;10:3959—68.

[102] Leveque I, Cusack M, Davis SA, Mann S. Promotion of fluora-patite crystallization by soluble-matrix proteins from LingulaAnatina shells. Angew Chem Int Ed Engl 2004;43:885—8.

[103] Hattab FN. Remineralisation of carious lesions and fluorideuptake by enamel exposed to various fluoride dentifrices invitro. Oral Health Prev Dent 2013;11(3):281—90.

[104] Qu H, Wei M. The effect of fluoride contents in fluori-dated hydroxyapatite on osteoblast behavior. Acta Biomater2006;2:113—9.

[105] Bhadang KA, Holding CA, Thissen H, McLean KM, Forsythe JS,Haynes DR. Biological responses of human osteoblasts andosteoclasts to flame-sprayed coatings of hydroxyapatite andfluorapatite blends. Acta Biomater 2010;6:1575—83.

[106] Theiszova M, Jantova S, Letasiova S, Palou M, CipakL. Cytotoxicity of hydroxyapatite, fluorapatite and

fluor-hydroxyapatite: a comparative in vitro study. Neoplasma2008;55:312—6.

[107] Gross KA, Berndt CC, Dinnebier R, Stephens P. Oxyap-atite in hydroxyapatite coatings. J Mater Sci Mater Med1998;33:3985—91.

[108] Alberius-Henning P, Adolfsson E, Grins J, Fitch A. Triclinic oxy-hydroxyapatite. J Mater Sci 2001;36:663—8.

[109] de Leeuw NH, Bowe JR, Rabone JAL. A computational inves-tigation of stoichiometric and calcium-deficient oxy- andhydroxy-apatites. Faraday Disc 2007;134:195—214.

[110] Moseke C, Gbureck U. Tetracalcium phosphate: synthe-sis, properties and biomedical applications. Acta Biomater2010;6:3815—23.

[111] Ellinger RF, Nery EB, Lynch KL. Histological assessmentof periodontal osseous defects following implantation ofhydroxyapatite and biphasic calcium phosphate ceramics: acase report. Int J Periodont Restor Dent 1986;3:22—33.

[112] Nery EB, Lynch KL, Hirthe WM, Mueller KH. Bioceramicimplants in surgically produced infrabony defects. J Periodon-tol 1975;46:328—47.

[113] Li Y, Weng W, Tam KC. Novel highly biodegradable biphasictricalcium phosphates composed of �-tricalcium phosphateand �-tricalcium phosphate. Acta Biomater 2007;3:251—4.

[114] Fleet M. Carbonated hydroxyapatite: materials, synthesis,and applications. Singapore: Pan Stanford; 2015 [278 pp].

[115] García-Tunón E, Couceiro R, Franco J, Saiz E, Guitián F.Synthesis and characterisation of large chlorapatite single-crystals with controlled morphology and surface roughness. JMater Sci Mater Med 2012;23:2471—82.

[116] Lin K, Wu C, Chang J. Advances in synthesis of calcium phos-phate crystals with controlled size and shape. Acta Biomater2014;10:4071—102.

[117] Zhou H, Lee J. Nanoscale hydroxyapatite particles for bonetissue engineering. Acta Biomater 2011;7:2769—81.

[118] Zakaria SM, Zein SHS, Othman MR, Yang F, Jansen JA.Nanophase hydroxyapatite as a biomaterial in advanced hardtissue engineering: a review. Tiss Eng B 2013;19:431—41.

[119] Dorozhkin SV. Calcium orthophosphate-containing biocompos-ites and hybrid biomaterials for biomedical applications. JFunct Biomater 2015;6:708—832.

[120] Dorozhkin SV. Nanodimensional and nanocrystalline calciumorthophosphates. Int J Chem Mater Sci 2013;1:105—74.

[121] Marya IR, Soniaa S, Vijia S, Mangalaraja D, ViswanathanaC, Ponpandian N. Novel multiform morphologies of hydroxy-apatite: synthesis and growth mechanism. Appl Surf Sci2016;361:25—32.

[122] Dorozhkin SV. Green chemical synthesis of calcium phosphatebioceramics. J Appl Biomater Biomech 2008;6:104—9.

Morphologie (2017) 101, 143—153


ScienceDirect


GENERAL REVIEW

A history of calcium orthophosphates(CaPO4) and their biomedical applicationsHistorique des orthophosphates de calcium (CaPO4)et de leurs applications biomédicales

S.V. Dorozhkin

Kudrinskaja sq. 1-155, Moscow 123242, Russia

Available online 5 June 2017

KEYWORDSApatite;Calciumorthophosphates;Calc phosphate;Lime phosphate;History

Summary The historical development of a scientific knowledge on calcium orthophosphates(CaPO4) from 1770-s till 1950 is described. Many forgotten and poorly known historical facts andapproaches have been extracted from old publications and then they have been analyzed, sys-tematized and reconsidered from the modern point of view. The chosen time scale starts withthe earliest available studies of 1770-s (to the best of my findings, CaPO4 had been unknownbefore), passes through the entire 19th century and finishes in 1950, because since then theamount of publications on CaPO4 rapidly increases and the subject becomes too broad. Fur-thermore, since publications of the second half of the 20th century are easily accessible, thesubstantial amount of them has been already reviewed by other researchers. The reportedhistorical findings clearly demonstrate that the substantial amount of the scientific facts andexperimental approaches has been known for very many decades and, in fact, the considerablequantity of relatively recent investigations on CaPO4 is just either a further development of theearlier studies or a rediscovery of the already forgotten knowledge.© 2017 Elsevier Masson SAS. All rights reserved.

Introduction

By virtue of abundance in the nature and presence in the liv-ing organisms, calcium apatites1 and other CaPO4 (Table 1)

E-mail address: [email protected] As a mineral species, apatite was first recognized by the father

of German geology Abraham Gottlob Werner (1750—1817) in 1786and named by him from the ancient Greek ��´� (apatao) — ‘‘tomislead’’ or ‘‘to deceive’’, because it had previously been mistakenfor other minerals, such as beryl, tourmaline, chrysolite, amethyst,

appear to be the chemical compounds of a special interest inmany fields of science, including geology, chemistry, biologyand medicine [1,2]. As follows from the designation, CaPO4

contain both calcium (Ca, atomic number 20) and phos-phorus (P, atomic number 15) as the major constituencies.Concerning the history of both chemical elements, according

fluorite, etc. Currently, apatite is the name for a group of mineralswith the same crystallographic structure and does not indicate onechemical composition. That is why the term ‘‘calcium apatite’’ isused in this review.







144 S.V. Dorozhkin

Table 1 Existing CaPO4 and their major properties [1,2].

Ca/P molarratio

Compound Formula Solubility at25 ◦C,-log(Ks)

Solubility at25 ◦C, g/L

pH stabilityrange inaqueoussolutions at25 ◦C

0.5 Monocalcium phosphatemonohydrate (MCPM)

Ca(H2PO4)2·H2O 1.14 ∼ 18 0.0—2.0

0.5 Monocalcium phosphateanhydrous (MCPA or MCP)

Ca(H2PO4)2 1.14 ∼ 17 c

1.0 Dicalcium phosphate dihydrate(DCPD), mineral brushite

CaHPO4·2H2O 6.59 ∼ 0.088 2.0—6.0

1.0 Dicalcium phosphate anhydrous(DCPA or DCP), mineralmonetite

CaHPO4 6.90 ∼ 0.048 c

1.33 Octacalcium phosphate (OCP) Ca8(HPO4)2(PO4)4·5H2O 96.6 ∼ 0.0081 5.5—7.01.5 �-Tricalcium phosphate

(�-TCP)�-Ca3(PO4)2 25.5 ∼ 0.0025 a


�-Ca3(PO4)2 28.9 ∼ 0.0005 a

1.2—2.2 Amorphous calcium phosphates(ACP)

CaxHy(PO4)z·nH2O,n = 3—4.5; 15—20% H2O

b b ∼ 5—12d

1.5—1.67 Calcium-deficienthydroxyapatite (CDHA orCa-def HA)e

Ca10-x(HPO4)x(PO4)6-x(OH)2-x

(0 < x < 1)∼ 85 ∼ 0.0094 6.5—9.5

1.67 Hydroxyapatite (HA, HAp orOHAp)

Ca10(PO4)6(OH)2 116.8 ∼ 0.0003 9.5—12

1.67 Fluorapatite (FA or FAp) Ca10(PO4)6F2 120.0 ∼ 0.0002 7—121.67 Oxyapatite (OA, OAp or OXA)f Ca10(PO4)6O ∼ 69 ∼ 0.087 a

2.0 Tetracalcium phosphate (TTCPor TetCP), mineralhilgenstockite

Ca4(PO4)2O 38—44 ∼ 0.0007 a

a These compounds cannot be precipitated from aqueous solutions.b Cannot be measured precisely. However, the following values were found: 25.7 ± 0.1 (pH = 7.40), 29.9 ± 0.1 (pH = 6.00), 32.7 ± 0.1

(pH = 5.28). The comparative extent of dissolution in acidic buffer is: ACP » �-TCP » �-TCP > CDHA » HA > FA.c Stable at temperatures above 100 ◦C.d Always metastable.e Occasionally, it is called ‘‘precipitated HA (PHA)’’.f Existence of OA remains questionable.

to Wikipedia, the free encyclopedia, calcium (from Latincalx, genitive calcis, meaning ‘‘lime’’) compounds wereknown as early as the first century, when the ancient Romansprepared lime as calcium oxide. However, calcium sulfate(also known as plaster of Paris or lime plaster) had beenknown much earlier: three statues were discovered in aburied pit at ′Ain Ghazal in Jordan those were sculptedwith lime plaster over armatures of reeds and twine. Theywere made in the pre-pottery Neolithic period, around 7200BC. However, calcium was not isolated until 1808, whenSir Humphry Davy (1778—1829) electrolyzed a mixture oflime and mercuric oxide [3,4]. Phosphorus is a bit younger.The discovery of this chemical element (its name givenfrom Greek mythology, ��óо meaning ‘‘light-bearer’’(Latin Lucifer), referring to the ‘‘Morning Star’’, the planetVenus) is credited to a German merchant and alchemistHennig Brand (ca. 1630—ca. 1710) in 1669, although otheralchemists might have discovered phosphorus around thesame time. Brand experimented with urine, which contains

considerable quantities of dissolved phosphates from nor-mal metabolism. However, it was Antoine Laurent Lavoisier(1743—1794), who recognized phosphorus as a chemical ele-ment in 1777. Interestingly, but phosphorus appears to bethe first element discovered since antiquity. To concludethese introductive exercises, the earliest research paper, Ihave been able to find, containing the word ‘‘phosphorus’’in the title was written by Robert Boyle (1627—1691) andpublished in 1693 after his death [5].

Knowledge on CaPO4 in the 18th century

Due to the big problems with accessing to the scientific liter-ature published in the first half of the 19th century and evenearlier, a deep invasion into the history of the subject stillremains to be both fragmental and incomplete. That time,the scientific concepts were rather different from the mod-ern ones and chemical formulae of the substances had not

A history of calcium orthophosphates (CaPO4) and their biomedical applications 145

been introduced yet. Furthermore, scientific journals wererare; luckily, many scientific books published before the 20thcentury have been scanned by Google as a part of its projectto make the world’s books discoverable online. This timelyproject by Google combined with the power of the modernelectronic databases of scientific publications allows recons-tructing the major historical milestones on CaPO4, whichwas often impossible for earlier writers. For example, apaper of 1994 by Driskell entitled: ‘‘Early history of cal-cium phosphate materials and coatings’’ [6] starts from theclassical publication of 1920 by Albee assisted byMorrison [7]. In 1999, Shackelford published a paper:‘‘Bioceramics — an historical perspective’’ [8], in which thesame publication by Albee and Morrison [7] was mentionedas the earliest reference. The same is valid for the historicalpapers by Hulbert et al. [9,10] and Shepperd [11]. Thus, itseemed that calcium phosphates had been unknown before1920. Certainly, this is not the case; nevertheless, the pre-cise sequence of the scientific events happened in the 18thcentury still remain poorly restorable, while the historicaltime scale of even earlier scientific events remain almostirrecoverable. This is mainly due to a lack of the citationpractice existed in the scientific literature published in the19th century and before.

To the best of my findings, according to Shepperd [11],as early as in the end of the 18th century, a Germanchemist Martin Heinrich Klaproth (1743—1817) and a Frenchchemist Joseph-Louis Proust (1754—1826) proposed that cal-cium phosphates were the major inorganic component ofbones. Unfortunately, Shepperd has not provided any refer-ences to the publications by those great chemists. However,according to Roscoe and Schorlemmer [12], other researcheshad discovered this fact a bit earlier: ‘‘Gahn2, in 1769, dis-covered the existence of calcium phosphate in bones, but itwas not until this fact was published by Scheele3 in 1771 thatphosphorus was obtained from bone ash, which has from thattime invariably served for its preparation.’’ (p. 458). Fur-thermore, let me cite a publication of 17774: ‘‘I have onlybeen informed of this discovery, by the Gazette, Salutairede Bouillon, October, 1775. It is there said, that Mr. HenryGahn, a physician at Stockholm, has communicated a pro-cess for extracting from bones the saline matter in question;and that Mr. Scheele had ascertained, that the earth of ani-mals was composed of a calcareous substance united withthe phosphoric acid. This discovery, continues the author

2 Johan Gottlieb Gahn (1745—1818) was a Swedish chemist andmetallurgist, who discovered manganese in 1774.

3 Carl Wilhelm Scheele (1742—1786) was a German-Swedish phar-maceutical chemist, who discovered oxygen (although JosephPriestley published his findings first), as well as identified molyb-denum, tungsten, barium and chlorine before Humphry Davy.

4 A dictionary of chemistry. Containing the theory and practiceof that science: its application to natural philosophy, natural his-tory, medicine, and animal economy: with full explanations of thequalities and modes of action of chemical remedies: and the funda-mental principles of the arts, trades, and manufactures, dependenton chemistry. Translated from the French. With notes, additions,and plates. The second edition. To which is added, as an appendix, atreatise on the various kinds of permanently elastic fluids, or gases.Vol. III. Printed for T. Cadell, and P. Elmsly, in the Strand. London,1777, 666 pp.

of the article of the Gazette, belongs to Mr. Gahn, and hasbeen confirmed by later experiments.’’ (p. 383). Presum-ably, this citation might be considered as one of the earliestmentioning on calcium phosphates.

Furthermore, as written in a book by Lavoisier [13], theproduction process of orthophosphoric acid by decompo-sition of calcined bones in sulfuric acid has been knownsince, at least, 1790: ‘‘The bones of adult animals beingcalcined to whiteness, are pounded, and passed througha fine silk sieve; pour upon the fine powder a quantity ofdilute sulphuric acid, less than is sufficient for dissolvingthe whole. This acid unites with the calcareous earth of thebones into a sulphat of lime, and the phosphoric acid remainsfree in the liquor.’’ (p. 205). Further, the production pro-cess of white phosphorus has been described: ‘‘The liquid isdecanted off, and the residuum washed with boiling water;this water which has been used to wash out the adheringacid is joined with what was before decanted off, and thewhole is gradually evaporated; the dissolved sulphat of limecristallizes in form of silky threads, which are removed, andby continuing the evaporation we procure the phosphoricacid under the appearance of a white pellucid glass. Whenthis is powdered, and mixed with one third its weight ofcharcoal, we procure very pure phosphorus by sublimation.’’(p. 206).

Chemical investigations on CaPO4 in the 19thand the first half of the 20th centuries

In the 19th century, various investigations of calciumapatites and other CaPO4 were performed by Jöns JacobBerzelius (1779—1848) [14,15], M. Baruel [16], C. Morfit [17],Robert Warington (there were 2 chemists with this name,presumably, a father (1807—1867) and a son (1838—1907)),who performed the earliest well-documented systematicstudies of the outstanding quality [18—21], and R. Fresenius[22]. Furthermore, a German chemist Eilhard Mitscherlich(1794—1863) also worked in this area (unfortunately, I failedto find any reference to his publications). To provide someproofs of this statement, let me cite a paragraph from Ref.[19]: ‘‘Mitscherlich tells us, that when chloride of calciumis added to ordinary disodic phosphate, the latter beingmaintained in excess, the precipitate formed is tricalcicphosphate, while the solution becomes acid from the pro-duction of monosodic phosphate. Berzelius, on the contrary,states, that the precipitate formed under these conditionsis not tricalcic phosphate, but the octocalcic triphosphate,which lie has elsewhere described. All experimenters agree,that when the operation is reversed, and discc1.c. phos-phate is poured into an excess of chloride of calcium, theprecipitate is neither tricalcic nor octocalcic, but dicalcicphosphate.’’ (pp. 296—297). Thus, TCP, DCP and OCP havebeen known since, at least, 1866, while, in fact, a bit ear-lier. Furthermore, the standard preparation procedure ofthe sparingly soluble CaPO4 has been known since the sametime.

Among the available publications written by two RobertWaringtons [19—21], Ref. [23] by Robert Warington Jr.deserves both a special attention and extensive citations.For example, to prove, that OCP indeed was known in 1866,let me make another citation from Ref. [19]: ‘‘Octocalcic

146 S.V. Dorozhkin

phosphate can only be produced by the simultaneous for-mation of monosodic phosphate:

8CaCl2 + 5Na4H2P2O8

= Ca8H2P6O24 + 16NaCl + 2Na2H4P2O8.′ ′(p. 300).

One can see a balanced chemical equation, fully iden-tical to the modern ones. It is hard to believe, that it waspublished in 1866! More to the point, the hydrated forms ofCaPO4 were known in 1866: ‘‘8·73 grs. of the vacuum-driedsalt, lost on ignition 12·30 grains, or 26·35 per cent.; the for-mula Ca2H2P2O8·4H2O, demands 26·16 per cent. of water.’’(p. 99). Needless to explain, that ‘‘Ca2H2P2O8·4H2O’’ repre-sents 2 molecules of DCPD (see Table 1). Furthermore: ‘‘Itis interesting to observe that while disodic phosphate is ofan alkaline nature, dicalcic phosphate possesses faint acidproperties.’’ (p. 300). The form and shape of DCPD crystalswere described as well: ‘‘The crystalline form of the dical-cic tetrahydrated phosphate has been examined by ProfessorChurch. He describes the crystals as thin rhomboïdal plates,of which the diagonally opposite acute angles are sometimestruncated, hexagonal forms being thus produced. This trun-cation seems to be occasionally hemihedral, and then mayproceed up to the diagonal between obtuse angles; fromthis change triangular forms arise. Other modifications arealso met with.’’ (pp. 300—301). Another interesting conclu-sion might be found here: ‘‘We may then safely affirm thatwhenever dicalcic phosphate, octocalcic triphosphate, orany phosphate of intermediate composition, is precipitatedfrom solution by ammonia, the salt obtained will be theoctocalcic triphosphate; a tricalcic phosphate cannot beobtained in this manner. The following is probably a typeof the reaction:

— 4Ca2H2P2O8 + 6NH3

= Ca8H2P6O24 + 6NH4.P2O8.′ ′(pp. 301—302).

This seems to be the earliest mentioning on the fact thatTCP cannot be precipitated from the aqueous solutions (cur-rently we know that ACP or CDHA are precipitated instead).However, the following citation from the same publication:‘‘It is quite possible that precipitated tricalcic phosphatemay possess somewhat different solubilities, when preparedby different methods; this difference can, however, scarcelybe great.’’ (p. 304) means that this fact was not quite clearin 1866.

