calcium phosphate graft substitute: when the impact of innovation is in the form rather than content

26nd European Conference on Biomaterials, 31th August – 03rd September, 2014, Liverpool, UK

CALCIUM PHOSPHATE GRAFT SUBSTITUTE: WHEN THE IMPACT

OF INNOVATION IS IN THE FORM RATHER THAN CONTENT

Francisco Braga1, Antonio Carlos da Silva

2, Sérgio Allegrini Jr.

3, Cyro Ottoni

4

1Instituto de Pesquisas Energéticas e Nucleares (IPEN) – Av. Prof. Lineu Prestes, 2242, São Paulo (SP), Brasil

2Consulmat Produtos Técnicos Indústria e Comércio Ltda. – Rua Juan Lopes, 159, São Carlos, SP, Brasil

3Universidade Ibirapuera (UNIB) – Av. Interlagos, 1329, São Paulo (SP), Brasil 4 Universidade de São Paulo/FMVZ, Av. Prof. Orlando Marquês Paiva, 87 – São Paulo, (SP), Brasil

E-mail: [email protected]

Abstract. Amorphous Calcium Phosphate (ACP) is the first mineral phase formed in hard tissues. ACP has low

ordering in the crystal lattice compared to other calcium phosphates. The X-ray diffraction pattern is broad and

diffuse with a maximum at 250 ( 2ϴ), being considered as a precursor of well crystallized hydroxyapatite (HA),

reference bone grafting material in odontology and medicine. The ACP graft has high capacity to be resorbable

and has been widely studied as a filler for bone defects. However, it is rare to find articles where the ACP is

presented in the form of fibers and yet always containing silicon. This research paper presents the ACP fibers

obtained by the process Hager-Rosengarth, confirming its amorphous condition by means of X-ray diffraction

and its capacity of resorption and ability to conduct and formation of new bone tissue proven by test "in vivo"

with subsequent analysis by light microscopy. ACP in the form of fibers is considered innovation as a bone

grafting material, having already its exclusive rights of patent claimed by a Brazilian company that is which is

being funded by CAPES / FINEP in a program of R & D.

Keywords: Amorphous Calcium Phosphate, Bone Grafting, Fiber, Osteo-Conductive Biomaterial.

1. INTRODUCTION

Four main factors are relevant in implantable bioceramics: a) bioactivity level, b) reactive

surface area, c) easy handling, and d) be scaffold for cell culture. The amorphous calcium

phosphate (ACP) graft composed of entangled fibers morphology suggest to meet such

requirements.

The ACP is pointed in the literature as a precursor stage of the hydroxyapatite in bone

tissue formation1. ACP has been used in cements with application to the skull and as base

material in covering dentin before close with resin, suggesting better osteoconductivity and

biodegradability than tricalcium phosphate and hydroxyapatite during the formation of

mineralized tissues2. It is feasible to assert that ACP increases alkaline phosphatase activities

of mesoblasts, enhance cell proliferation and promote cell adhesion.

Similar to a cotton swab, a set of micrometer fibers of amorphous calcium phosphate

graft is obtained by Hager-Rosengarth process3 (Figure 1). The advantage of the shape in

entangled fibers, makes possible the graft fits any configuration of defect, combining

hemostatic function to its main characteristic, that is, be biocompatible, osseointegrated, resorbable and assist in bone remodeling. The fibers presented high-purity composition, non-

cytotoxicity, resorbable in months and osteo-conductive as any other graft of amorphous

calcium phosphate2,4,5

. It's rare to find scientific literature regarding ACP fibers, perhaps due

to difficulties of fiber small-volume production using possible industrial processes.


Figure 1. Hager-Rosengarth machine: 1-furnace; 2- outlet mouth; 3-burner; 4-air pressure

pipe; 5-circular plate; 6- centrifuging disc.