The latest available publication by Warington [21] wasdevoted to the hydrolysis of a freshly precipitated TCP(i.e., either ACP or CDHA) to the stoichiometric HA undercontinuous (up to 50 hours) boiling in distilled water. Fromthe results of numerous chemical analyses, the author con-cluded that during boiling an aqueous suspension of the TCPwas slowly transformed to a suspension of 3Ca3P2O8.CaOH2O(i.e., HA — see Table 1) and soluble acidic CaPO4. Thefollowing conclusion was made: ‘‘Since it appears that allphosphates of calcium less basic than apatite are unstableunder the continued action of pure water, it seems probablethat a more exact examination of natural phosphates wouldshow that many phosphates now regarded as tricalcic are infact of a more basic nature.’’ (p. 989). Thus, the apatiticnature of the majority of natural CaPO4 has been predicted

in 1873. The next available study on the TCP hydrolysis waspublished in 1929 only [23].

To give the appropriate tribute to other researchers, oneshould mention that existence of acidic CaPO4, currentlyknown as MCP and DCP (it is impossible to discover, whetherthey were hydrated or anhydrous), has been known, since, atleast 1806: ‘‘In this way were distinguished among the saltstwo combinations, one neutral, and one with an excess ofacid; and these were supposed to be determinate, as in sul-phate and super-sulphate of potassa, or the phosphate andsuperphosphate of lime.’’ [24, p. 38]. Furthermore, in 1849,it was written that ‘‘M.J.L. Lassaigne, at the meeting of theAcademy of Sciences of Paris, of the 15th January, presenteda memoir upon this subject, showing by experiments thatthe phosphate and carbonate of lime are introduced intoplants in solution in water containing carbonic acid, whichhad before been shown as to the phosphate by M. Dumas,and has long been known as to the carbonate.’’ [25]. Thus,a higher solubility of both CaPO4 and calcium carbonate inweak acids was already known in 1849. The first accessiblepaper on detection and preservation techniques of variousdeposits, including CaPO4, was published shortly afterwards[26]. Presence of CaPO4 in teakwood was established in1862 [27]. Various analytical topics on CaPO4 chemistry hasbeen studied since, at least, 1863 [28] and remained to bea subject of active research approx. until 1910-s [29—38].A popular fertilizer superphosphate, which represents ‘‘amixture of the last-mentioned compound and sulphate oflime’’ [39] has been known since, at least, 1868 [40]. Morethan 20 research papers on superphosphate were publishedby the beginning of the 20th century, which indicated tothe importance of this chemical for the human being. Togive the appropriate tribute to those early investigatorsand restore the historical perspective, it is worth citingthem all [41—62]. One can see that almost all of thesestudies were performed and published by single researchers(see individual comments to Refs. [41,48]), while themajority of the investigations were devoted to analyticalchemistry.

In Chemistry by Wilson, published in 1850 [63], one canread the following: ‘‘797. Phosphates of Lime. — There aremany compounds of lime and phosphoric acid, owing to thepeculiarity of that acid in relation to the number of equiva-lents of base it combines with at once. The most interestingphosphate of lime is that which occurs in bones, and is distin-guished as the bone-earth phosphate, 3CaO,PO5.’’ (p. 219).Thus, various CaPO4 were already known in 1850. However,the preparation technique sounds unusual to the modernreaders: ‘‘The phosphorus combines in part with the oxy-gen of the lime, CaO, to form phosphoric acid, and this withundecomposed lime, to form phosphate of lime, CaO,PO5.At the same time another portion of the phosphorus com-bines with the calcium of the lime, forming phosphuret ofcalcium, CaP.’’ (p. 164). In Chemistry by Brande and Taylor,published in 1863 [64], one can find the following state-ments: ‘‘Common Phosphate of Lime; Tribasic Phosphate of Lime; Bone

Phosphate; (3(CaO),PO5). — This salt occurs abundantly inbone ash, and is found as a mineral product.’’ (p. 331).Furthermore, ‘‘Native phosphate of lime (bone phosphate)occurs in apatite, moroxite, phosphorite, and asparagusstone; its primitive form is a six-sided prism: it also occursin some volcanic products.’’ (p. 332). Thus, a similarity


between the inorganic phase of bones and CaPO4 rocks ofnatural origin (apatite and phosphorites) was already knownin 1863. ‘‘When a solution of bone-earth in hydrochloric ornitric acid is boiled to expel all carbonic acid, and decom-posed by caustic ammonia, the bone phosphate separatesin the form of a bulky precipitate, which, when perfectlydried, is white and amorphous.’’ (p. 331). This statementis really astonishing because it might be considered as thefirst mention on ACP (reviewed in Ref. [65]), 32 years beforeWilhelm Conrad Röntgen (1845—1923) discovered X-rays in1895! Furthermore, the presence of carbonates in bones wasalready known. Next citation: ‘‘The substance known underthe name of coprolites, and which appear to be the excre-ments of fossil reptiles, also abound in phosphate of lime.’’(p. 332) means that already in 1863 researchers were awareon this fact.

Furthermore, let me make a citation from a publicationby Wells of 1906 [66]: ‘‘The apparent constancy of the pro-portion of carbonate and phosphate of calcium in bonesmade an impression on Hoppe-Seyler in I862, and we findhim speculating on the possibility of the components of thetwo salts being joined together to form one giant molecule:3 (Ca3 (Po4)2)-CaCo3, which he imagined might be united insome such way’’ (p. 522) as shown on Fig. 1. Further, Wellsmentions: ‘‘This formula is interesting chiefly from the his-torical standpoint, but it serves to emphasize the tendencyof these salts to appear in nearly constant proportions in theanimal body, a fact possibly of some importance.’’ (p. 523).Obviously, the atomic arrangement shown on Fig. 1 repre-sents the earliest structural drawing of a single molecule ofcarbonateapatite. An attentive reader will notice two dif-ferent types of calcium (currently known as Ca(1) and Ca(2))in that structure.

Vitrification properties of some forms of CaPO4 at heatinghave been known since, at least, 1877 [67]. The mod-ern chemical formula of perfectly transparent crystals ofnatural FA as Ca5(PO4)3F has been known since, at least,1873 [68], while the major crystallographic faces of a nat-ural calcium apatite were described in 1883 [69]. Chemical

Figure 1 The first available structure of a bone mineral (car-bonateapatite). Reprinted from Ref. [66].

formulae of DCP (as ‘‘Mono-Hydrogen CaPO4, HCaPO4’’) andMCP (as ‘‘Tetra-Hydrogen Calcium Phosphate, H4Ca(PO4)2’’)have been known since, at least, 1879 [43, pp. 205—206].Interestingly that in a publication of 1871, the chemicalformulae of CaPO4 were written in different ways: 3CaO PO5

for apatite and CaO 2HO PO5 for ‘‘some acid phosphate oflime’’ [42] (Table 2).

Neutral phosphates of lime have been known since, atleast, 1872 [70]. Besides, in the 19th century, calciumapatites were considered as combined compounds, whichresults from this citation: ‘‘Calcium phosphate, combinedwith calcium chloride or calcium fluoride, occurs in thewell-known minerals, apatite and osteolite.’’ [39, p. 188].One might guess that, in the 19th century, the atomicarrangement of single molecule of carbonateapatite (Fig. 1)could have inspired researchers to compose similar draw-ings for the single molecules of FA, HA and/or chlorapatite;however, I have not succeeded to find anything on thismatter.

Chemical equations, describing various interactionsbetween calcium phosphates and other chemicals havebeen known since, at least, 1863. For example, theafore-cited production processes of both orthophosphoricacid and white phosphorus from the Lavoisier book [13],in 1863 were written using chemical equations [64]:‘‘When bone phosphate is digested in dilute sulphuricacid, it is resolved into sulphate of lime and (if asufficiency of sulphuric acid be used) phosphoric acid:3(CaO),PO5 + 3SO3 = 3[CaO,SO3] + PO5.’’ (p. 331). Further-more, various types of phosphates (namely, metaphosphate,acid phosphate) and differences in their solubility have beenalready known. Let me cite: ‘‘In order to prepare phospho-rus, the bone ash is first mixed with so much dilute sulphuricacid as to form the acid phosphate:

— Ca3(PO4)2 + 2H2SO4 = CaH4(PO4)2 + 2CaSO4.

The solution of this soluble acid phosphate is nextpoured off from the precipitated gypsum, and evaporated to

Table 2 The chemical composition of two bone samplestaken from a publication of 1871 [20].

Commercial bone ash Pure ox bone ash

Moisture andvolatilematter

6.70 1.86

Siliceousmatter

9.69 0.51

Oxide of iron 0.58 0.17Lime 43.37 52.46Magnesia 1.14 1.02Phosphoric acid 33.68 39.55Carbonic acid,

alkalies, andothersubstancesundeter-mined

4.84 4.43

Total 100.00 100.00

148 S.V. Dorozhkin

dryness, after which, the solid residue being heated to red-ness, water is given off and calcium metaphosphate formed:

— CaH4(PO4)2 = Ca(PO3)2 + 2H2O.

This salt is then carefully mixed with charcoal, andheated to bright redness in earthenware retorts shown inFig. 147, when the following change takes place:

— Ca(PO3)2 + 10C = P4 + Ca3(PO4)2 + 10CO.′ ′[12][12, p. 460].

The quantitative analysis of a CaPO4 was performed in1884 [71], followed by remarks by C. Glaser in 1885 [72].In 1880-s, occurrence of a calcium apatite [73] and TTCP[74—76] in metallurgical slags was discovered. A chemicalinteraction of TCP with carbon dioxide and iron hydrox-ide was investigated in 1891 [77], while, at the beginningof the 20th century, the systematic studies of CaPO4 wereperformed by F.K. Cameron with co-workers [62,78—84]and H. Bassett [85—88]. Namely, the first version of thesolubility diagram CaO—P2O5—H2O was composed by Bas-sett in 1908 [87]. As follows from the available literature[12,39,89], by that time, the researchers already operatedwith individual CaPO4. Binary salts of CaPO4 with orthophos-phates of other chemical elements have been known since,at least, 1911 [90]. Currently well-known chemical route ofCDHA preparation by mixing of aqueous solutions of calciumnitrate and K2HPO4 (now (NH4)2HPO4 is used instead) wasinvestigated conductometrically in 1915 [91], while a neu-tralization reaction of H3PO4 by Ca(OH)2 was investigated in1923 [92]. The first paper on in vitro mineralization usingaqueous solutions containing ions of calcium and orthophos-phate was published in 1926 [93], followed by a publicationof 1933 [94]. The crystal structure of FA was established in1931 [95], followed by a great study on the structural char-acteristics of apatite-like CaPO4 of various origin in 1933[96]. Such terms as hydroxyfluorapatite, Ca10(F,OH)2(PO4)6,OA and carbonateapatite were already known. Besides,currently unknown substance ‘‘a hydrate of tricalcium phos-phate, Ca9(H2O)2(PO4)6’’ with the molecular weight 966.4and apatite-like diffraction pattern (obviously, it was CDHAwith x = 1, see Table 1) was mentioned as well [96]. Deter-mination of the amounts of CaPO4 in spinal fluid [97] andserum [98,99] were performed in the 1920-s, while thatin saliva — in the beginning of 1930-s [100]. Furthermore,the solubility data of several CaPO4 were updated in 1931[101,102]. Very popular at the turn of the millennium silicon-(or silica-) contained CaPO4, in fact, appear to be knownsince, at least, 1937 [103], while the earliest available paperon application of CaPO4 in chromatography was published in1942 [104].

In 1940, the available level of knowledge on basic CaPO4

(TCP, TTCP and apatites) was summarized into a big review[105], which might be considered as the first comprehen-sive review on the subject. Interestingly, that the authorsof this review have written about HA that ‘‘Existence asa unique stoichiometric compound doubtful’’ [105, p. 259(table)]. Furthermore, according to this review, �- and �-modifications of TCP (see Table 1) have been differentiatedin 1931—1933 by Schneiderhöhn and Bredig et al., who alsocreated the initial versions of the high-temperature diagram

for the binary system CaO—P2O5. That time, the existenceof OA was uncertain [105]. Besides, in 1940, a fundamentalstudy on the equilibrium in the system CaO—P2O5—H2O waspublished [106].

To finalize this chemical section, one should mention thatby 1928 it was clearly known that TCP could not be pre-pared by wet-precipitation. Let me cite: ‘‘We have beenunable to find any evidence of the existence of a molec-ular species with the formula Ca3(PO4)2’’. Precipitates ofthis ‘‘substance’’ rarely have the correct empirical compo-sition and they cannot be purified by recrystallization. Theevidence is such as to lead us to suspect that there maybe no such chemical entity as Ca3(PO4)2. No one has suc-ceeded in preparing it by precipitation [11,31—44], bearingout the theoretical objections to such a reaction on thegrounds that fifth order reactions do not occur [45]. We donot take the position that there can be no compound withthis formula. It may be found possible to synthesize it byother methods, but so far no one has succeeded in prepar-ing it by precipitation.’’ [107, p. 128]. Similar conclusionswere made by other researchers [108]. Nevertheless, thisknowledge was not common yet, since, in 1935, a reportwas published that ‘‘Tricalcium phosphate monohydrate wasprepared by the slow addition of calcium chloride solutionto a constantly agitated alkaline solution of disodium phos-phate, maintained at 65◦ to 70 ◦C.’’ [109]. This controversyhas been explained in Ref. [105] by a matter of definitions:‘‘The terms ‘‘tricalcium phosphate’’ and ‘‘hydroxyapatite’’are very widely and very loosely used. For example, someauthors use the former for any precipitate more basic thandicalcium phosphate, although such precipitates have beenfrequently shown to have an apatite lattice or to be mixturesof dicalcium phosphate and an apatite. Others confine theuse of the term to those precipitates with P2O5:CaO ratiosapproaching that of Ca3(PO4)2.’’ (p. 265).

More recent (after 1940) publications on chemistry ofCaPO4 are not considered, since they are well-known.

CaPO4 as bone graft substitutes

Historically, plaster of Paris (calcium sulfate) was the firstwidely tested artificial bioceramics. For example, accord-ing to Wikipedia, the free encyclopedia, literature datingback to 975 AD notes that calcium sulfate was useful forsetting broken bones. However, those were ex vivo appli-cations. According to the available literature, by the endof the 19th century, surgeons already used plaster of Parisas a bone-filling substitute [110]. Nevertheless, it was afamous German surgeon Themistocles Gluck (1853—1942),who, amongst his range of contributions, on 20 May 1890performed the first well-documented ivory (virtually, purebiological apatite) knee replacement bedded in a calciumsulfate based cement, which was followed by a total wristreplacement in another patient three weeks later [111].Later in 1890, Gluck presented a further case of a totalknee replacement to the Berlin Medical Society: at only35 days after operation, the patient was pain free withactive knee flexion and extension. All the joint arthro-plasties performed by Gluck were remarkably successful inthe short term; however, all ultimately failed because ofchronic infections [112,113]. After the abovementioned case


Figure 2 An advertisement of the S.S. White company for ‘‘Lacto-Phosphate of Lime’’ 1873. Reprinted from Dent. Cosmos 1873,15, 683.

of lacto-phosphate of lime (Fig. 2), this seems to be thesecond well-documented grafting application of CaPO4.

However, in the aforementioned cases, either thebiomedical applications of biologically produced CaPO4

(Gluck) or dental applications, not requiring any surgery(Cravens) have been described. According to both theelectronic databases and previous reviews on the subject[6,8—11], the first attempt to implant a laboratory producedCaPO4 (it was TCP) as an artificial material to repair surgi-cally created defects in rabbit bones was performed in 1920[7] by an US surgeon Fred Houdlette Albee (1876—1945),who invented bone grafting [114] and some other advancesin orthopedic surgery. The researchers injected either 0.5or 1 c.c. of 5% slurries of TCP in distilled water (whichwas then sterilized for three successive days in the Arnoldsterilizer, at 60 ◦C) into surgically created radial bone gapsof rabbits, leaving the periosteum intact [7]. Radiographicanalysis revealed that the TCP injected defect demonstratedmore rapid bone growth and union than the control. Theaverage length of time for bony union with TCP was 31days, compared to 41 days for the controls. No apprecia-ble bone growth was stimulated by injecting TCP beneaththe periosteum in non-defective radii or into subcutaneoustissues. Although this seems to be the first scientific studyon use of an artificially prepared CaPO4 for in vivo repairingof bone defects, it remains unclear whether that TCP was aprecipitated or a ceramic material and whether it was in apowder or a granular form. Unfortunately, the researcherspublished nothing further on this topic. In 1927, Hey Groves(1872—1944) described pure ivory hip hemi-arthroplasty forfracture [115]. In 1931, Murray also reported an accelerationof healing following implantation of calcium salts composedof 85% TCP and 15% CaCO3 in canine long bone defects[116,117].

At the beginning of 1930-s, the classic osteoinductivephenomenon was defined well by Huggins [118], who demon-strated that autoimplantation of transitional epitheliumof the urinary bladder to abdominal wall muscle in dogsprovoked ectopic bone formation. A bit later, Levanderdemonstrated that crude alcoholic extracts of bones induceda new bone formation when injected into muscle tissue[119,120].

Simultaneously, in 1930-s, Haldeman and Moore [121],Stewart [122], Key [123] and Shands [124] discovered thefact that only certain types of CaPO4 mentioned in Table 1really influenced the bone healing process. Namely, Halde-man and Moore implanted various CaPO4 such as MCP andDCP (it remains unclear whether they were in hydratedor anhydrous forms), TCP, as well as calcium glycerophos-phate as dry powdered salts into 0.5 to 1.0 cm defectsin radii of 17 rabbits, while the opposite side served as

control. Radiographic analysis demonstrated that in no casedid the presence of MCP, DCP or calcium glycerophosphatehad a favorable influence delayed healing compared to con-trol, while the presence of TCP at the site of the fractureappeared to favor the union [121]. Furthermore, Key [123]suggested that ‘‘if a defect in bone could be filled by a non-irritating, slowly soluble mass, which was porous and whichcontained calcium phosphate and carbonate in a form inwhich they could be resorbed, it would be reasonable toexpect osteoblasts to invade the mass, utilize the calcium,and build new bone which would replace the mass of calciumand cause the bone to be restored to its original form. Theideal material would appear to be rather dense cancellousbone from which a large percentage of the organic mate-rial had been removed.’’ (p. 176). However, Key found that‘‘Neither calcium phosphate and carbonate in the propor-tions in which they occur in bone, nor bone powder, made byremoving the organic matter from bone, appear to stimulateosteogenesis of bone when implanted in a bone defect.’’(p. 184). Stewart [122] concluded that ‘‘1. Lime salts andboiled bone when placed into a bone defect with either trau-matized muscle or fascia do not serve as a source of availablecalcium resulting in supersaturation of connective tissue andregeneration of missing bone. 2. Live bone chips placed inbone defects regenerate the missing bone.’’ (p. 871). Shands[124] also reported conflicting effects of several calciumsalts (calcium glycerophosphate, a mixture of TCP (3 parts)and CaCO3 (1 part), bone ash and calcium gluconate) on bonerepair. Namely, in defects in the ulna of dogs, the investi-gated calcium salts appeared to stimulate bone formation,while in operations upon the spine, calcium glycerophos-phate did not stimulate bone formation and appeared ratherto exert an inhibiting influence. In 1948, Schram and Fos-dick confirmed the fact that only certain types of CaPO4

influence the bone healing process [125]. Similar conclusionswere obtained in 1951 by Ray and Ward [126].

In 1950, the history of CaPO4 cements was started[127]. The author of that important publication investi-gated mixtures of both oxides and hydroxides of variousmetals with aqueous solutions of orthophosphoric acid anddiscovered a number of cold-setting formulations. For exam-ple, he found that CaO, sintered at 1100 ◦C, did not setin H3PO4, while that in liquid containing 9.6% CaO wasfound to set after ∼ 12 h in presence of H3PO4 [127].The latter mixture might be considered as the first proto-type of self-setting CaPO4 formulations (reviewed in Ref.[128]); however, the real history of this subject started in1982.

Due to the reasons, mentioned in the abstract, morerecent historical events are reported very briefly. Namely,the modern history of ACP started in 1955 [65], while more

150 S.V. Dorozhkin

than 20 years afterwards the first dental application of aCaPO4 (erroneously described as TCP) in surgically createdperiodontal defects [129] and the use of dense HA cylindersfor immediate tooth root replacement [130] were reported.The history of CaPO4 based biocomposites and hybrid bioma-terials (reviewed in Ref. [131]) started in 1981, while thatof CaPO4 deposits (reviewed in Ref. [132]) started in 1980.An extensive commercialization of the dental and surgicalapplications of CaPO4 (mainly, HA) bioceramics (reviewed inRef. [133]) occurred in the 1980’s, largely due to the pio-neering efforts by Jarcho in the USA, de Groot in Europeand Aoki in Japan. Shortly afterwards HA has become abioceramic of reference in the field of CaPO4 for biomed-ical applications, while the history of nanodimensional andnanocrystalline CaPO4 (reviewed in Ref. [134]) started in1995.

The interested readers are able to get further details on arecent history of CaPO4, bioceramics and biomaterials fromother reviews on the subject [6,8—11].

Conclusions

Even though CaPO4 have been investigated for morethan two centuries, commercial implants containing CaPO4

appeared only a few decades ago [133]. However, the advan-tages and obvious benefits of these chemical compounds arestill both an inspiration and a hope of many researchers andclinicians. The reported historical findings should encourageboth scientists and clinicians to reinvestigate the alreadyforgotten and poorly known facts and approaches in furtherbiomedical applications of CaPO4.


The author declare that they have no competing interest.

References

[1] Dorozhkin SV. Calcium orthophosphates: applications innature, biology, and medicine. Singapore: Pan Stanford; 2012[850 p.].

[2] Dorozhkin SV. Calcium orthophosphate-based bioceramics andbiocomposites. Weinheim, Germany: Wiley-VCH; 2016 [405p.].

[3] Davy H. The Bakerian lecture: on some new phenomena ofchemical changes produced by electricity, particularly thedecomposition of the fixed alkalies, and the exhibition ofthe new substances, which constitute their bases; and onthe general nature of alkaline bodies. Phil Trans R Soc Lond1808;98:1—44.

[4] Davy H. Electro-chemical researches on the decomposition ofthe earths; with observations on the metals obtained from thealkaline earths, and on the amalgam procured from ammonia.Phil Trans R Soc Lond 1808;98:333—70.

[5] Boyl R. A paper of the honourable Robert Boyl’s, depositedwith the secretaries of the Royal Society, Octob. 14. 1680.and opened since his death; being an account of his makingthe phosphorus, etc. Phil Trans 1693;17:583—4.

[6] Driskell TD. Early history of calcium phosphate materials andcoatings. In: Horowitz E, Parr JE, editors.Characterization

and performance of calcium phosphate coatings for implants.ASTM STP 1196. Philadelphia, USA: American Society for Test-ing and Materials; 1994. p. 1—9.

[7] Albee FH. Studies in bone growth. Triple calcium phosphate asa stimulus to osteogenesis. Ann Surg 1920;71:32—9 [Assistedby Morrison HF].

[8] Shackelford JF. Bioceramics — an historical perspective. MaterSci Forum 1999;293:1—4.

[9] Hulbert SF, Hench LL, Forbers D, Bowman LS. History of bio-ceramics. Ceram Int 1982;8:131—40.

[10] Hulbert SF, Hench LL, Forbers D, Bowman LS. His-tory of bioceramics. In: Vincenzini P, editor. Ceramicsin surgery. Amsterdam, The Netherlands: Elsevier; 1983.p. 3—29.

[11] Shepperd J. The early biological history of calcium phos-phates. In: Epinette JA, Manley MT, editors. Fifteen yearsof clinical experience with hydroxyapatite coatings in jointarthroplasty. France: Springer; 2004. p. 3—8.

[12] Roscoe HE, Schorlemmer C. A treatise on chemistry. Volume I:the non-metallic elements. London: Macmillan and Co.; 1881[751 p.].

[13] Lavoisier. Elements of chemistry, in a new systematic order,containing all the modern discoveries. Translated from theFrench, by Robert Kerr. Edinburgh: printed for WilliamCreech, and sold in London by G.G. and J.J. Robinsons; 1790[511 p.].

[14] Berzelius J. Untersuchungen über die Zusammensetzung derPhosphorsäure, der phosphorigen Säure und ihrer Salze. AnnPhysik 1816;53:393—446.

[15] Berzelius J. Ueber basische phosphorsaure Kalkerde. JustusLiebigs Annalen der Chemie 1845;53:286—8.

[16] Baruel M. Analysis of a double phosphate of lead and lime. JFranklin Inst 1838;25:343.

[17] Morfit C. On Colombian guano; and certain peculiarities in thechemical behavior of ‘‘bone phosphate of lime’’. J FranklinInst 1855;30:325—9.

[18] Warington R. CXIX. On a curious change in the composi-tion of bones taken from the guano. Mem Proc Chem Soc1843;2:223—6.

[19] Warington Jr R. XXVII. — Researches on the phosphates of cal-cium, and upon the solubility of tricalcic phosphate. J ChemSoc 1866;19:296—318.

[20] Warington R. X. — On the solubility of the phosphatesof bone ash in carbonic water. J Chem Soc 1871;24:80—3.

[21] Warington R. XL. — On the decomposition of tricalcic phos-phate by water. J Chem Soc 1873;26:983—9.

[22] Fresenius R. Ueber die Bestimmung der Phosphorsäure imPhosphorit nebst Mittheilung der Analysen des Phosphoritsund Staffelits aus dem Lahnthal. Zeitschrift für AnalytischeChemie 1867;6:403—9.

[23] Lorah JR, Tartar HV, Wood L. A basic phosphate of calciumand of strontium and the adsorption of calcium hydroxide bybasic calcium phosphate and by tricalcium phosphate. J AmChem Soc 1929;51:1097—106.

[24] Murray J. A system of chemistry. Vol. I. Edinburgh. Printed forLongman, Hurst, Rees & Orme, London; and William Creech,and A. Constable & Co.; 1806 [592 p.].

[25] F. On the method by which the phosphate and carbonate oflime is introduced into the organs of plants. J Franklin Inst1849;48:156.

[26] Hassall A. On the detection and preservation of crystallinedeposits of uric acid, urate of ammonia, phosphate of lime,triple phosphate, oxalate of lime, and other salts. Lancet1852;59:466—7.

[27] Abel FA. XVIII. — On the occurrence of considerable depositsof crystallized phosphate of lime in teakwood. J Chem Soc1862;15:91—3.


[28] Stammer C. Bestimmung kohlensauren Kalkes nebenphosphorsaurem Kalk. Zeitschrift für Analytische Chemie1863;2:96—7.

[29] Roussin Z. Prufung des Wismuthsubnitrats auf eine Ver-fälschung mit Kalkphosphat. Zeitschrift für AnalytischeChemie 1868;7:511.

[30] Janovsky JV. Ueber die verschiedenen Methoden derPhosphorsäure-Bestimmung neben Eisenoxyd, Thonerde,Kalk und Magnesia. Zeitschrift für Analytische Chemie1872;11:153—67.

[31] Pellet H. Die Zusammensetzung des Niederschlags, welcherdurch Ammoniak aus sauren Lösungen von Phosphorsäure,Baryt, Kalk und Magnesia gefällt wird. Zeitschrift für Ana-lytische Chemie 1882;21:261.

[32] Stokvis BJ, Salkowski E, Smith WG. Ueber die Löslichkeitsver-hältnisse des phosphorsauren Kalks im Harn. Zeitschrift fürAnalytische Chemie 1884;23:273—4 [in reality, this publica-tion represents a set of 3 short independent studies performedby 3 individual authors but combined under a general title].

[33] Ott A. Die Löslichkeitsverhältnisse des phosphorsauren Kalksim Harn. Zeitschrift für Analytische Chemie 1886;25:279—80.

[34] Kennepohl G. Zur Bestimmung von Eisenoxyd und Thonerdeneben Kalk und Phosphorsäure. Zeitschrift für AnalytischeChemie 1889;28:343.

[35] Immendorff H, Reitmair O. Zur Bestimmung des Kalks inGegenwart von Phosphorsäure, Eisen, Thonerde und Mangan.Zeitschrift für Analytische Chemie 1892;31:313—6 [in reality,this publication represents a set of 2 short independent stud-ies performed by 2 individual authors but combined under ageneral title].

[36] Fingerling G, Grombach A. Eine neue Modifikation derBestimmung der zitratlöslichen Phosphorsäure in den Fut-terkalken nach Petermann. Zeitschrift für Analytische Chemie1907;46:756—60.

[37] Schulze B. Untersuchung des phosphorsauren Futterkalkes.Zeitschrift für Analytische Chemie 1911;50:126—7.

[38] Hinden F. Anreicherungsmethode zur Bestimmung der Phos-phorsäure in phosphorsäurearmen Kalksteinen. Zeitschrift fürAnalytische Chemie 1915;54:214—6.

[39] Roscoe HE, Schorlemmer C. A treatise on chemistry. VolumeII: metals. Part 1. London, UK: Macmillan and Co.; 1879 [504p.].

[40] Fresenius R. Zur Analyse der Superphosphate. Zeitschrift fürAnalytische Chemie 1868;7:304—9.

[41] Chesshire JA, Hughes J, Sutton F, Sibson A. Ueber dieBestimmung des Betrags an ‘‘reducirten’’ Phosphatenin Superphosphaten. Zeitschrift für Analytische Chemie1870;9:524—7 [in reality, this publication represents a set of 4short independent studies performed by 4 individual authorsbut combined under a general title].

[42] Williams CP. On the solubility of some forms of phosphate oflime. J Franklin Inst 1871;92:419—23.

[43] Rümpler A. Ueber eisen- und thonerdehaltige Superphosphateund deren analytische Untersuchung. Zeitschrift für Analytis-che Chemie 1873;12:151—63.

[44] Albert H, Siegfried L. Beiträge zur Werthbestimmungder Superphosphate. Zeitschrift für Analytische Chemie1877;16:182—8.

[45] Albert H, Siegfried L. Beiträge zur Werthbestimmungder Superphosphate. Zeitschrift für Analytische Chemie1879;18:220—4.

[46] Pavec A. Zur maassanalytischen Bestimmung der Phospho-rsäure im Superphosphat und Spodium mittelst Uranlösung.Zeitschrift für Analytische Chemie 1879;18:360—1.

[47] Mohr C. Ein maassanalytisches Bestimmungsverfahren der inRohphosphaten und Superphosphaten enthaltenen Phospho-rsäure mit Uran bei Gegenwart von Eisenoxyd. Zeitschrift fürAnalytische Chemie 1880;19:150—3.

[48] Erlenmeyer E, Wattenberg H, Wein E, Rösch L, LehmannJ, Johnson SW, et al. Zur Analyse der Superphosphate.Zeitschrift für Analytische Chemie 1880;19:243—6 [in reality,this publication represents a set of 7 short independent stud-ies performed by 7 individual authors but combined under ageneral title].

[49] Meyer CF. Weitere Mittheilungen über das Zurückgehen dereisen- und thonerdehaltigen Superphosphate — Berichtigung.Zeitschrift für Analytische Chemie 1880;19:309—11.

[50] Drewsen S. Zur Bestimmung der löslichen Phosphorsäurein Superphosphaten. Zeitschrift für Analytische Chemie1881;20:54—7.

[51] Mollenda A. Eine neue Methode zur maassanalytischenBestimmung der Phosphorsäure in den Superphosphaten.Zeitschrift für Analytische Chemie 1883;22:155—9.

[52] Phillips WB. Rate of reversion in superphosphates preparedfrom red navassa rock. J Am Chem Soc 1884;6:224—8.

[53] Wagner P. Eine neue Methode zur Feststellung des Han-delswerthes der Superphosphate. Zeitschrift für AnalytischeChemie 1886;25:272—8.

[54] Emmerling A. Eine Methode zur Bestimmung der wasser-löslichen Phosphorsäure in Superphosphaten auf maas-sanalvtischem Wege. Zeitschrift für Analytische Chemie1887;26:244—7.

[55] Stoklasa J. Bestimmung des Wassers in den Superphosphaten.I. Zeitschrift für Analytische Chemie 1890;29:390—7.

[56] Crispo D. Belgische Methode zur Bestimmung der in Wasserlöslichen Phosphorsaure in den Superphosphaten. Zeitschriftfür Analytische Chemie 1891;30:301—3.

[57] Güssefeld O. Eine Schüttelmaschine für die Analyse von Super-phosphaten. Zeitschrift für Analytische Chemie 1892;31:556.

[58] Keller A. Eine Schüttelmaschine für Superphosphaten.Zeitschrift für Analytische Chemie 1893;32:590—1.

[59] Kalmann W, Meissels K. Eine Methode zur maassanalytischenSchätzung der wasserlöslichen Phosphorsaure in Super-phosphaten. Zeitschrift für Analytische Chemie 1894;33:764—6.

[60] Glaser C. Zur maassanalytischen Bestimmung der wasser-löslichen Phosphorsäure in Superphosphaten. Zeitschrift fürAnalytische Chemie 1895;34:768—9.

[61] Seib O. Bestimmung der zitratlöslichen Phosphorsäurein Superphosphaten. Zeitschrift für Analytische Chemie1905;44:397—8.

[62] Cameron FK, Bell JM. The phosphates of calcium, III; Super-phosphate. J Am Chem Soc 1906;28:1222—9.

[63] Wilson G. Chemistry. Edinburgh, UK: William and RobertChambers; 1850 [316 p.].

[64] Brande WT, Taylor AS. Chemistry. Philadelphia, USA: Blan-chard and Lea; 1863 [696 p.].

[65] Dorozhkin SV. Amorphous calcium orthophosphates: nature,chemistry and biomedical applications. Int J Mater Chem2012;2:19—46.

[66] Wells HG. Pathological calcification. J Med Res1906;14:491—525.

[67] C. Vitreous phosphate of lime. J Franklin Inst 1877;104:315.[68] Church AH. New analyses of certain mineral arseniates and

phosphates. 1. Apatite; 2. Arseniosiderite; 3. Childrenite;4. Ehlite; 5. Tyrolite; 6. Wavellite. J Chem Soc 1873;26:101—11.

[69] LWJ. On a fine specimen of apatite from Tyrol, lately in thepossession of Mr. Samuel Henson. Nature 1883;27:608—9.

[70] Reichardt E. Ueber neutralen phosphorsauren Kalk, Darstel-lung und Löslichkeit desselben. Zeitschrift für AnalytischeChemie 1872;11:275—7.

[71] Mohr C. Ueber die quantitative Bestimmung der zurück-gegangenen Phosphorsäure und der Phosphorsäure imDicalciumphosphat. Zeitschrift für Analytische Chemie1884;23:487—91.

152 S.V. Dorozhkin

[72] Glaser C. Bemerkungen zu der Abhandlung des Herrn CarlMohr über die quantitative Bestimmung der zurückgegan-genen Phosphorsäure und der Phosphorsäure im Dicalci-umphosphat. Zeitschrift für Analytische Chemie 1885;24:180.

[73] Hutchings WM. Occurrence of apatite in slag. Nature1887;36:460.

[74] Hilgenstock G. Eine neue Verbindung von P2O5 und CaO. Stahlund Eisen 1883;3:498.

[75] Hilgenstock G. Das vierbasische Kalkphosphat und die Basic-itätsstufe des Silicats in der Thomas-Schlacxke. Stahl undEisen 1887;7:557—60.

[76] Scheibler C. Ueber die Herstellung reicher Kalkphosphate inVerbindung mit einer Verbesserung des Thomasprocesses. BerDtsch Chem Ges 1886;19:1883—93.

[77] Georgievics GV. Über das Verhalten des Tricalciumphos-phats gegen Kohlensäure und Eisenhydroxyd. Monatshefte fürChemie 1891;12:566—81.

[78] Cameron FK, Hurst LA. The action of water and saline solu-tions upon certain slightly soluble phosphates. J Am Chem Soc1904;26:885—913.

[79] Cameron FK, Seidell A. The action of water upon the phos-phates of calcium. J Am Chem Soc 1904;26:1454—63.

[80] Cameron FK, Seidel A. The phosphates of calcium. I. J AmChem Soc 1905;27:1503—12.

[81] Cameron FK, Bell JM. The phosphates of calcium. II. J AmChem Soc 1905;27:1512—4.

[82] Cameron FK, Bell JM. The phosphates of calcium. IV. J AmChem Soc 1910;32:869—73.

[83] Cameron FK, McCaughey WJ. Apatite and spodiosite. J PhysChem 1911;15:463—70.

[84] Mebane WM, Dobbins JT, Cameron FK. The solubility of thephosphates of calcium in aqueous solutions of sulfur dioxide.J Phys Chem 1929;33:961—9.

[85] Bassett Jr H. Beiträge zum Studium der Calciumphosphate. I.Die Hydrate der Calcium-Hydroorthophosphate. Z Anorg Chem1907;53:34—48.

[86] Bassett Jr H. Beiträge zum Studium der Calciumphos-phate. II. Die Einwirkung von Ammoniakgas auf Calcium-Hydroorthophosphate. Z Anorg Chem 1907;53:49—62.

[87] Bassett Jr H. Beiträge zum Studium der Calciumphosphate.III. Das System CaO — P2O5 — H2O. Z Anorg Chem 1908;59:1—55.

[88] Bassett Jr H. The phosphates of calcium. Part IV. The basicphosphates. J Chem Soc 1917;111:620—42.

[89] Norton TH, Newman HE. LV. — On a soluble compound ofhydrastine with monocalcium phosphate. J Am Chem Soc1897;19:838—40.

[90] Rolfe BH. Autunite (hydrated uranium-calcium phosphate).Lancet 1911;177:766.

[91] Withers WA, Field AL. A conductivity study of the reactionbetween calcium nitrate and dipotassium phosphate in dilutesolution. J Am Chem Soc 1915;37:1091—105.

[92] Wendt GL. An electrometric study of the neutralizationof phosphoric acid by calcium hydroxide. J Am Chem Soc1923;45:881—7.

[93] Shipley PG, Kramer B, Howland J. Studies upon calcificationin vitro. Biochem J 1926;20:379—87.

[94] Benjamin HR. The forms of the calcium and inorganicphosphorus in human and animal sera II. The nature andsignificance of the filtrable, adsorbable calcium-phosphoruscomplex. J Biol Chem 1933;100:57—78.

[95] Náray-Szabó S. The structure of apatite (CaF)Ca4(PO4)3.Zeitschrift für Kristallographie 1931;75:387—98.

[96] Hendricks SB, Hill WL, Jacob KD, Jefferson ME. Structuralcharacteristics of apatite-like substances and composition ofphosphate rock and bone as determined from microscopicaland X-ray diffraction. Ind Eng Chem 1931;23:1413—8.

[97] Barrio NG. Comparative studies in the chemistry of blood andcerebrospinal fluid. II. Calcium, magnesium, and phosphorus.J Labor Clin Med 1923;9:54—6.

[98] Wang CC, Felsher AR. The effect of hemolysis on the cal-cium and inorganic phosphorus content of serum and plasma.J Labor Clin Med 1925;10:269—72.

[99] Nitschke A. Darstellung Einer den Calciumgehalt und Einerden Phosphatgehalt des Serum Senkenden Substanz — II.Mitteilung. Nachweis in Milz und Lymphknoten. KlinischeWochenschrift 1929;8:794.

[100] Krasnow F, Karshan M, Krejci LE. The determination of calciumand phosphorus in saliva. J Labor Clin Med 1932;17:1148—52.

[101] Clark NA. The system P2O5—CaO—H2O and the recrys-tallization of monocalcium phosphate. J Phys Chem1931;35:1232—8.

[102] Lugg JWH. A study of aqueous salt solutions in equilibriumwith solid secondary calcium phosphate at 40 ◦C. Trans Fara-day Soc 1931;27:297—309.

[103] Nagelschmidt G. A new calcium silicophosphate. J Chem Soc1937:865—7.

[104] Moore LA. Activation of dicalcium phosphate for the chro-matographic determination of carotene. Ind Eng Chem1942;14:707—8.