The biocompatibility of bone grafts is evaluated mainly by the type of bone/biomaterial

reaction process and the ideal condition conduces to a total osteogenesis that promotes a

physical-chemistry union of the bone to the biomaterial surface, and where the process

follows some steps that define the osseointegration phenomenom: (10) The biomaterial

surface energy related to the surface wetability by blood will define the type and amount of

contact that the formed bone tissue will have with the biomaterial; (20) Then, the biomaterial

surface electrochemical potential will determine the adhesion capacity level of the biomaterial

to the formed bone tissue; (30) The type of ionic exchange between the biomaterial surface

and the body will define the organic/mineral type of the formed tissue around the biomaterial

surface; e (40) The biomaterial surface topography will define the retention capacity by the

new formed tissue6. In such mechanism, the biocompatibility and biofunctionality are

properties that give the right concept of biomaterials, i. e., the capacity to fulfill properly the

desired function with the necessary acceptance by the body7.

To reconstruct of a bone region using a graft material, it is necessary incite a local inflamatory process. This inflammatory process is characterized by the excess of blood

irrigation that conduce to clot formation into the free spaces between the traumatized bone surface and graft surface. Macrophage cells digest the clot given space to fibroblast and

osteoblast cells competitively begin the new tissue formation. The formed tissue characteristic

depends on intrinsic factors like metabolic condition of the body and type of local bone

matrix, and extrinsic factors like surgical condition and grafting characteristics. These factors

define the model of distribution and quantity of the final fibrous and calcified formed tissue.

The calcium phosphate structural properties like chemical purity, crystalline degree and

molecular stoichiometry, are fundamental to define the resistance to degradation action of the

metabolism, as well its morphologies and dimensions due to the surface area that is in contact

to the cells.

The purpose of this work is present a new bone graft biomaterial, not innovative in its

chemical composition but in its morphological characteristic similar to cotton fibers, which

demonstrated keep their biocompatible and osteoconductive properties added to hemostatic capacity and greater range of traumo-orthopedic applications, some cases currently with no

grafting option.


2. MATERIALS AND METHODS

The starting material is obtained by hydrothermal reaction of CaO and P2O5 in a Ca/P >

1. The material is dried and ground and then heated to 1350 Celsius when flows into the

rotational base of the Hager-Rosengarth machine forming the fibers with diameters ranging

from 5 µm to 200 µm that can be seen in Figure 2.

Figure 2. Obtained ACP fibers.

The ACP fibers were analyzed by X-Ray Diffraction (XRD), Scanning Electronic Microscopy (SEM), Electronic Dispersive Scanning (EDS) and Polarized Light Microscopy

(PLM).

Two biological assays were done:

1. “In Vitro” Assay: Cytotoxicity Test (CT)

Cytotoxicity assay is a procedure adopted as method of screening of toxicity in vitro of

materials. The assay was carried out following International Standardization Organization -

ISO 1099317 and methodology according Rogero et al8. Monolayer cell line of NCTC clone

929 obtained from American Type Culture Collection (ATCC) was used in 96 microplate

wells which received serially diluted extracts of ACP fiber samples. The extract was obtained

by ACP fibers immersion in a flask with cell culture medium MEM (minimum Eagle´s

medium) with 5% calf fetal serum plus amino acids and incubated at 37oC during 24h. PVC

pellets were used as negative control and the extract was obtained by the same way as ACP

fibers. 0.02% phenol solution was used as positive control and received the same treatment of extract dilution. The aim of controls is to verify the assay performance, positive control has to

be a material which causes toxicity and negative has to be no toxic. The cytotoxic effect of each material was measured by the percentage of viability of cells

when in contact with extract of testing material and was quantitatively assessed by measuring


the uptake of neutral red by the viable cells. The optical densities (DO) were measured in a

microplate ELISA reader spectrophotometer Sunrise – Tecan in 540nm filter and calculated

cell viability % in relation to cell control.

Plotting the cell viability % against extract concentration it could obtain the cytotoxicity

index represented by IC50% in the viability curves. The cytotoxicity index represents the

concentration of the extract which kills or injury 50% of cell population in the assay.