[105] Elsenberger S, Lehrman A, Turner WD. The basic calcium phos-phates and related systems. Some theoretical and practicalaspects. Chem Rev 1940;26:257—96.

[106] Elmore KL, Farr TD. Equilibrium in the system cal-cium oxide—phosphorus pentoxide—water. Ind Eng Chem1940;32:580—6.

[107] Shear MJ, Kramer B. Composition of bone. III. Physicochemicalmechanism. J Biol Chem 1928;79:125—45.

[108] Trömel G, Möller H. Die Bildung schwer löslicher Calci-umphosphate aus wäßriger Lösung und die Beziehungen dieserPhosphate zur Apatitgruppe. Zeitschrift für anorganische undallgemeine Chemie 1932;206:227—40.

[109] Larson HWE. Preparation and properties of mono-, di-, andtricalcium phosphates. Ind Eng Chem Anal Ed 1935;7:401—6.

[110] Dreesmann H. Ueber Knochenplombierung. Beitr Klin Chir1892;9:804—10.

[111] Gluck T. Referat über die durch das moderne chirurgis-che. Langenbecks Archiv für Klinische Chirurgie 1891;41:187—239.

[112] Muster D. Themistocles Gluck, Berlin 1890: a pioneer ofmultidisciplinary applied research into biomaterials for endo-prostheses. Bull Hist Dent 1990;38:3—6.

[113] Eynon-Lewis NJ, Ferry D, Pearse MF. Themistocles Gluck: anunrecognised genius. BMJ 1992;305:1534—6.

[114] Albee FH. Bone graft surgery. Philadelphia and London:W.B. Saunders Company; 1915 [417 p.].

[115] Hey Groves EW. Some contributions to the reconstructivesurgery of the hip. Br J Surg 1927;14:486—517.

[116] Murray CR. Delayed and non-union in fractures in the adult.Ann Surg 1931;93:961—7.

[117] Murray CR. The modern conception of bone formation and itsrelation to surgery. J Dent Res 1931;11:837—45.

[118] Huggins C. The formation of bone under the influence ofepithelium of the urinary tract. Arch Surg 1931;22:377—408.

[119] Levander G. On the formation of new bone in bone transplan-tation. Acta Chir Scand 1934;74:425—6.

[120] Levander G. A study of bone regeneration. Surg GynecolObstet 1938;67:705—14.

[121] Haldeman KO, Moore JM. Influence of a local excess of calciumand phosphorus on the healing of fractures. An experimentalstudy. Arch Surg 1934;29:385—96.

[122] Stewart WJ. Experimental bone regeneration using lime saltsand autogenous grafts as sources of available calcium. SurgGynec Obstet 1934;59:867—71.


[123] Key JA. The effect of a local calcium depot on osteogen-esis and healing of fractures. J Bone Joint Surg 1934;16:176—84.

[124] Shands Jr AR. Studies in bone formation: the effect of thelocal presence of calcium salts on osteogenesis. J Bone JointSurg 1937;19:1065—76.

[125] Schram WR, Fosdick LS. Stimulation of healing in longbones by use of artificial material. J Oral Surg 1948;6:209—17.

[126] Ray RD, Ward Jr AA. A preliminary report on studies ofbasic calcium phosphate in bone replacement. Surg Form1951;3:429—34.

[127] Kingery WD. II, cold-setting properties. J Am Ceram Soc1950;33:242—6.

[128] Dorozhkin SV. Self-setting calcium orthophosphate formula-tions. J Funct Biomater 2013;4:209—311.

[129] Nery EB, Lynch KL, Hirthe WM, Mueller KH. Bioceramicimplants in surgically produced infrabony defects. J Periodon-tol 1975;46:328—47.

[130] Denissen HW, de Groot K. Immediate dental root implantsfrom synthetic dense calcium hydroxylapatite. J ProsthetDent 1979;42:551—6.

[131] Dorozhkin SV. Calcium orthophosphate-containing biocompos-ites and hybrid biomaterials for biomedical applications. JFunct Biomater 2015;6:708—832.

[132] Dorozhkin SV. Calcium orthophosphate deposits: prepara-tion, properties and biomedical applications. Mater Sci EngC 2015;55:272—326.

[133] Dorozhkin SV. Calcium orthophosphate bioceramics. CeramInt 2015;41:13913—66.

[134] Dorozhkin SV. Nanodimensional and nanocrystalline calciumorthophosphates. Am J Biomed Eng 2012;2:48—97.

Morphologie (2017) 101, 154—163


ScienceDirect


ORIGINAL ARTICLE

Behavior of macrophage and osteoblast celllines in contact with the �-TCP biomaterial(beta-tricalcium phosphate)Comportement de lignées cellulaires de macrophages etd’ostéoblastes en contact avec le biomatériau �-TCP(phosphate bêta tricalcique)

B. Arbez, H. Libouban ∗

GEROM Groupe études remodelage osseux et biomatériaux, IRIS-IBS institut de biologie en santé,université d’Angers, CHU d’Angers, 49933 Angers cedex, France

Available online 19 September 2017

KEYWORDS�-TCP;Macrophages;Osteoblasts;Osteoconduction;Resorption

Summary Beta-tricalcium phosphate (�-TCP) is a synthetic ceramic used for filling bonedefects. It is a good alternative to autologous grafts since it is biocompatible, resorbable andosteoconductive. Previous in vivo studies have shown that macrophages are one of the first cellscoming in contact with the biomaterial followed by osteoclasts and osteoblasts that will elab-orate new bone packets. Studies have focused on osteoclast morphology and very few of themhave investigated the role of macrophages. The aims of this study were to characterize (i) thebiomaterial surface; (ii) the in vitro behavior of macrophages (J774.2 and Raw264.7 cells) usingthe description of cell morphology by scanning electron microscopy (SEM) at 7 and 14 days; (iii)the behavior of osteoblasts (SaOs-2 and MC3T3-E1 cells) seeded at the surface of the biomate-rial 24, 48 and 72 hours by SEM and confocal microscopy. Cell proliferation was analyzed by MTTassays. Viability and affinity of the macrophages for �-TCP were found significantly increasedafter 7 and 14d. MC3T3-E1 cells were anchored and stretched onto the �-TCP surface as early as24 h with a high proliferation rate (+ 190%) when compared to the surface of a well plate. SaOs-2 exhibited the same morphological profile at 72 h. Proliferation became significantly highercompared to the plastic surface at only 72 h (+129%). This study emphasises the importance ofchoice of the cell line used in exploring the osteoconductive and osteoinductive properties of abiomaterial. Additional studies are needed to analyze differentiation of macrophages into giantmultinucleated cells and how the biomaterial surface influences osteoblast differentiation.© 2017 Elsevier Masson SAS. All rights reserved.

∗ Corresponding author. GEROM — LHEA IRIS-IBS, CHU d’Angers 49933, Cedex - FRANCE.E-mail address: [email protected] (H. Libouban).







Morphology of macrophages and osteoblasts on �-TCP 155

MOTS CLÉS�-TCP ;Ostéoconduction ;Résorption ;Macrophages ;Ostéoblastes

Résumé Le bêta-tricalcium phosphate (�-TCP) est une céramique synthétique utilisée pourle comblement de defects osseux. Étant biocompatible, résorbable et ostéoconducteur, ilreprésente une bonne alternative aux greffes autologues. Des précédentes études in vivo ontmontré que les macrophages étaient l’un des premiers types cellulaires en contact avec lebiomatériau avec des cellules mésenchymateurs et des capillaires. Il sont suivis par les ostéo-clastes puis les ostéoblastes apposent de la matrice osseuse. Les études se sont centrées surla caractérisation des ostéoclastes et la morphologie des macrophages a été très peu étudiée.Les objectifs de cette étude ont été (i) de caractériser la surface du biomatériau ; (ii) la mor-phologie in vitro des macrophages déposés sur la biomatériaux (lignées J774.2 et Raw264.7)par microscopie électronique à balayage (MEB) à 7 et 14 jours ; (iii) d’analyser le comporte-ment cellulaire de 2 lignées ostéoblastiques (SaOs-2 et MC3T3-E1) en MEB et en microscopieconfocale à 24, 48 et 72 h après ensemencement La prolifération a été analysée par un test auMTT. Les résultats ont montré une bonne survie et une bonne affinité des macrophages sur le�-TCP à 7 et 14 jours. Les cellules MC3T3-E1 ont présenté un aspect aplati et très étiré à lasurface du �-TCP dès 24 h avec une prolifération plus élevée (+ 190 %) par rapport celle obtenuesur une surface plastique. Les cellules SaOs-2 ont montré le même profil morphologique à 72 h.La prolifération est devenue significativement plus élevée par rapport à la prolifération surune surface plastique à 72 h (+ 129 %). L’étude met en évidence l’importance du choix de lalignée cellulaire dans l’étude des propriétés inductives et ostéconductives d’un biomatériau.Des études supplémentaires sont nécessaires afin de mieux appréhender les mécanismes impli-quant la différenciation des macrophages en cellules géantes multinuclées ainsi que l’influencedu biomatériau sur la différenciation ostéoblastique.© 2017 Elsevier Masson SAS. Tous droits reserves.

Introduction

Beta-tricalcium phosphate (�-TCP) is a synthetic ceramicthat belongs to the calcium orthophosphate family. Its chem-ical composition (�-Ca3(PO4)2), close to the mineral phaseof bone, allows it to be used as a bone substitute forfilling defects in neurosurgery, maxillofacial, reconstruc-tive, orthopedics and spinal surgeries. TCP are known tobe biocompatible since almost a century. In 1920, Albeeand Morrison have reported for the first time the use cal-cium orthophosphate as bone graft in the rabbit radius [1].No adverse reaction, inflammation or toxic symptoms wereobserved. Osteogenesis was stimulated by TCP leading to afaster bone healing. The authors concluded that this mate-rial was suitable for clinical applications and could be usedin further studies on human subjects. Studies on �-TCPincreased in the 70s, showing the bioresorption ability of�-TCP with the first histologic observations in 1971: biore-sorption occurred simultaneously with the apposition of newbone packets after local recruitment of osteoblasts [2].Reliable methods of �-TCP production were subsequentlyproposed that lead to the commercialization of the materialin the 80s [3,4]. �-TCP is now recognized as osteoconduc-tive as it provides a resorbable template for the formationof new bone [5]. Histological studies show a marked apposi-tion of lamellar bone directly in contact with �-TCP within aperiod of 6 to 24 months [6,7]. In a rat model, resorption of�-TCP granules occurs 2 weeks after implantation associatedwith new bone formation inside �-TCP pores after 5 weeks[2]. In a rabbit model, new bone trabeculae invading thegrafted biomaterials were evidenced as early as 8 days bymicrocomputed tomography (microCT) [8]. Bone formationoccurring directly onto a biomaterial surface necessitatesdifferent types of cells: recruitment of osteoprogenitor cellsfrom surrounding mesenchymal cells, adhesion of osteogenic

cells followed by survival, proliferation and differentiation[9].

However, the degradation mechanisms of �-TCP remainunclear. Degradation of calcium/phosphate biomaterialsin the body is composed of two stages: cellular resorp-tion and the dissolution of the material [10,11]. Ca/Pbiomaterials can be eroded, phagocytized or degradedby pH modifications caused by osteoclasts which lead tothe demineralization of the material. Besides osteoclasts,macrophages (or their derived giant cells formed by fusion)are involved at an early stage of resorption [12,13]. Somestudies on �-TCP granules grafted in oral surgery suggestthat resorption of the biomaterial may happen by phago-cytosis with macrophages together with osteoclasts oncenew bone trabeculae are formed [6,14]. A double mech-anism of cellular degradation of �-TCP by osteoclasts andmacrophages is most probable.

Surface topography and porosity of implants and graftscan also influence bioresorption and the behavior of cellscoming in direct contact with the materials [10,15—17].Interactions of osteoblasts and macrophages with �-TCPsurface remains unclear. The aim of the study was to char-acterize: (i) the surface of plates made with �-TCP; (ii)the morphology of macrophages seeded onto the plates byscanning electron microscopy (SEM); (iii) the behavior ofosteoblast-like cells seeded on these plates by SEM, confocalmicroscopy and proliferation assay.

Material And methods

Characterization and preparation of �-TCP

�-TCP samplesPlates of 3D-printed �-TCP (Sinus-UpTM) were obtain fromKasios (Kasios, L’Union, France). Sinus-UpTM plates are

156 B. Arbez, H. Libouban

prepared by rapid prototyping by using elementary �-TCPpowder in hydroxypropyl-methylcellulose with water asbinder, plates are then subsequently sintered at high tem-perature and the hydroxypropylmeythylcellulose is burntoff during sintering. Sinus-UpTM are commercially availableand sold for sinus floor elevation. Sinus-UpTM have a cen-tral macroporous area (which was discard in this study) andflat lateral sides which were cut in plates for experimentalpurposes (0.8 cm by side).

Scanning Electron Microscopy (SEM)Surface morphology of the �-TCP plates was analyzed bySEM on a JEOL 6301F (JEOL Paris, France). All samples werecoated with a 20 nm layer of platinum by sputtering witha high vacuum coater (Leica EM ECA600, Leica, France).Images were captured in the secondary electron mode withan acceleration tension of 3 kV.

Energy-Dispersive X-ray Spectroscopy (EDS)An elemental analysis was performed on the �-TCP plates byenergy-dispersive X-ray spectroscopy (EDS) on a Zeiss, EVOLS10 SEM. The samples were not carbon or gold coated. Theworking pressure was 50 Pa and the acceleration tension was5 kV.

Raman spectroscopyThe chemical spectrum of the �-TCP plates was analyzedby Raman spectroscopy on a Senterra microscope with OPUS5.5 software (Bruker optic, Ettlingen). The excitation laserwavelength was 532 nm with an excitation power of 25 mWand 3—5 cm−1 resolution. The final spectrum was obtainedby averaging five scans of 20 sec each. A concave rubberbandbase line correction was applied (11 iterations, 64 points).

Cell culture reagents and preparation

All cell culture consumables were obtained from GIBCO(Thermofisher Scientific, Illkirch, France). Four culture celllines were used: two monocyte/macrophage cell linesJ774.2 (European Collection of Authenticated Cell CultureECACC #85011428, Salibury, UK) and Raw264.7 (AmericanType Culture Collection ATCC #TIB-71, Molsheim, France),Human SaOs-2 osteoblast-like cells (ATCC #HTB-85) andpre-osteoblast cell line MC3T3-E1 subclone 4 (ATCC #CRL-2593). J774.2, Raw264.7 and MC3T3-E1 cells were culturedin �-MEM (Minimum Essential Medium, alpha Modification)and SaOs-2 cells were cultured in DMEM (Dulbecco’s Mod-ified Eagle Medium). For all cultures, the medium wassupplemented with 10% heat-inactivated fetal calf serum,100 IU/ml penicillin and 100 �g/ml streptomycin. Mediumwas replaced every 2—3 days and the cultures were main-tained in humidified atmosphere of 5% CO2 at 37 ◦C. At80% confluence, MC3T3-E1 and SaOs-2 cells were detachedusing trypsin-EDTA (trypsin/ethylenediamine tetraaceticacid) and J774.1/Raw264.7 cells were harvested by scrap-ping.

Prior to cell seeding, �-TCP plates were sterilized in 70%ethanol during 24 hours and dried for an hour. They weresubsequently immerged during a night in the media (�-MEMsupplemented with 10% fetal calf serum) to remove any

trace of ethanol and to allow proteins from the medium toadhere onto the �-TCP surface.

Macrophage culture and seeding on �-TCP

J774.2 and Raw264.7 cell lines were seeded onto �-TCPplates at a density of 3.104 cells/cm2 and cultured dur-ing 7 and 14 days (two samples/time). �-TCP plates wereimmerged in 1 mL of medium in a 24-well plate andmacrophages were seeded in a homogenous way in themedium above the samples. At time of seeding, the mediumwas supplemented with 25 ng/mL Macrophage Colony Stim-ulating factor (M-CSF, Biotechne brand, R&D systems, Lille,France). Preparation of cells for SEM observation was thendone (see below).

Osteoblasts culture and seeding on �-TCP

MC3T3-E1 and SaOs-22 osteoblast cells were seeded onto �-TCP plates in 24 well plates at a density of 2 104 cell/cm2

and cultured during 24, 48 and 72 h. The �-TCP plates wereimmerged in 1 mL of culture medium and the cells wereseeded in a homogenous way in the medium above thesamples. Experiments for analysis of cell spreading, cellmorphology and proliferation were done in duplicate at eachtime point.

Cell spreading analyzed by confocal microscopyCells were fixed in 4% paraformaldehyde for 20 minutes at4 ◦C. They were rinsed 3 times 5 min in PBS and stained with2 �g/mL 4,6-diamidino-2-phenylindole (DAPI, Sigma, SaintQuentin-Fallavier, France) for 2 min at room temperature inthe dark. After rinsing 6 times in PBS for 5 min each, cellswere labeled with 6.6 �M Alexa Fluor 488-conjugated phal-loidin (Thermofisher Scientific Illkirch, France) for 45 min atroom temperature in the dark and rinsed in PBS (6 times,5 min each) and distilled water (6 times 5 min). The �-TCPplates with labeled cells were mounted between glass slideswith 30% glycerol. Labeled cells were observed on a LeicaTCS SP8 laser-scanning confocal microscope (Leica Microsys-tems, Heidelberg, Germany) with a HXC PL APO 63X CS2oil immersion objective (N.A. 1.40). Excitation and emis-sion wavelengths were set at 405 nm for DAPI labelling and488 nm for phalloidin labelling.

Some slides were counterstained with xylenol orange0.5 mg (Sigma) for 10 min in distilled water after the doublelabelling phalloidin/DAPI to label the �-TCP surface,

Cell proliferation by MTT assayThe number of total and viable cells on the surface of �-TCP plates was measured with a colorimetric MTT assayand compared with that of cells cultured directly ontothe well surface. MTT assay is based on the reductionof the yellow tetrazolium salt MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich) bythe mitochondrial succinate deshydrogenase. At each timepoint, �-TCP plates were transferred in a new 24 wells plate;cells were incubated with MTT at 0.5 mg/mL for 2 hours in ahumidified atmosphere of 5% CO2 at 37 ◦C. MTT was reducedto purple formazan crystals which were then dissolved in500 �L acidified isopropanol per well. Each supernatant was


transferred in 96 well plates for absorbance reading at570 nm with a spectrophotometric plate reader (SpectraMaxM2, Molecular Devices, Sunnyvale, CA) because absorbanceis proportional to the number of viable cells. Number of cellswas determined using a standard curve with a range of eachcell type concentration between 0 and 10 cells/cm2 and theywere reported to the seeded surface area (well surface or�-TCP plate surface). Prior to cell culture, the �-TCP plateswere imaged with a numeric radiography equipment (Fax-itron X-Ray LX-60, Edimex, France) and the surface area (incm2) was measured using the ImageJ software 1.45.

SEM of cells seeded on �-TCP plates

Samples were rinsed with cacodylate buffer (37 ◦C, pH 7.4)and fixed during one night at 4 ◦C with glutaraldehyde (2.5%in cacodylate buffer 0.2 M). Samples were subsequentlypostfixed with osmium tetroxide (1% in distilled water for1 hour). They were dehydrated with a gradient of ethanoland desiccated with hexamethyldisilazane and examined asabove described.

Statistical analysis

Statistical analysis was performed with the statistical soft-ware MedCalc version 8.2.10 (Ostend, Belgium). All datawere reported as mean ± standard error of the mean (SEM).Statistical significance between groups for MTT test wasdetermined by a non-parametric Kruskal-Wallis analysis ofvariance and comparison between groups was determined bypost-hoc test. A difference was considered significant whenP < 0.05.

Results

�-TCP characterization

SEMThe raw surface of the �-TCP samples appears on Fig. 1A.Although the surface of the biomaterial seemed rather flatmacroscopically, the SEM analysis revealed in fact a roughsurface with valleys and hills. At higher magnifications, thematerial surface showed a polycrystalline pavement withdifferent polygonal crystallites separated by grain bound-aries. Defect lines were also present on the surface of thematerial. Some crystallites showed a hexagonal pattern atthe surface of the polygonal pavement (Fig. 1B). A micro-porosity was evidenced at the surface of the biomaterialbetween the sintered grains, it was also visible of fracturedsections (data not shown).

Raman spectroscopyThe Raman spectrum of the �-TCP plates appears on Fig. 2for a wavenumber ranging from 50 to 1550 cm−1. The labeledpeaks are characteristic of the internal vibration of thePO4

3 tetrahedric groups of the �-TCP molecule. The sym-metric stretching (�1) of P O bonds of the tetrahedroncorresponds to the peaks with the highest intensity at around950 cm−1 and 970 cm−1. The asymmetric stretching (�3) hasa lower intensity and is located in the 1015—1090 cm−1

Figure 1 (A) SEM image of the surface of a �-TCP sinteredplate from a Sinus UpTM showing grain boundaries (white arrows)and defect lines (black arrows). Note the presence of a microp-orosity between the sintered grains. (B) SEM image of the �-TCPsurface showing shear bands with a hexagonal pattern.

1089

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200 40 0 60 0 800 100 0 1200 1400

Wavenumber (c m-1 )

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Figure 2 Raman spectrum of the �-TCP plate.

range. The other vibrational modes (�2 and �4) correspondto O P O bending deformations of the tetrahedron. Theyare respectively located at 407 and 548 cm−1.

Monocyte/macrophage cells morphology by SEM

After 7 and 14 days, RAW 264.7 and J774.2 cells have sur-vived on �-TCP surface. At 7 days of culture (Fig. 3A), themajority of Raw264.7 cells had a round shape. After 14days, some RAW cells appeared flattened when some othershad maintained a round shape (Fig. 3B). These cells have


Figure 3 RAW 264.7 cells after (A) 7 days and (B) 14 days of culture. At 14 days, some cells had an elongated shape (white arrows)and some others had a round shape (black arrows). Higher magnification of a RAW 264.7 cell on �-TCP after (C) 7 days and (D) 14days of culture.

emitted long filopodia that anchor them onto the �-TCP sur-face (Fig. 3D). The number of filopodia increased between 7days (Fig. 3 C) to 14 days; even if they cannot be counted onsuch a rough material, this corresponds to a firmer anchorcapacity of the cells to the biomaterial.

At 7 and 14 days, the osteoblastic J774 cells seededon the �-TCP had a round shape (Fig. 4). They appearedanchored to the surface with less filopodia than RAW 264.7cells in the same conditions; cytoplasmic veil-like structureswere observed on their surface (Fig. 4C-D). No difference inmorphology between the two times of culture was observed.

Morphological aspect of SaOs-2 and MC3T3-E1 cellson �-TCP

SEMSEM analysis of SaOs-2 and MC3T3-E1 cells in contact with �-TCP at 24, 48 and 72 h appears on Fig. 5. At 24 h, SaOs-2 cellshad a round shape; at 48 h, they flattened and stretched onthe �-TCP pavement (Fig. 5A). Cells exhibited pseudopodiaallowing a direct anchorage to the biomaterial surface. At72 h, SaOs-2 cells appeared flat and more stretched than at48 h. Cells exhibited long cytoplasmic extensions (pseudopo-dia with some filopodia) that allowed them to be anchoredon the rough surface of the �-TCP (Fig. 5B). At 72 h, SaOs-2cells showed a morphological adaptation to the relief madeof valleys and hills.

At 24 h, MC3T3-E1 cells were flat and affixed ontothe �-TCP surface (Fig. 5 C). Cells were anchored byboth filo podia and larger pseudopodia. At 48 h, cells had

proliferated and formed a dense layer. At 72 h, they wereflat, with an enlarged surface and had established numerouscontact between each other; thus a monolayer of MC3T3-E1 cells covered almost all the surface of the biomaterial(Fig. 5D).

Confocal microcopyFig. 6 shows confocal images obtained after a doublelabelling DAPI/phalloidin of SaOs-2 and MC3T3 cells. At 24 hafter cells seeding, phalloidin labelling showed that SaOs-2cells were attached on the �-TCP surface but appeared lessspread than MC3T3-E1 cells (Fig. 6A-C). The actin networkappeared clearly much more developed in the cytoplasm ofMC3T3-E1 cells compared to SaOs-2 cells. As early as 24 h,MC3T3-E1 cells appeared in contact and overlapped; confo-cal images evidenced cytoplasmic extensions that interactwith neighboring cells (Fig. 6 C). At 48 h, the actin cytoskele-ton of SaOs-2 cells remained poorly developed and thesecells were not well spread. On the contrary, MC3T3-E1cells appeared well spread and cytoplasmic extensions wereobserved. Confocal observation at a smaller magnification,clearly showed a dense layer of MC3T3-cells on the �-TCPsurface (that appeared in red after xylenol orange counter-staining) (Fig. 7).

At 72 h, SaOs-2 cells formed a dense layer on the �-TCP surface and their morphological aspect clearly showedimprovement by exhibiting round shaped nuclei, a devel-oped actin network and cytoplasmic extensions (Fig. 6B).MC3T3-E1 cells covered almost the �-TCP surface and these


Figure 4 J774 cells on �-TCP after (A) 7 days and (B) 14 days of culture. Higher magnification of J774 cells on �-TCP surface after(C) 7 days and (D) 14 days of culture.

Figure 5 SEM observations of SaOs-2 cells behavior in contact with �-TCP plates at 48 h (A) and 72 h (B); MC3T3-E1 cells behaviorin contact with �-TCP plates at 24 h (C) and 72 h (D).


Figure 6 Confocal microscopy observations of SaOs-2 cells adhesion (A-B) and MC3T3-E1 cells adhesion (C-D) on �-TCP surface at24 and 72 h. The actin filaments are stained in green with phalloidin and the nuclei are stained in blue with DAPI.

cells appeared well spread with long cytoplasmic extensions(Fig. 6D).

Proliferation of osteoblast-like cells on �-TCP

Proliferation kinetics of SaOs-2 and MC3T3-E1 cells on the�-TCP surface at 24, 48 and 72 hours appears on Fig. 8.The number of SaOs-2 cells significantly increased at 48 h(P < 0.05 vs 24 h) and at 72 h (P < 0.05 vs 48 h). At 24 and48 h SaOs-2 proliferation was not significantly higher thanon the plastic surface. At 72 h proliferation of SaOs-2 cellson the biomaterial had increased by 129% compared to con-trol conditions on the plastic surface (P < 0.05). The numberof MC3T3-E1 cells on �-TCP increased from 24 to 72 h but itbecame statistically significant only at 72 h vs 48 h (P < 0.05).Proliferation on the �-TCP surface was significantly higherwhen compared to controls on the plastic surface ateach time point at 24, 48 and 72 h (resp. + 190%, + 177%,+ 181%).

Discussion

The surface of the �-TCP plates observed by SEM presentedthree main characteristics: micropores, a polygonal pave-ment of polycrystalline tessels limited by grain joints anddefect lines. The defect lines observed on our SEM images,represent shear bands, a characteristic surface defect onceramics. During sintering at high temperature, the motionof atoms allows the material to form crystallites. Whenforming, these crystallites are submitted to high shear stressthat leads to the formation of structural defects called dis-locations; they are 2D linear plastic deformations of thecrystal. Under the shear stress, they have the ability tomove over the crystallite leading to the formation of defectlines, also called shear bands. Some shear bands show anhexagonal pattern characteristic from the hexagonal lat-tice of the �-TCP structure [18]. These specificities of thesurface topography is of the upmost importance and mayinfluence adhesion of macrophages and expression of dif-ferent cytokines [19]. It has been shown that the chemical


Figure 7 Confocal microcopy observation of MC3T3-E1 cellscultures 48 h on �-TCP surface stained in red with xylenolorange. The actin filaments are stained in green with phalloidinand the nuclei are stained in blue with DAPI.

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Figure 8 Proliferation of SaOs-2 and MC3T3-E1 cell cultured

directly on 24 well plates ϒ and on �-TCP plates , expressedin number of cells per cm2 at 24, 48 and 72 hours. aP < 0.05 vsplastic surface; *P < 0.05 vs 24 h on �-TCP surface; #P < 0.05 vs48 h on �-TCP surface.

and roughness surface of �-TCP favored the adhesion processosteoblast cells in vitro [20].

The present study focused on cells morphology whichhave developed in vitro on �-TCP. Several studies have beendone using macrophages cultured on �-TCP [10,16,17]. Inmost of them, macrophages were cultured in presence ofreceptor activator of nuclear factor kappa-B ligand (RANK-L)to form osteoclast-like cells but none of them have focusedon the macrophage morphology. In a previous study, we havefound by a time-laps in vitro study that macrophages wereable to resorb �-TCP granules and that osteoblast-like cellscould climb at the surface of the biomaterial [21].

The two mouse monocyte/macrophage cell lines usedin this study were cultured with M-CSF. This cytokine isknown to regulate and control the survival, proliferationand differentiation of phagocytic macrophages from undif-ferentiated precursors [22,23]. Adding M-CSF in the culturesallows cells to differentiate into fully mature macrophages.RAW 264.7 and J774 cells survived between 7 and 14 days.They had cytoplasmic veil-like expansions on their surfacecharacteristic of healthy macrophages. The cells presentedpseudopodia and numerous filopodia that anchored them atthe surface of the �-TCP. Previous work on rabbit bone biop-sies showed that cellular resorption of �-TCP occurred in twosteps [24]. Giant nucleated TRAcP-negative cells first colo-nized the surface of the biomaterial from 7 to 14 days. Thesecells contained a great amount of mineral crystals from thecalcium-phosphate material inside their vacuoles suggest-ing degradation by phagocytosis. As new bone is formed,multinucleated TRAcP positive cells with a ruffled border(characteristic of osteoclasts) are evidenced on the surfaceof Ca/P ceramics [24]. The number of osteoclasts increasesupon time. So, a double population of multinucleated cellis responsible for the cellular resorption of ceramics: giantTRAcP-negative cells that erode the biomaterial and osteo-clasts that resorb the biomaterial and remodel the newlyformed bone [24,25].

Besides osteoclasts, macrophages could also be involvedat an early stage of biomaterial resorption. In a seriesof 14 patients that had sinus lift augmentation in oralsurgery with �-TCP granules, TRAcP-positive multinucleatedcells were observed in contact with granules [6]. However,slightly TRAcP-positive cells (characteristic of macrophageactivation) were also observed with �-TCP grains insidetheir cytoplasm after phagocytosis. Similar findings werealso reported by others [8,14,26,27]. This suggests thatresorption happens by phagocytosis due to macrophagestogether with osteoclasts. The early vascularization aroundthe grafted �-TCP particles allows in situ migration of pre-cursor cells, macrophages and osteoprogenitors [28]. In ourstudy, no giant multinucleated cells were observed meaningthat macrophages did not fuse into giant cells in vitro. Inthe future, it could be interesting to analyze the expressionof TRAcP by macrophages in presence of �-TCP and how itvaries over time.

It is admitted that the resorption in case of a biodegrad-able material occurs simultaneously with apposition of newbone packets after recruitments of osteoblasts. Numerousstudies have focused on the osteoconductive characteris-tics of a biomaterial in culture using an osteoblast cellline and/or bone marrow stroma cells (BMSC) [20,29,30].The choice of a cell type in an in vitro study is of theupmost importance. BMSCs are interesting because they candifferentiate into osteoblasts. Indeed, such an osteogenicdifferentiation in contact with a biomaterial can reflect itsosteoinductive potential [30]. In the present study, SaOs-2are mature osteoblast derived from a human osteosar-coma as they express alkaline phosphatase [31]. In contrast,MC3T3-E1 cells are pre-osteoblasts as they do not express-ing alkaline phosphatase in the absence of ascorbic acidand �-glycero phosphate [31]. MC3T3-E1 have been shownto be the most appropriate model in biomaterial studies[31]. Culture of MC3T3-E1 on calcium phosphate ceramicsinduces alkaline phosphatase gene expression after 14 days


of culture without any medium supplementation [30]. In ourstudy MC3T3-E1 cells adhered and spread out on the �-TCPsurface more rapidly than SaOs-2 cells. At 72 h, the two celllines occupied most of the surface and exhibited a developedcytoskeleton with a marked actin network [32]. Interac-tion between cells and the biomaterial surface is crucial toinduce proliferation, followed by differentiation. Our resultsshowed an influence of the �-TCP surface on cell prolifera-tion as early as 24 h which could be correlated with a rapidadhesion process at 24 h. Interaction with an extracellularmatrix or a biomaterial involves is mediated by integrinsthat interacted with the matrix. Another study has shownthat spreading of SaOs-2 osteoblastic cells occurred within1 day on �-TCP (as also found here) and that focal adhesionare observed at 4 days [20]. In our study, cytoplasmic exten-sions were observed after 48 hours allowing a firm anchorageof the cells onto the biomaterial surface.

In conclusion, the present study emphasises theimportance of the choice of a cell line in exploring the osteo-conductive and osteoinductive properties of a biomaterial.Additional studies are needed to better understand theresorption process involving differentiation of macrophageinto giant multinucleated cells. The topographical, chemicaland physicochemical characteristics of �-TCP may accountfor its excellent capacity of inducing a regenerative boneformation associated with progressive resorption of the bio-material.


B.A. received a PhD scholarship from Kasios SAS.

Acknowledgments

This work was made possible by grants, from ANR, pro-gram LabCom ‘‘NextBone’’. SEM and confocal analysis wereperformed at Service Commun d’Imagerie et d’AnalysesMicroscopiques (SCIAM), Université d’Angers, thanks to R.Perrot and R. Mallet. Many thanks for Kasios SAS, 18, cheminde la Violette 31240 L’UNION—France for providing the Sinus-LiftTM devices.

References

[1] Albee FH. Studies in bone growth: triple calcium phosphate asa stimulus to osteogenesis. Ann Surg 1920;71:32.

[2] Bhaskar SN, et al. Biodegradable ceramic implants inbone: electron and light microscopic analysis. Oral Surg1971;32:336—46.

[3] Jarcho M ea. Synthesis and fabrication of �-tricalciumphosphate (whitlockite) ceramics for potential prostheticapplications. J Mater Sci 1979;14:142—50.

[4] Akao M, et al. Dense polycrystalline B-tricalcium phosphate forprosthetic applications. J Mater Sci 1982;17:343—6.

[5] LeGeros RZ. Properties of osteoconductive biomaterials: cal-cium phosphates. Clin Orthop Relat Res 395, 2002:81—98.

[6] Chappard D, Guillaume B, Mallet R, Pascaretti-Grizon F, BasleMF, Libouban H. Sinus lift augmentation and beta-TCP: amicroCT and histologic analysis on human bone biopsies. Micron2010;41:321—6.

[7] Tanaka T, Kumagae Y, Saito M, Chazono M, Komaki H, KikuchiT, et al. Bone formation and resorption in patients after

implantation of beta-tricalcium phosphate blocks with 60% and75% porosity in opening-wedge high tibial osteotomy. J BiomedMater Res B Appl Biomater 2008;86:453—9.

[8] Nyangoga H, Aguado E, Goyenvalle E, Basle MF, Chap-pard D. A non-steroidal anti-inflammatory drug (ketoprofen)does not delay beta-TCP bone graft healing. Acta Biomater2010;6:3310—7.

[9] Sai Nievethitha S, Subhapradha N, Saravanan D, SelvamuruganN, Wei-Bor T, Srinivasan N, et al. Nanoceramics on osteoblastproliferation and differentiation in bone tissue engineering. IntJ Biol Macromol 2017;98:67—74.

[10] Schaefer S, Detsch R, Uhl F, Deisinger U, Ziegler G. Howdegradation of calcium phosphate bone substitute materials isinfluenced by phase composition and porosity. Adv Eng Mater2011;13:342—50.

[11] Legeros RZ, et al. Biphasic calcium phosphate bioceramics:preparation, properties and applications. J Mater Sci Mater Med2003;14:201—9.

[12] Sheikh Z, Abdallah M-N, Hanafi A, Misbahuddin S, Rashid H,Glogauer M. Mechanisms of in vivo degradation and resorp-tion of calcium phosphate based biomaterials. Materials2015;8:7913—25.

[13] Baslé MF, Chappard D, Grizon F, Filmon R, Delecrin J, Daculsi G,et al. Osteoclastic resorption of Ca-P biomaterials implantedin rabbit bone. Calcif Tissue Int 1993;53:348—56.

[14] Kucera T, Sponer P, Urban K, Kohout A. Histological assess-ment of tissue from large human bone defects repairedwith beta-tricalcium phosphate. Eur J Orthop Surg Traumatol2014;24:1357—65.

[15] Samavedi S, Whittington AR, Goldstein AS. Calcium phosphateceramics in bone tissue engineering: a review of proper-ties and their influence on cell behavior. Acta Biomater2013;9:8037—45.

[16] Detsch R, Schaefer S, Deisinger U, Ziegler G, Seitz H, Leukers B.In vitro-Osteoclastic activity studies on surfaces of 3D printedcalcium phosphate scaffolds. J Biomater Appl 2010;26:359—80.

[17] Roy M, Fielding G, Bandyopadhyay A, Bose S. Effects of zinc andstrontium substitution in tricalcium phosphate on osteoclastdifferentiation and resorption. Biomater Sci 2013:1.

[18] Yashima M, Sakai A, Kamiyama T, Hoshikawa A. Crystal struc-ture analysis of �-tricalcium phosphate Ca3(PO4)2 by neutronpowder diffraction. J Solid State Chem 2003;175:272—7.

[19] Miron RJ, Bosshardt DD, OsteoMacs:. Key players around bonebiomaterials. Biomaterials 2016;82:1—19.

[20] dos Santos EA, Farina M, Soares GA, Anselme K. Chemical andtopographical influence of hydroxyapatite and beta-tricalciumphosphate surfaces on human osteoblastic cell behavior. JBiomed Mater Res A 2009;89:510—20.

[21] Beuvelot J, Pascaretti-Grizon F, Filmon R, Moreau MF, Basle MF,Chappard D. In vitro assessment of osteoblast and macrophagemobility in presence of beta-TCP particles by videomicroscopy.J Biomed Mater Res A 2011;96:108—15.

[22] Stanley ER, et al. Biology and action of colony—stimulatingfactor-1. Mol Reprod Dev 1997;46:4—10.

[23] Stanley ER, Berg KL, Einstein DB, Lee PS, Pixley FJ, Wang Y,et al. Biology and action of colony-stimulating factor-1. MolReprod Dev 1997;46:4—10.

[24] Baslé MF, et al. Osteoclastic resorption of Ca-P biomaterialsimplanted in rabbit bone. Calcif Tissue Int 1993;53:348—56.

[25] Chazono M, Tanaka T, Kitasato S, Kikuchi T, Marumo K. Electronmicroscopic study on bone formation and bioresorption afterimplantation of beta-tricalcium phosphate in rabbit models. JOrthop Sci 2008;13:550—5.

[26] Lu J, Descamps M, Dejou J, Koubi G, Hardouin P, Lemaitre J,et al. The biodegradation mechanism of calcium phosphatebiomaterials in bone. J Biomed Mater Res A 2002;63:408—12.

[27] Ghanaati S, Barbeck M, Detsch R, Deisinger U, Hilbig U, RauschV, et al. The chemical composition of synthetic bone substitutes


influences tissue reactions in vivo: histological and histomor-phometrical analysis of the cellular inflammatory response tohydroxyapatite, beta-tricalcium phosphate and biphasic cal-cium phosphate ceramics. Biomed Mater 2012;7:015005.

[28] Anderson JM, Rodriguez A, Chang DT. Foreign body reaction tobiomaterials. Semin Immunol 2008;20:86—100.

[29] Liu G, Zhao L, Cui L, Liu W, Cao Y. Tissue-engineered boneformation using human bone marrow stromal cells and novelbeta-tricalcium phosphate. Biomed Mater 2007;2:78—86.

[30] Zhang J, Sun L, Luo X, Barbieri D, de Bruijn JD, van Blitter-swijk CA, et al. Cells responding to surface structure of calcium

phosphate ceramics for bone regeneration. J Tissue Eng RegenMed 2017.

[31] Czekanska EM, Stoddart MJ, Richards RG, Hayes JS. In searchof an osteoblast cell model for in vitro research. Eur Cell Mater2012;24:1—17.

[32] Demais V, Audrain C, Mabilleau G, Chappard D, Baslé MF. Diver-sity of bone matrix adhesion proteins modulates osteoblastattachment and organization of actin cytoskeleton. Morpholo-gie 2014;98:53—64.

Morphologie (2017) 101, 164—172


ScienceDirect


GENERAL REVIEW

Basic research and clinical application ofbeta-tricalcium phosphate (�-TCP)Recherche fondamentale et application clinique dubêta-tricalcium phosphate (ˇ-TCP)

T. Tanaka a,b,∗, H. Komaki a,b, M. Chazono a,b, S. Kitasatob,A. Kakutab, S. Akiyamab, K. Marumob

a Department of Orthopaedic Surgery, NHO Utsunomiya National Hospital, 2160 Shimo-Okamoto, 329-1193Utsunomiya city, Tochigi, Japanb Department of Orthopaedic Surgery, Jikei University School of Medicine, 3-25-8, Nishishinbashi,Minato-ku, Tokyo, Japan

Available online 24 April 2017

KEYWORDSBone substitute;�-TCP;Bone formation;Osteoclasts;Coupling

Summary The mechanism of bone substitute resorption involves two processes: solution-mediated and cell-mediated disintegration. In our previous animal studies, the main resorptionprocess of beta-tricalcium phosphate (�-TCP) was considered to be cell-mediated disintegrationby TRAP-positive cells. Thus, osteoclast-mediated resorption of �-TCP is important for enablingbone formation. We also report the results of treatment with �-TCP graft in patients since 1989.Two to three weeks after implantation, resorption of �-TCP occurred from the periphery, andthen continued toward the center over time. Complete or nearly complete bone healing wasachieved in most cases within a few years and was dependent upon the amount of implantedmaterial, the patient’s age, and the type of bone (cortical or cancellous). We have previouslyreported that an injectable complex of �-TCP granules and collagen supplemented with rhFGF-2 enabled cortical bone regeneration of rabbit tibiae. Based on the experimental results, weapplied this technique to the patients with femoral and humeral fractures in elderly patients,and obtained bone union.© 2017 Elsevier Masson SAS. All rights reserved.

∗ Corresponding author. Department of Orthopaedic Surgery, NHO Utsunomiya National Hospital, 2160 Shimo-Okamoto, 329-1193Utsunomiya city, Tochigi, Japan.

E-mail address: [email protected] (T. Tanaka).







Basic research and clinical application of beta-tricalcium phosphate (�-TCP) 165

MOTS CLÉSSubstituts osseux ;�-TCP ;Formation osseuse ;Ostéoclastes ;Couplage

Résumé Le mécanisme de résorption des matériaux de substitution osseux implique deux pro-cessus : une destruction en solution et une médiée par les cellules. Dans nos études précédenteschez l’animal, le principal processus de résorption du phosphate bêta-tricalcique (�-TCP) étaitconsidéré comme une résorption cellulaire par des cellules TRAP-positives. Ainsi, la résorptiondu �-TCP due aux ostéoclastes est importante pour permettre la formation osseuse (couplage).Nous rapportons également les résultats du traitement par greffe avec du �-TCP chez despatients depuis 1989. Deux à trois semaines après l’implantation, la résorption de �-TCP estsurvenue à partir de la périphérie, puis a continué au cours du temps de facon centripète. Lacicatrisation osseuse complète (ou presque complète) a été obtenue dans la plupart des cas enquelques années et dépendait de la quantité de matériel implanté, de l’âge du patient et du typed’os (cortical ou trabéculaire). Nous avons déjà rapporté qu’un complexe injectable de gran-ules de �-TCP et de collagène supplémenté avec du rhFGF-2 a permis la régénération osseusecorticale des tibias de lapin. Sur la base des résultats expérimentaux, nous avons appliqué cettetechnique aux patients âgés atteints de fractures fémorale et humérale et obtenu une unionosseuse.© 2017 Elsevier Masson SAS. Tous droits reserves.

Introduction

Autologous bone is the preferred graft material for treatingbone defects. However, disadvantages of autografting aresignificant and include procurement morbidities, increasedoperative time, and limited availability. In addition, autol-ogous bone is difficult to obtain when the patient is achild. Allografts have commonly been used as substitutesfor autogenous bone grafts in Europe and the United Statesbut not in Japan. Significant problems associated withallograft introduction include a low bone-fusion rate anddisease transmission [1,2]. Recently, bone substitute mate-rials have been advocated as alternatives to autografts andallografts. Hydroxyapatite (HAP) is widely used as a bonesubstitute because of its excellent biocompatibility andosteoconductive properties [3]. However, HAP has severaldisadvantages, such as slow biodegradation during the repairof bone defects [4]. Calcium phosphate cement (CPC) hasgood biocompatibility, bioactivity, low setting temperature,adequate stiffness, and easy shaping into complicated geo-metrics [5]. However, the resorption rate is slow. �-TCP hasrecently received considerable attention as a bone graft sub-stitute because of its biocompatibility and biodegradability[6—10]. In animal experiments, �-TCP is gradually degradedduring bone remodeling and is finally replaced by maturenew bone [11—13]. In this study, we report experimental ani-mal results and the representative clinical results of �-TCPimplantation in patients with bone defects.

Materials and methods

The �-TCP products used in this study was synthesized with amechanochemical method and provided by Olympus TerumoBiomaterials Co. (Tokyo, Japan). Briefly, CaHPO4/H2O andCaCO3 at a molar ratio of 2:1 were mixed into slurry withpure water and zirconia ball in a pot mill for 24 hours anddried at 80 ◦C. The calcium-deficient HAP was converted to�-TCP by calcinations at 750—900 ◦C. Following the prepara-tion of forming slurry of �-TCP with forming agent and drying

process, the preforming porous body of �-TCP was obtained.After the preforming body was sintered at 1050 ◦C for 1 hour,a porous �-TCP block with 75% porosity was obtained. Abimodal pore size distribution in the �-TCP block with 75%porosity was observed on the evaluation by porosimeter,where one peak existed in a region of more than 100 �m andthe other peak existed in a region of less than 5 �m. The �-TCP block with 60% porosity was synthesized by the samemethod as 75% porosity except that the amount of formingagent was changed.

Animal experiments

Cylindrical cancellous bone defects were created by drillingof the rabbit distal femur. Bone defects were filled withcylindrical �-TCP blocks. Rabbits were sacrificed aftersurgery, and the distal part of the femur was removed.Decalcified sections were obtained for HE and tartrate-resistant acid phosphatase (TRAP) staining.

Clinical application

From 1989 to 2004, we have used �-TCP with 75% poros-ity. Since 2005 we have used �-TCP with both 75 and 60%porosity. Patients ranged in age from 2 to 91 years at thetime of surgery. In several cases, �-TCP was placed on theperiosteum after fibula harvest.

A new evaluation system of new bone formationand �-TCP resorption

We have recently established a new evaluation system tomeasure bone formation and �-TCP resorption in openinghigh tibial osteotomy. All patients underwent CT examina-tion at 2 weeks and 6 years. The CT image data were dividedinto 3 areas by tracing manually on the screen of the com-puter. CT attenuation values (in Hounsfield units [HU]) ofthe area implanted with �-TCP of 60% porosity, the area

166 T. Tanaka et al.

Figure 1 Decalcified histological sections stained with HE 2 weeks after implantation of the �-TCP block with 75% porosity. Newbone formation is found in the defect. Osteoblasts (arrows) are present on the newly formed bone in a monolayer (A). TRAP-positivecells were present on the surface of the �-TCP. The arrows indicate TRAP-positive cells (B).

Figure 2 The macroscopic appearance and scanning electron micrographs (original magnification, 3000 ×) of the two types of�-TCP block. The compression strengths of �-TCP blocks with 60 and 75% porosity are 22 and 3 MPa, respectively.


implanted with �-TCP of 75% porosity, and hinged cancellousbone were analyzed using the imaging software, Osirix.

Results

Basic research (animal experiment)

Two weeks after implantation of �-TCP block with 75% poros-ity, new bone formation was already found. A mono-layerlining of osteoblast was found on the surface of newlyformed bone (Fig. 1A). Serial sections with TRAP stainingshowed that most of the multinuclear cells were TRAP-positive and were present on the surface of the �-TCP butsome of them were present on the newly formed bone(Fig. 1B). New bone formation and �-TCP resorption wereprogressed over time, and most of �-TCP with 75% porositywas resorbed at 12 weeks (Fig. 2).

Case presentation

Case 1. A 13-year-old girl. An 18-cm-long fibula graft wasobtained for spinal fusion, and �-TCP blocks were placedon the remaining periosteum. One year and 6 months afterimplantation, the fibula had been almost completely recon-structed. Twenty years after surgery, the bone and bonemarrow had been kept as an original shape of the fibula(Fig. 3).

Case 2. A 5-year-old girl with multiple enchondroma ofthe phalanx. HAP with low porosity (42%) was implantedafter curettage of the bone tumor. Twenty years aftersurgery, most of HAP still remained (Fig. 4).

Figure 3 Radiographs of a 13-year-old girl. Left) An 18-cm-long section of the fibula was obtained for spinal fusion, and�-TCP blocks were placed on the remaining periosteum. Mid-dle) Resorption of �-TCP and formation of cortical bone canbe observed 18 months after surgery. Right) Twenty years afterimplantation, the fibula is fully reconstructed.

Figure 4 Left) A 5-year-old girl with multiple enchondromaof the phalanx. Bone tumor curettage was followed by HAPimplantation. Right) Most of HAP still remained even after 20years.

Case 3. A 56-year-old man with tibia plateau fracture.Open reduction and internal fixation were performed, andCPC was injected in the cancellous bone defects. Tenyears after surgery, partial resorption of CPC was observed(Fig. 5).

Case 4. A 24-year-old woman with hip dysplasia. Rota-tional acetabular osteotomy was performed and the �-TCPblocks with 60% porosity were implanted between therotated acetabular and remaining bone. Ten years aftersurgery the �-TCP blocks with 60% porosity were completelyresorbed and replaced by bone. Osteoarthritic changes ofthe hip were minimal (Fig. 6).

Case 5. A 58-year-old woman with medial compartmentalknee osteoarthritis. Opening high tibial osteotomy (HTO)was performed using �-TCP blocks with 60 and 75% porosity

Figure 5 Radiographs of a 56-year-old man with depressedfracture of the lateral tibia plateau. Left) Two weeks afteropen reduction and internal fixation with filling of the resul-tant defect with CPC. Right) Ten years after surgery, partialresorption of CPC was observed.


Figure 6 Left) Radiographs of a 24-year-old woman with hip dysplasia. Middle) Rotational acetabular osteotomy was performedand the �-TCP blocks with 60% porosity were implanted between the rotated acetabular and remaining bone. Ten years after surgerythe �-TCP blocks with 60% porosity were completely resorbed and replaced by bone. Right) Osteoarthritic changes of the hip wereminimal. The arrow indicates �-TCP block with 60% porosity.

without autogenous bone grafting. After placement of aPuddu plate, the cancellous bone defect was filled withwedge-shaped �-TCP blocks with 75% porosity. Followingthat, three wedge-shaped �-TCP blocks with 60% porositywere implanted in front (2 blocks) and back of the plate (1block) (Figs. 7, 8). Six years after surgery the �-TCP blockswith 75% porosity were completely resorbed and replacedby bone, but a small amount of �-TCP with 60% porosityremained (Figs. 7, 8).

Case 6. A 88-year-old woman with subtrochanteric frac-ture. The complex of �-TCP granules with 60% porosity, 1%of hyaluronic acid, and rhFGF-2 (Fig. 9) was injected beforeinsertion of the nail. Eight weeks after surgery, marked newbone formation was found (Fig. 10).

No adverse reactions to �-TCP or disturbances of woundhealing were observed in the postoperative period. Boneformation was observed in all cases except 5 cases. Threepatients with recurrence of a bone cyst showed �-TCPresorption. A similar phenomenon was observed in twopatients with postoperative recurrence of osteomyelitis.Resorption of �-TCP occurred from the periphery of the �-TCP implant at 2 to 3 weeks and then continued towardthe center over time. In most adult patients, completeor nearly complete healing of bone defects had occurredwithin several years. All 5 patients younger than 10 yearsshowed complete resorption of �-TCP within 1.5 yearsafter surgery. Resorption of �-TCP was dependent upon theamount implanted, the patient’s age, and the type of bone

Figure 7 Anteroposterior radiographs of a 58-year-old womanwith medial compartmental knee osteoarthritis. Opening hightibial osteotomy (HTO) was performed using �-TCP blocks with60 and 75% porosity. Left) After placement of a Puddu plate,the cancellous bone defect was filled with wedge-shaped �-TCPblocks with 75% porosity, and then three wedge-shaped �-TCPblocks with 60% porosity were implanted in front (2 blocks) andback of the plate (1 block). Right) Six years after surgery mostof �-TCP were resorbed. Broken lines indicate the center ofosteotomy plane.


Figure 8 Left) A CT image of a 58-year-old woman showingthe center of the osteotomy plane at 2 weeks. The mean CTvalues (HU) of the area implanted with �-TCP of 60% porosity(60%), the area implanted with �-TCP of 75% porosity (75%), andhinged cancellous bone (B) were 1635, 1002, and 98, respec-tively. Right) Six years after surgery, the values of the areaimplanted with �-TCP of 60% porosity in front of the plate (A),the area implanted with �-TCP of 60% porosity at the back of theplate (C), the area implanted with �-TCP of 75% porosity (D),and hinged cancellous bone (B) were 171, 578, 113, and 99,respectively. The CT value of the 60% porosity �-TCP implantedarea, especially at the back of the plate (C) was much higherthan that of bone, indicating that approximately 1/3 of the�-TCP with 60% porosity still remained.

(cortical or cancellous bone). The larger the implant wasand the older the patient was, the slower the healing was,similarly, cortical bone defects healed more slowly thancancellous bone defects. However, elderly patients withosteoporosis showed faster resorption of �-TCP.

Figure 9 An injectable complex of �-TCP granules, hyaluronicacid, and rhFGF-2 was prepared in a 1-cm diameter cylinderused for osteochondral grafts.

A new evaluation system of new bone formationand �-TCP in opening HTO

CT image analysis including the center of the osteotomyplane in case 5 (Fig. 7) showed that the mean CT value(HU) of hinged cancellous bone, the implanted area of �-TCP with 75% porosity, and 60% porosity at 2 weeks were 98,1002, 1635 respectively. At 6 years, the �-TCP of 60% poros-ity implanted area in front of the plate was 161, which was

Figure 10 Right) Radiographs of an 88-year old woman with subtrochanteric fracture. Middle) The injected complex (Fig. 9)remained in its original place (arrows) and marked callus formation was found in the lateral cortical bone defect (arrow head)8 weeks after surgery (right).


slightly higher than that of cancellous bone, indicating mostof �-TCP was resorbed and replaced by bone. In contrast,CT value of the �-TCP of 60% porosity implanted area at theback of the plate was 578, which was much higher than can-cellous bone, indicating incomplete resorption of �-TCP wasoccurred in this area.

Discussion

HAP has been widely used to repair bone defects due totumor and trauma surgery. Long-term results of HAP implan-tation in bone tumor surgery was reported [14]. In theirreport, HAP was incorporated well into host bone, butnone of the patients showed complete resorption. Thus,HAP might interfere with remodeling and create a locus ofincreased stress owing to its slow resorption.

CPC has good biocompatibility, bioactivity, adequatestiffness, and easy shaping into complicated geometrics [5].CPC set and harden in vivo under physiological conditions.The final setting product of CPC is HAP. However, this HAP isnot sintered, thus it was considered to be resorbed becauseof low crystallization. However, clinical results showed thatresorption was observed in comparison to sintered HAP, butits rate was very slow. In contrast, the present study showedthat �-TCP was resorbed and replaced by bone. Thus, the�-TCP implanted site can be remodeled. Moreover, bonereconstructed (Fig. 3) after implantation of �-TCP may bereusable for bone grafting. This advantage does not appearwith HAP-implanted bone.

The mechanism of bone substitute resorption involvestwo processes: solution-mediated disintegration and cell-mediated disintegration [15]. An example of the first processis calcium sulfate and alpha TCP resorption. The �-TCPresorption is thought to involve both solution- and cell-mediated disintegration [16,17]. In contrast, poor resorptionof �-TCP has been reported. One of the reasons for differ-ences in �-TCP resorbability is differences in pore structure.Handschel et al. [18] reported that �-TCP is poorly resorbedafter being implanted in a non-load-bearing environment.However, the �-TCP granules (Cerasorb, Curasan, Kleinos-theim, Germany) used in their study was different in porestructure from the �-TCP used in our study. Altermatt et al.[19] have reported that most implanted �-TCP remained incalcaneal defects even after 7 years. They used highly puri-fied �-TCP with 60% porosity. We speculate that the numberand size of macropores and micropores and the interconnec-tions between pores were not suitable for �-TCP resorptionand bone formation. Although implant purity and surfacearea are important, �-TCP resorption appears to depend onpore structure. The �-TCP used in our study had both macro-pores and micropores, most of which were interconnected.This structure facilitates the entry of proteins and cells forbone formation and �-TCP resorption. De Groot [20] has sug-gested that the rate of degradation is determined by implantmicroporosity. Macroporous materials without microporosityallow only bone ingrowth, whereas dense materials with-out pores of either type show little degradation. The �-TCPused in the present study contained numerous micropores.Recently, Davison et al. [21] reported that resorption of�-TCP was controlled by microporous structure. Our previ-ous electron microscopic study [22] showed that collagenfibrils secreted from the monocytic cells invaded �-TCP

micropores at 2 weeks. This result demonstrated that themicropores of �-TCP block may provide an environment forcollagen formation, leading to the deposition of apatite crys-tals. Therefore, the micropores facilitate bone ingrowth aswell as �-TCP resorption. Electron microscopic analysis alsoshowed that multinucleated giant cells were in contact withthe surface of �-TCP. Some of them had a ruffled border atthe cell—substrate interface, characteristic of osteoclasts.These findings suggest that cell-mediated disintegration byosteoclasts played a role in the bioresorption of �-TCP. Thisresult was obtained 2 weeks after implantation and is con-sistent with the present clinical results in which the outermargin of the implanted �-TCP was unclear 2 to 3 weeksafter implantation. We also found clusters of multinucle-ated giant cells and osteoblasts at the boundary betweennew bone and ceramic 2 to 4 weeks after surgery in arabbit model. Ogose et al. [23] have reported a lining ofosteoblastic cells on the surface of �-TCP and new bone,and a considerable number of osteoclast-like giant cells sur-rounding �-TCP in a human specimen 4 weeks after surgery.

In order to support the role of osteoclasts on �-TCPresorption, we investigated the effects of alendronate (ALN)on osteoclastic resorption of �-TCP and bone formation.Cyrindrical �-TCP bocks with ALN treatment were implantedinto bone defects of rabbit distal femur. The results showedthat local application of ALN reduced the number ofosteoclasts. In addition, high-dose of ALN inhibited �-TCPresorption and bone formation [24]. This result suggestedthat osteoclast-mediated resorption play an important rolein bone formation and a coupling-like phenomenon couldoccur in �-TCP implanted area.

Resorption of �-TCP and replacement by bone are alsoinfluenced by the host environment as well as by the inter-nal structure of TCP. Despite a similar amount of �-TCPhaving been implanted after surgery, the younger patientshowed greater resorption of �-TCP. A 2-year-old boy and a3-year-old boy showed greater resorption (data not shown).These results suggest that �-TCP resorption and replace-ment by bone are dependent on age. However, elderlypatients with osteoporosis also showed faster resorption.We also found that the type of bone affected �-TCP resorp-tion. The rates of �-TCP resorption and bone formation weregreater in cancellous bone defects than in cortical bonedefects. This difference in resorption may be due to dif-ferences in blood flow between cancellous bone and corticalbone. Thus, it is not enough to fill cortical bone defects with�-TCP alone. Additional stimulations such as growth fac-tors and ultrasound administration are necessary to repaircortical bone defects. We have previously reported thata complex of �-TCP granules and collagen supplementedwith 200 �g of FGF-2 induced cortical bone regenerationand repaired 5-mm cortical bone defects in rabbit tibiaeby 12 weeks post-treatment [25]. Based on the experimen-tal results, we applied this technique to the patients withfemoral and humeral fractures with bone defects in elderlypatients. The results showed that the complex of �-TCPand hyaluronate combined with FGF-2 induced marked cal-lus formation around the injected sites in trochanteric andhumeral fractures [9].

Bone substitutes have been used to fill original bonedefects. However, as better bone substitutes have beendeveloped, they have also been used for newly created


bone defects, such as in opening osteotomies. Recently,opening HTO has become popular because of its severaladvantages. Koshino T et al. [26] have reported good long-term results after opening HTO using HAP as a bone filler;no correction loss was reported. However, if severe varusor valgus knee deformity were to occur, fixing a componentin the tibia during total knee arthroplasty might be diffi-cult. In contrast, most of the porous �-TCP can be resorbedwithin a few years. The �-TCP with 75% porosity had acompression strength of only 3 MPa, which is inadequatefor weight-bearing sites until bone incorporation occurs.Compression strength can be increased by reducing porosity.Thus, we developed wedge-shaped �-TCP with 60% porosityfor opening HTO. This TCP has a compression strength of22 MPa, which is approximately seven-fold greater than thatof TCP with 75% porosity. During opening HTO, the openeddefect was fixed with a Puddu plate, after which TCP with75% porosity was used to fill the cancellous bone defect,except the medial side where a wedge-shaped TCP blockwith 60% porosity was implanted in front and back of theplate (Fig. 8). The use of a �-TCP block with 60% porosityavoided autogenous bone grafting and shortened the surgi-cal time. We have used this technique since 2005 [8,10,27].Resorption of �-TCP with 60% porosity occurred but requiredmore time than that of �-TCP with 75% porosity.

To our knowledge, no radiological rating system to moni-tor remodeling of �-TCP using CT images has been reported.We have recently developed a novel evaluation system tomonitor bone formation and �-TCP resorption in openingHTO [10]. �-TCP was resorbed from the periphery, butnot uniformly resorbed. Thus, measurements focused on acertain area (region of interest; ROI) of �-TCP were not ade-quate to evaluate �-TCP resorption. Thus, monitoring of thewhole �-TCP implanted area is necessary. The imaging soft-ware, Osirix, enabled scanning of the whole area to measureCT values. This system is the first to quantitatively evalu-ate �-TCP resorption and bone formation. In addition, thissystem can be useful in any �-TCP implanted area.



References

[1] Aurori BF, Weierman RJ, Lowell HA, Nabel CI, Parsons JR.Pseudoarthrosis after spinal fusion for scoliosis. A compar-ison of autogenic and allogenic bone grafts. Clin Orthop1985;199:153—8.

[2] Karcher HL. HIV transmitted by bone graft. BMJ 1997;314:1300.[3] Kitsugi T, Yamamoto T, Nakamura T, Kotani S, Kokubo T,

Takeuchi H. Four calcium phosphate ceramics as bone substi-tutes for non-weight-bearing. Biomaterials 1993;14:216—24.

[4] Hoogendoorn HA, Renooij W, Akkermans LMA, Visser DDS, Wit-tebol P. Long-term study of large ceramic implants in dogfemora. Clin Orthop 1984;187:281—8.

[5] Driessens FCM, Boltong mg, Bermudez O, Planell JA. For-mulation and setting time of some calcium orthophosphatecements: a pilot study. J Mater Sci Mater Med 1993;4:503—8.

[6] Dong J, Uemura T, Shirasaki Y, Tateishi T. Promotion ofbone formation using highly pure porous beta-TCP combined

with bone marrow-derived osteoprogenitor cells. Biomaterials2002;23:493—502.

[7] Ogose A, Hotta T, Kawashima H, Kondo N, Gu W, Kamura T, et al.Comparison of hydroxyapatite and beta tricalcium phosphateas bone substitutes after excision of bone tumors. J BiomedMater Res 2005;72B:94—101.

[8] Tanaka T, Kumagae Y, Saito M, Chazono M, Komaki H, Kikuchi T,et al. Bone formation and resorption in patients after implan-tation of beta-tricalcium phosphate blocks with 60% and 75%porosity in opening wedge high tibial osteotomy. J BiomedMater Res B Appl Biomater 2008;86B:453—9.

[9] Tanaka T, Kitasato S, Chazono M, Kukagae Y, Iida T, MitsuhashiM, et al. Use of an injectable complex of beta-tricalciumphosphate granules, hyakuronate, and FGF-2 on repair ofunstable intertrochanteric fractures. Open Biomed Eng J2012;6:98—103.

[10] Tanaka T, Kumagae Y, Chazono M, Kitasato S, Kakuta A, MarumoK. A novel evaluation system to monitor bone formationand beta-tricalcium phosphate resorption in opening wedgehigh tibial osteotomy. Knee Surg Sports Traumatol Arthrosc2015;23(7):2007—11.

[11] Ozawa M. Experimental study on bone conductivity andabsorbability of the pure beta-TCP. J Jpn Soc Biomater1995;13:167—75.

[12] Chazono M, Tanaka T, Komaki H, Fujii K. Bone formation andbioresorption after implantation of injectable beta-tricalciumgranules-hyaluronate complex in rabbit bone defects. J BiomedMater Res 2004;70A:542—9.

[13] Tanaka T, Komaki H, Chazono M, Fujii K. Use of a Biphasic GraftConstructed with Chondrocytes Overlying a Beta-TricalciumPhosphate Block in the Treatment of Rabbit OsteochondralDefects. Tissue Engineering 2005;11:331—9.

[14] Matsumine A, Myoui A, Kusuzaki K, Araki N, Seto M, YoshikawaH, et al. Calcium hydroxyapatite ceramic implants in bonetumor surgery. J Bone Joint Surg 2004;86B:719—25.

[15] Jarcho M. Calcium phosphate ceramics as hard tissue prosthe-sis. Clin Orthop 1981;157:259—78.

[16] Renooji W, Hoogendoorn A, Visser WJ, Lentferink RH, SchmitzMG, Van Ieperen H, et al. Bioresorption of ceramic strontium85-labeled calcium phosphate implants in dog femora. ClinOrthop 1985;197:272—85.

[17] Lu JX, Gallur A, Flautre B, Anselme K, Descamps M, Thierry B,et al. Comparative study of tissue reactions to calcium phos-phate ceramics among cancellous, cortical, and medullar bonesites in rabbits. J Biomed Mater Res 1998;42:357—67.

[18] Handschel J, Wiesmann HP, Stratmann U, Kleinheinz J, MeyerU, Joos U. TCP is hardly resorbed and not osteoconductive ina non-loading calvarial model. Biomaterials 2002;23:1689—95.

[19] Altermatt S, Schwobel M, Pochon JP. Operative treatment ofsolitary bone cysts with tricalcium phosphate ceramic. A 1 to7 year follow-up. Eur J Pediatr Surg 1992;2:180—2.

[20] De Groot K. Bioceramics consisting of calcium phosphate salts.Biomaterials 1980;1:47—50.

[21] Davison NL, ten Harkel B, Schoenmaker T, Luo X, YuanH, Everts V, et al. Osteoclast resorption of beta-tricalciumphosphate controlled by surface architecture. Biomaterials2014;35:7441—51.


[23] Ogose A, Hotta T, Hatano H, Kawashima H, Tokunaga K, EndoN. Histological examination of beta-tricalcium phosphate graftin human femur. J Biomed Mater Res 2002;63:601—4.

[24] Tanaka T, Saito M, Chazono M, Kumagae Y, Kikuchi T, Kitasato S,et al. Effects of alendronate on bone formation and osteoclas-tic resorption of beta-tricalcium phosphate. J Biomed MaterRes 2010;93A:469—74.


[25] Komaki H, Tanaka T, Chazono M, Kikuchi T. Repair of segmentalbone defects in rabbit tibiae using a complex of beta-tricalciumphosphate, type I collagen, and fibroblast growth factor-2. Bio-materials 2006;27:5118—26.

[26] Koshino T, Murase T, Saito T. Medial opening-wedge high tibiaosteotomy with use of porous hydroxyapatite to treat medial

compartmental osteoarthritis of the knee. J Bone Joint Surg2003;85A:75—85.

[27] Tanaka T. Opening wedge high tibial osteotomy using a Pudduplate and beta-tricalcium phosphate blocks. Tech Orthopaed2013;28(2):185—90.

Morphologie (2017) 101, 173—179


ScienceDirect


ORIGINAL ARTICLE

Beta-tricalcium phosphate for orthopedicreconstructions as an alternative toautogenous bone graftLe phosphate bêta-tricalcique comme alternative aux greffesd’os autogène pour les reconstructions orthopédiques

P. Hernigou ∗, A. Dubory, J. Pariat, D. Potage, F. Roubineau,S. Jammal, C.H. Flouzat Lachaniette

Department of Orthopaedic Surgery, University Paris East (UPEC), hôpital Henri-Mondor, avenue duMaréchal-de-Lattre-de-Tassigny, 94010 Créteil, France


KEYWORDSBeta-tricalciumphosphate;Orthopedic surgery;Bone defect;Bone graft

Summary Autogenous bone graft (autograft) remains the gold standard in the treatment ofmany orthopedic problems. However, graft harvest can lead to perioperative morbidity andincreased cost. We tested the hypothesis that an osteoconductive matrix, beta-tricalcium phos-phate (�-TCP), would be a safe and effective alternative to autograft alone. Beta-tricalciumphosphate (�-TCP) is considered as one of the most promising biomaterials for bone reconstruc-tion. This study analyzes the outcomes of patients who received �-TCP as bone substitutes inorthopedic surgery.Methods. — A total of 50 patients were enrolled in a controlled, non-inferiority clinical trial tocompare the safety and efficacy of �-TCP (25 patients) with those of autograft (25 patients)in indications requiring usually autograft. These 50 patients were categorized according to theetiology and morphology of the 54 bone defects resulting from elective surgical procedures,such as 34 open-wedge high tibial osteotomies, and 20 osteonecrosis treatments with coredecompression. Radiographic (healing process with or without integration of �-TCP), clinical(no other surgical procedure), functional outcomes and safety (with or without complications)were assessed through fifty-two weeks postoperatively.Results. — With regard to the primary endpoint (radiographic evolution), the fusion rate ofthe 34 open-wedge osteotomies was 100% (17 among 17) for patients in the group with �-TCP compared with 94% (16 among 17) for patients in the autograft group. For the 20 cavitarydefects (osteonecrosis), the radiographic union rates, as determined by the presence of osseousbridging, were 100% for patients in the group with �-TCP and 100% for those in the autograftgroup. Clinically at one year, all quality-of-life and functional outcome data supported non-inferiority of �-TCP compared with autograft, and patients in the �-TCP group were found tohave less pain and an improved safety profile.

∗ Corresponding author.E-mail address: [email protected] (P. Hernigou).







174 P. Hernigou et al.

Conclusions. — Treatment with �-TCP resulted in comparable fusion rates, less pain and fewerside effects as compared with treatment with autograft. This study established clinical param-eters where the �-TCP alone can successfully support the osteogenic process.© 2017 Elsevier Masson SAS. All rights reserved.

Résumé L’os autologue (autogreffe) reste la référence dans le traitement de nombreuxproblèmes orthopédiques. Toutefois, le prélèvement de greffons entraîne une morbidité péri-opératoire accrue et une augmentation du coût de l’intervention. Nous avons testé l’hypothèseselon laquelle une matrice ostéoconductrice, le phosphate bêta-tricalcique (�-TCP), pouvaitêtre une alternative sûre et efficace à l’autogreffe seule. Le �-TCP est considéré comme l’undes biomatériaux les plus prometteurs pour la reconstruction osseuse. Cette étude analyse lesrésultats de patients qui ont recu du �-TCP comme substitut osseux en chirurgie orthopédique.Un total de 50 patients a été inclus dans un essai clinique contrôlé de non-infériorité pour com-parer l’innocuité et l’efficacité du �-TCP (25 patients) avec ceux d’une autogreffe (25 patients)dans des indications nécessitant habituellement une autogreffe. Les 50 patients ont été classésen fonction de l’étiologie et de la morphologie de 54 défauts osseux résultant de procédureschirurgicales spécifiques : 34 ostéotomies tibiales et 20 traitements à d’ostéonécrose avecforage-décompression. Les analyses radiographiques (guérison avec/sans intégration de �-TCP),cliniques (aucune intervention chirurgicale additionnelle), fonctionnelles et suites opératoires(avec/sans complications) ont été évaluées pendant 52 semaines après l’intervention chirur-gicale. Le critère principal (évolution radiographique) a montré un taux de fusion de 100 %(17/17) des 34 ostéotomies pour les patients du groupe avec �-TCP par rapport à 94 % (16/17)pour les patients du groupe avec autogreffe. Pour les 20 défauts cavitaires (ostéonécrose), lestaux d’union radiographique, déterminés par la présence d’un pont osseux, ont été de 100 %pour les patients du groupe �-TCP et 100 % pour ceux du groupe autogreffe. Cliniquement àun an, la qualité de vie et les résultats fonctionnels ont montré une non-infériorité du �-TCPpar rapport à l’autogreffe et les patients du groupe �-TCP ont eu moins de douleurs et dessuites opératoires meilleures. Le traitement par greffe de �-TCP a entraîné des taux de fusioncomparables, moins de douleur et moins d’effets secondaires par rapport au traitement parautogreffe. Cette étude a établi des paramètres cliniques où le �-TCP seul a pu montrer sescapacités ostéogéniques.© 2017 Elsevier Masson SAS. Tous droits reserves.

Introduction

Surgeons use autograft to fill bone defects and promotefusion across osseous surfaces, particularly in higher-risksurgical sites, but its use comes with certain trade-offs.Important clinical complications [1] have been documentedat the autograft donor site, including hematoma, pain, frac-ture, infection, heterotopic ossification, hernia and nerveinjury. Furthermore, the quality and quantity of autograftare known to vary with patient age, body and overall healthstatus. Equally important, harvesting autograft also requiresadditional operative time, hospitalization and cost.

Commercial bioactive ceramics [2—4] used for bonerepair [5,6] include calcium carbonate (CaCO3, in ara-gonite form), calcium sulfate (CaSO4 + H2O, plaster ofParis), calcium phosphates and bioactive glasses. Calciumphosphate ceramics include beta-tricalcium phosphate (�-TCP, Ca3(PO4)2), hydroxyapatite [HA, Ca10(PO4)6(OH)2], andbiphasic calcium phosphate (BCP) (consisting of an intimatemixture of HA and �-TCP of varying HA/�-TCP ratios).

These synthetic biomaterials have been proposed formany indications. However, there is no consensus in theliterature if the biomaterial, when used alone, has the sim-ilar properties as autograft. In the present study, the boneloss was filled with pure beta-tricalcium phosphate in some

patients and compared with autograft. We report here theinvestigation and data regarding the safety and efficacy of�-TCP compared with autograft in segmental and cavitarydefects with a size lower than 9 cm3.

Material and methods

The calcium phosphate granules

The �-TCP granules (Kasios; L’union; France) were producedby using the foam technology [7]. Briefly, 25 g of a �-TCPslurry were used to infiltrate, with manual pressure, 1 gof polyurethane foam. After drying and sintering at veryhigh temperature (1200 degrees Celsius), �-TCP granules(size: 1 mm diameter) were obtained. The characteristicsof these granules are the association of macroporosity sur-face and a microporosity structure. The macroporosity iscontrolled by the mesh of the polyurethane foam that isused to prepare the granules. The size of macroporosity isin the order of 400 �m at the surface of the material. Thissurface macro-roughness is also associated with a surfacemicro-roughness in relation with the fused grains of sur-face separated with the small holes that constituted themicroporosity. The microporosity is in relation with holes

Beta-tricalcium phosphate for orthopedic reconstructions 175

between polyhedric grains (2.73 ± 1.0 �m in width) tightlypacked together in the number of 4 to 6 grains. Grains are2.73 ± 1.0 micrometer (�m) in width and micropores are1.01 ± 0.16 �m width.

Study design

Patients undergoing elective procedures such as open-wedgeosteotomies that create orthopedic defects [8—12] or recon-struction of cavitary bone defects (for example, filling thechannel after core decompression for treatment of hiposteonecrosis) were enrolled in the study. Eligible subjectsprovided informed consent, were proposed to receive �-TCP or autograft, and were managed with use of standardsurgical procedure plus either autograft or �-TCP. Tobaccoand diabetes were not exclusion criteria. A previous infec-tion at the site of surgery was an exclusion criterion. Allpatients who met the enrollment criteria had as part ofthe disease state that required up to 9 cc of graft and didnot have any defect that, in the opinion of the surgeon,would be treated best by another augmentation procedure.Patients were recruited and operated by several surgeons ofthe same team in the same orthopedic department. Therewas no real randomization. However, to equalize the numberof subjects on each treatment (25 patients with �-TCP and25 patients with autograft) blocking technique of 10 caseswas used. For each block of ten patients, the first patientswere proposed to receive �-TCP or autograft until 5 sub-jects were assigned to one treatment, and after the otherpatients received treatment for equalization. This methodallowed control safety of the procedure during each blockinclusion of 10 patients. In this study, only patients who hadno previous operation were included in order to eliminatethe influence of any additional soft-tissue compromise frommultiple operations.

Prior to any graft insertion at the surgical site(s), patientswho have chosen to receive autograft underwent a routinegraft harvest through a separate exposure of the iliac crestand those who had chosen to receive �-TCP had the gran-ules inserted at the site of operation during the surgicalprocedure.

Patients

These 50 patients corresponded to 54 surgical procedures(four patients had bilateral interventions). Twenty-twopatients were males and 28 females. The ages of the patientsranged between 18 and 65 years old with a mean age corre-sponding to 48 years.

The defects were classified according to the etiologyof the lesions and the morphology of the bone defect.Regarding the etiology, the 54 orthopedic defects resultedfrom elective surgical procedures, such as 34 open-wedgehigh tibial osteotomies [8—12] and 20 osteonecrosis treat-ments with core decompression.

Regarding the morphology, the defects were divided into:(1) cavitary bone defects (osteonecrosis treatment) and (2)segmental bone defects (osteotomies). The volume of thecavitary bone defects were measured when the shape wasregular and only evaluated when the shape was irregular.

Figure 1 Open-wedge osteotomy; the fixation is done with alocked plate and the gap filled with �-TCP granules.

Investigators were provided with sterile graduated surgicalcups to estimate the amount of autograft or granules used.

The technique of the osteotomy was exactly the samefor the two groups as described previously [8—11]: after theexposure of the proximal tibia, the osteotomy line, startingfrom 3 to 4 cm distal of the medial joint line, passing abovethe insertion of patellar tendon to the tibial tubercle. Thelevel and direction are checked under scopy (C-arm of theimage intensifier) towards the lateral border of the tibiajust at the level of the proximal tibiofibular joint. The cut ismade with osteotomes, leaving the lateral part of the cortexof the lateral tibial plateau intact. The bone at the site ofthe osteotomy is forced open. We used granules to fill thedefect and a plate was then applied to the medial aspect ofthe tibia (Fig. 1).

For the core decompression of osteonecrosis, the channelwas 4 mm diameter.

Radiographic evaluation

Specifically for the purpose of assessing patient outcomesin this trial, radiographs were performed at four, nine, six-teen, twenty-four and thirty-six weeks. In addition to beingviewed by each clinician as a part of routine follow-up care,these images were independently assessed by a radiolo-gist responsible for determining all radiographic endpoints.Percent of osseous bridging was assessed on the basis ofbenchmarks of percentage bridging bone across the defect.

Healing of the osteotomy was measured by the progres-sion of bone filling the triangular volume of the osteotomyfrom the lateral cortex to the medial cortex. The amount of


filling (increase in density of the osteotomy) and its progres-sion were assessed at each radiological session as previouslyreported [11]. The volume created by the opening techniquewas calculated as previously described: the volume of open-ing was calculated as half of the elliptic cylinder [11]. Forthe channel of the core decompression, the volume of thedefect was calculated as the volume of a cylinder.

Clinical endpoints

Clinical, functional and radiographic endpoints wereassessed to monitor safety, clinical healing status andprogression of fusion. Tracked metrics included recordingadverse events, complications, protocol deviations and revi-sion surgeries.

The primary effectiveness endpoint was fusion asassessed by means of radiographs at twenty-four weeks. Adefect was considered fused if 50% osseous bridging acrossthe bone was identified. The clinical healing status was alsoextrapolated from the clinical and composite success ratesas well as the therapeutic failure rate (full weight withoutcrutches allowed or failure requiring secondary therapeu-tic intervention). Secondary outcomes also included visualanalog scale (VAS) pain measurements at the surgical siteand at the graft harvest site, as well as quality-of-lifeand functional assessments using the Short Form-12 (SF-12)instrument. The SF-12 is a generic measure and does not tar-get a specific age or disease group. The SF-12 is weighted andsummed to provide easily interpretable scales for physicaland mental health. Physical and Mental Health CompositeScores are computed using the scores of twelve questionsand range from 0 to 100, where a zero score indicates thelowest level of health measured by the scales and 100 indi-cates the highest level of health. When only physical healthis recorded (as in this study), 50 indicates the highest levelof health. The data obtained with the SF-12 has been devel-oped, tested and validated by Quality Metric Incorporated.

Analysis of safety-related data included adverse eventfrequency, severity and potential relationship to the �-TCP;surgical site complications, including nonunion. Treatmentemergent adverse events are adverse events reported dur-ing or following treatment through completion of the study.Events were classified as serious if they required or pro-longed inpatient hospitalization or any serious problemassociated with the device that related to the safety orwelfare of the patients in the study.

Statistical methods

The goal of the trial was to establish non-inferiority of �-TCP relative to autograft. For binary endpoints (includingthe primary endpoint of fusion), non-inferiority tests werecarried out by fitting a ten-percentage point margin. The Pvalues and confidence intervals were based on normal the-ory computations. Because of small sample sizes, binaryendpoint data were reported as counts and percentages,with corresponding non-inferiority P values, with the excep-tion of the percent of patients with graft harvest site painand adverse events, which report difference test P valuesrather than non-inferiority P values. All scales used 10-point

non-inferiority margins except for the SF-12, which used amargin of 5 points.

The radiographs images were evaluated independently bytwo investigators. The two investigators re-evaluated thedata one more time at 2 months after the first analysis.Inter-observer variability as well as intra-observer variabil-ity during the first and second rounds were determined usingintraclass correlation coefficient, an appropriate summarystatistic for determining the reliability of measurement.The intraclass correlation coefficients of values of �-TCPand cancellous bone at 2 weeks and 36 weeks weredetermined. The intraclass correlation coefficients of theinter-observer measurements for the first and second roundswere 0.67—0.94 and 0.79—0.96, respectively. The intraclasscorrelation coefficients of the intra-observer measurementsfor the first and second investigators were 0.89—0.96 and0.66—0.94, respectively.

Results

Radiographic results

Radiographic effectiveness was similar in both groups. Non-inferiority was observed for the primary endpoint of bonehealing and new bone formation in the defects.

In patients with segmental bone defects, the fusion rate(Table 1) of the 34 open-wedge osteotomies was 100% (17among 17) for patients in the group with �-TCP comparedwith 94% (16 among 17) for patients in the autograft group(odd ratio 0.3143; 95% Confidence Interval 0.0119 to 8.2740;P = 0.4879). The fusion rates (Table 1) stratified by volumeof defect (Table 2) demonstrated quicker healing for smalldefects across treatment groups: for 1 to 3 cm3 defects (5defects), the fusion rate was obtained at 8 weeks for the �-TCP group as for the autograft group; for 4 to 6 cm3 defects(14 defects), it was obtained at 10 weeks for the �-TCP groupas for the autograft group; and for 7 to 10 cm3 defects (15defects), it was obtained at 12 weeks for �-TCP group com-pared to 2.5 months for the autograft group (except for onefailure).

For the 20 cavitary defects, the radiographic union rates,as determined by the presence of osseous bridging were100% for patients in the two groups (with �-TCP or with auto-graft). With autografts, bridging was obtained at 8 weeks(range 4 to 12 weeks). With �-TCP, new bone (Fig. 2)developed in all patients and the mean period required forbridging was nine weeks (range 4—16 weeks) and not signif-icantly different (P = 0.24).

Periodic radiographic assessment revealed replacementof �-TCP by newly formed bone. However, a higher radiopac-ity persisted at the implant site (Fig. 3). This radiopacitymay be due to a greater mineral concentration of thecomposite formed by the new bone tissue and the resid-ual granules rather to a non-resorption of the biomaterial.After 12 months post-implantation, nearly all with �-TCP,was replaced by newly formed bone in all cases. However,exact evaluation of the ratio of residual with �-TCP andnewly formed bone was impossible by radiograph.

Postoperative MR imaging was performed in patients withcore decompression for osteonecrosis. Duration of MR eval-uation from surgery ranged from 12 to 24 months. On both


Table 1 Radiographic results summary.

HTO analysis (n = 34) CD analysis (n = 20)

�-TCP Autologous graft �-TCP Autologous graft

At 8 weeksPrimary end pointFusion rates

2 (6%) 3 (9%) 10 (50%) 10 (50%)


8 (24%) 13 (38%)


7 (20%)

Failure 1 (3%)

�-TCP: beta-tricalcium phosphate; CD: core decompression channel; HTO: high tibial osteotomy. The values are given as the numberof defects, with the percentage in parentheses.

Table 2 Demographic, clinical results and safety.

HTO analysis (n = 34) CD analysis (n = 20)

�-TCP Autologous Graft �-TCP Autologous Graft

Age (years): mean (range) 57 (40—65) 51 (42—64) 30 (18—41) 32 (21—46)Gender

Men 6 7 5 4Women 11 8 4 5

Volume of bone defect1 to 3 cm3 2 34 to 6 cm3 8 6 10 107 to 10 cm3 7 8

SF-12 Physical Component Score 44.1 ± 11.4 39.5 ± 10.2 41.9 ± 8.3 40.1 ± 10.6Graft harvest site pain 0 5 0 0

CD: core decompression channel; HTO: high tibial osteotomy. The values are given as the number.

Figure 2 Radiograph of the osteotomy at 2 months.

T1- and T2-weighted images, all areas implanted with �-TCPexhibited intensity that was nearly identical to surround-ing cancellous bone with a scattered low-intensity area,which probably represented small amounts of residual with�-TCP.

Figure 3 Radiograph of the osteotomy at 2 years.

Clinical results and functional outcomes

Non-inferiority of �-TCP was established at fifty-two weeks.At twenty-four weeks, the achievement of clinical union inthe osteotomies was 100% (17 among 17) for patients in the


group with �-TCP compared with 94% (16 among 17) forpatients in the autograft group. There was no significantrelation with age and gender.

The SF-12 questionnaire demonstrated improvementfrom baseline in both groups for all outcome measures. Allquality-of-life and functional outcome data supported non-inferiority of �-TCP compared with autograft. SF-12 PhysicalComponent Score (maximum 50 points) was 42.4 ± 10.3points in the �-TCP group and 41.0 ± 9.4 in the autograftgroup.

Safety

Few complications were observed in the �-TCP group com-pared with the autograft group. As regards the autograftharvest site, one patient in the autograft group requiredhospitalization for the treatment of an infection at theharvest site. Another patient in the autograft group devel-oped hematoma at the graft harvest site requiring additionalhospitalization. Obviously, no patients in the �-TCP groupexperienced any autograft harvest site pain or other suchcomplications as no bone graft harvest was required on theinitial surgery. Of the patients in the autograft group (forwhom a graft harvest procedure was required), 5 among 25patients (20%) reported clinically significant graft harvestsite pain at fifty-two weeks.

As regards the implantation site, in the present patientseries, no adverse reactions in the �-TCP group, such asexcessive postoperative drainage, dermatitis allergic reac-tion or other local complications, were observed.

Discussion

This study evaluated the efficacy of �-TCP bioceramicsin the reconstruction of bone defects caused by electiveprocedures such as open-wedge osteotomies (that cre-ate orthopedic defects) or reconstruction of cavitary bonedefects.

This study was designed to demonstrate that granules�-TCP in these indications are as effective as autograftbecause prior data [13—15] have suggested that it offers theadvantages of being safer and less painful for the patient asa result of eliminating the bone graft harvest site. In fact,the results demonstrate that the two treatment groups hadhighly similar radiographic, clinical, functional and quality-of-life outcomes but that the patients in the �-TCP grouphad fewer serious treatment emergent adverse events andcomplications. Patients in the �-TCP group obviously alsohad no donor site pain when compared with patients in theautograft group. In addition to the risk-benefit assessment,the economic value of any novel technology must also beseriously considered today by surgeons, hospitals, payersand patients. Studies have demonstrated notable resourceutilization related to using autograft, including additionaloperating room time, supply and personnel costs, additionalmedications, added lengths of stay, donor site complicationsand short and long-term side effects following graft harvest.Our good results are in agreement with those reported byothers [16—20] in orthopedic studies.

We have shown that the �-TCP was successful in regen-erating bone in all the cavitary and in segmental bone

defects where the gaps were around 10 cubic centimetersin volume. None of the patients presented acute inflamma-tory reactions or infections. One of the factors that has beendescribed as eliciting response of cells [21] such as mono-cytes, which are among the first cells to contact the implantsurface and to colonize the surgical site, is the ratio betweenthe surface area of cells and the surface area of the bioma-terial. The granule’s porosity size used in these clinical casesappears correct for clinical treatment of defects.

Some limitations to this study are as follows. First, it wasdifficult to distinguish newly formed bone from residual �-TCP. Second, obtaining completely matched images at eachtime was a problem, even though we tried to perform radio-graphs with the same radiologist. Third, the images weredivided into areas by tracing manually. Although this manualprocess caused inter- and intra-observer differences, intr-aclass correlation coefficients analysis showed substantialto almost perfect agreement between the two independentinvestigators.

Our radiographic analysis showed a higher radiopacityat the implant site even at 36 weeks evaluation. This is inagreement with other studies [22—27] that showed the sameimages. These authors demonstrated that the high radiopac-ity was due to the greater mineral concentration and thecomposite formed by the new bone tissue and the residualgranules and not due to the non-resorption of the bioma-terial [22—27]. Since the first successful clinical applicationof commercial calcium phosphate bioceramics in the early1970s, modifications [2—4] on their properties gave rise toalternatives with improved biological and mechanical prop-erties [28,29]. The bioceramics used in this study belongs tothe third generation of biomaterials, namely, those havingappropriate micro- and macroporosities, good mechanicalproperties and promoting not only bone substitution but alsobone regeneration [5,30—32] with different formulations asgranules, blocks, injectable form [33,34] in many surgicalsites of orthopedic surgery [6,18,19,35] as spine [16], hip,knee [9,20], fractures. . .

In conclusion, the results demonstrated that �-TCP treat-ment produced equivalent rates of healing, clinical success,functional patient improvement and radiographic outcomescompared with autograft treatment. In addition, patients inthe �-TCP group exhibited fewer therapeutic failures, lesspain and fewer serious treatment emergent adverse eventsand complications compared with patients in the autograftgroup. Importantly, patients in the �-TCP group were sparedany autograft donor site pain and morbidity, which, in nearly15% of patients in the autograft group, lasted for at least oneyear postoperatively.



References

[1] Goulet JA, Senunas LE, DeSilva GL, Greenfield ML. Autogenousiliac crest bone graft. Complications and functional assess-ment. Clin Orthop Res 1997;339:76—81.

[2] Daculsi G, Passuti N, Martin S, Deudon C, Legeros RZ, Raher S.Macroporous calcium phosphate ceramic for long bone surgery


in human and dogs. Clinical and histological study. J BiomedMater Res 1990;24:379—96.

[3] Daculsi G, Laboux O, Malard O, Weiss P. Current state of the artof biphasic calcium phosphate bioceramics. J Mater Sci MaterMed 2003;14:195—200.

[4] Daculsi G. Smart scaffolds: the future of bioceramic. J MaterSci Mater Med 2015;26(4):154.

[5] LeGeros RZ. Properties of osteoconductive biomaterials: cal-cium phosphates. Clin Orthop 2002;395:81—98.

[6] Passuti N, Daculsi G, Rogez JM, Martin S, Bainvel JV. Macropo-rous calcium phosphate ceramic performance in human spinefusion. Clin Orthop 1989;248:1169—76.

[7] Filmon R, Retailleau-Gaborit N, Brossard G, Grizon-PascarettiF, Baslé MF, Chappard D. Preparation of �-TCP granular mate-rial by polyurethane foam technology. Image Anal Stereol2009;28:1—10.

[8] Hernigou P, Medevielle D, Debeyre J, Goutallier D. Proximaltibial osteotomy for osteoarthritis with varus deformity. Aten to thirteen-year follow-up study. J Bone Joint Surg Am1987;69(3):332—54.

[9] Hernigou P, Roussignol X, Flouzat-Lachaniette CH, Filippini P,Guissou I, Poignard A. Opening wedge tibial osteotomy for largevarus deformity with Ceraver resorbable beta tricalcium phos-phate wedges. Int Orthop 2010;34(2):191—9.

[10] Hernigou P, Queinnec S, Picard L, Guissou I, Naanaa T, Duffiet P,et al. Safety of a novel high tibial osteotomy locked plate fix-ation for immediate full weight-bearing: a case-control study.Int Orthop 2013;37(12):2377—84.

[11] Hernigou P, Flouzat Lachaniette C, Delambre J, Guissou I,Dahmani O, Ibrahim Ouali M, et al. Full weight bearing anddynamisation with Limmed

®locked plate fixation accelerates

bone regeneration in the volume of opening wedge high tibialosteotomy. Int Orthop 2015;39(7):1295—300.

[12] Poignard A, Flouzat Lachaniette CH, Amzallag J, HernigouP. Revisiting high tibial osteotomy: fifty years of experiencewith the opening-wedge technique. J Bone Joint Surg Am2010;92(Suppl. 2):187—95.

[13] Hossain MZ, Kyomen S, Tanne K. Biologic responses ofautogenous bone and beta-tricalcium phosphate ceramicstransplanted into bone defects to orthodontic forces. CleftPalate Craniofac J 1996;33:277—83.

[14] Wada T, Hara K, Ozawa H. Ultrastructural and histochemicalstudy of betatricalciumphosphate resorbing cells in periodon-tium of dogs. J Periodontal Res 1989;24:391—401.

[15] Zerbo IR, Zijderveld SA, de Boer A, Bronckers AL, de Lange G,ten Bruggenkate CM, et al. Histomorphometry of human sinusfloor augmentation using a porous beta-tricalcium phosphate:a prospective study. Clin Oral Implants Res 2004;15:724—32.

[16] Aunoble S, Clément D, Frayssinet P, Harmand MF, Le Huec JC.Biological performance of a new b-TCP/PLLA composite mate-rial for applications in spine surgery: in vitro and in vivo studies.J Biomed Mater Res A 2006;78:416—22.

[17] Chappard D, Retailleau-Gaborit N, Legrand E, Baslé MF, AudranM. Comparison insight bone measurements by histomorphom-etry and microCT. J Bone Miner Res 2005;20:1177—84.

[18] Galois L, Mainard D, Delagoutte JP. Beta-tricalcium phosphateceramic as a bone substitute in orthopaedic surgery. Int Orthop2002;26:109—15.

[19] Ozawa M, Tanaka K, Morikawa S, Chazono M, Fujii K. Clinicalstudy of the pure �-tricalcium phosphate: reports of 167 cases.J East Jpn Orthop Traumatol 2000;12:409—13.

[20] Tanaka T, Kumagae Y, Chazono M, Kitasato S, Kakuta A,Marumo K. A novel evaluation system to monitor bone forma-tion and �-tricalcium phosphate resorption in opening wedgehigh tibial osteotomy. Knee Surg Sports Traumatol Arthrosc2015;23(7):2007—11.

[21] Dos Santos EA, Farina M, Soares GA, Anselme K. Chemical andtopographical influence of hydroxyapatite and beta-tricalciumphosphate surfaces on human osteoblastic cell behavior. JBiomed Mater Res A 2009;89:510—20.

[22] Baslé MF, Chappard D, Grizon F, Filmon R, Delecrin J, Daculsi G,et al. Osteoclastic resorption of Ca—P biomaterials implantedin rabbit bone. Calcif Tissue Int 1993;53:348—56.

[23] Chappard D, Terranova L, Mallet R, Mercier P. 3D porous archi-tecture of stacks of �-TCP granules compared with that oftrabecular bone: a microCT, vector analysis, and compressionstudy. Front Endocrinol (Lausanne) 2015;6:161.


[25] Ogose A, Hotta T, Hatano H, Kawashima H, Tokunaga K, EndoN, et al. Histological examination of beta-tricalcium phosphategraft in human femur. J Biomed Mater Res B Appl Biomater2002;63:610—4.

[26] Renooji W, Hoogendoorn A, Visser WJ, Lentferkink RHF,Schimitz MGJ, Ieperen HV, et al. Bioresorption of ceramicstrontium-85-labeled calcium phosphate implants in dogfemora. Clin Orthop 1985;197:272—85.

[27] Saito M, Shimizu H, Beppu M, Takagi M. The role of beta-tricalcium phosphate in vascularized periosteum. J Orthop Sci2000;5:275—82.

[28] Chappard D. New microscopies, biomaterials: two newaxes for morphologies. Morphologie 2016, http://dx.doi.org/10.1016/j.morpho.2016.07.055 [pii: S1286-0115(16)30084-4].

[29] Hsu YH, Turner IG, Miles AW. Fabrication and mechanical testingof porous calcium phosphate bioceramic granules. J Mater SciMater Med 2007;18:1931—7.

[30] Knabe C, Koch C, Rack A, Stiller M. Effect of beta-tricalciumphosphate particles with varying porosity on osteogene-sis after sinus floor augmentation in humans. Biomaterials2008;29:2249—58.

[31] Kotani S, Fujita Y, Kitsugi T, Nakamura T, Yamamuro T, OhtsukiC, et al. Bone bonding mechanism of beta-tricalcium phos-phate. J Biomed Mater Res 1991;25:1303—15.

[32] Nicholas RW, Lange TA. Granular tricalcium phosphategrafting of cavitary lesions in human bone. Clin Orthop1994;306:197—203.

[33] Blouin S, Moreau MF, Weiss P, Daculsi G, Baslé MF,Chappard D. Evaluation of an injectable bone substitute(betaTCP/hydroxyapatite/hydroxypropyl-methyl-cellulose) inseverely osteopenic and aged rats. J Biomed Mater Res A2006;78:570—80.

[34] Terranova L, Libouban H, Mallet R, Chappard D. Analysisof �-tricalcium phosphate granules prepared with differentformulations by nano-computed tomography and scanningelectron microscopy. J Artif Organs 2015;18(4):338—45.

[35] Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: anupdate. Injury 2005;36(Suppl. 3):20—7.



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