2. “In Vivo” Assay: Implantation with bone void filler in two situations

(a) In rats: The ACP fiber material was implanted in dental alveoli for the observation

of new bone formation in bone repair of Wistar rats alveoli subjected to extraction. Eight

animals of the species Rattus Novergicus Albinus – male Wistar lineage rats were used. This population was divided into two distinct groups: control group and experimental group. In

both groups, the animals were subjected to avulsion of the right upper incisor. The control group was made only to occlusive suture of the alveoli after visual confirmation that it was

filled with blood clot. In the experimental group the alveoli were first filled by the graft material and then also had their suture occlusive manner. At the end of 28 days, the rats were

sacrificed and their alveoli analyzed under conventional and polarized light microscopy. Semi-serial 10µm samples slices were cutted and subsequently stained with hematoxylin-

eosin and picrosirius. The samples slices were demineralized and analyzed to observe the

osteiod matrix formation in bone repair under conventional and polarized light microscopy.

(b) In human: The ACP fiber material was implanted in a fibrotic region by peri-

implantitis around an implant in one patient, 62 years old man for observation its

osteoconductive capacity and resorbable characteristic. This implant element, with more than

20 years in function, cylindrical shape and titanium plasma spray surface treatment, was

disabled due loosening of the prosthetic component. For the surgical procedure, the patient

was treated with antibiotic, a non-steroidal anti-inflammatory and an analgesic as preoperative

and after the explantation procedure. After anesthesia in the vestibular bottom and atrium

gingiva, the site was incised with small preventive removal of diseased epithelium and

connective tissue with total displacement of the contaminated fibrous tissue. The site was

curetted, until no remaining fibrous tissue and only could verify the presence of the local hard tissue, washed with physiological saline, the ACP fibers graft inserted slightly condensed and

then the place was sutured using nylon suture. After 15 weeks the site was anesthetized again using the same procedures described

above. The area was then exposed and with the aid of a cutter shaped trephine with a diameter of 2 mm it was removed a bone fragment from the central area where the graft was

performed. Furthermore in the place of intersection between the bone graft area and the primary bone matrix area, another fragment was removed using a trephine with diameter of 4

mm. The samples were immersed in paraformaldehyde solution for 4 hours and then sent for

histological analysis. These samples were subsequently washed in sucrose solution. After the

dehydration process non decalcified samples were immersed in xylene and then included in

resin. The samples were cut by microtomy fitted with tungsten disposable blade to give slices

of 5µm thick. Then, the slices were accommodated on glass slides previously packed with

binders for evaluation by PLM and SEM.

3. RESULTS AND DISCUSSION

The non-crystalline condition of the obtained fibers is confirmed by X-ray diffraction

(XRD) as shown in Figure 3. The obtained ACP fiber XRD pattern is in agreement with


literature2 where shows a broad and diffuse profile with a maximum at 25 degree in 2ϴ scale,

and no other different features compared with crystallized calcium phosphates.

Figure 3. ACP fiber X-ray diffraction spectrum.

However, these fibers suggest to have various amorphous compositions in a coaxial

condition, that were formed as a result of melted liquid super-cooling, according the phase diagram of P2O5:CaO showed in Figure 4.

Figure 4. Phase diagram of P2O5:CaO where possible different compositions can be formed

due to high cooling rate.

A test of dissolution in simulated body fluid (SBF) proved this conjecture. After one

week in SBF, the ACP fibers were analyzed by SEM and EDS. The Figure 5 shows the dissolution effect on regions chemically analyzed. The corroded core of the fibers, shows a

configuration that can behave as scaffold to cell adhesion (Figure 6).


Figure 5. Fiber exposed to SBF showing different aspects and Ca/P in its structure.

Figure 6. Intermediate layer of fiber exposed to SBF showing a cancellous configuration.

The cell viability data (%) against extract concentration (%) projected in the Figure 7

shows the cell viability curves of cytotoxicity assay. All tested samples showed the same


behavior of negative control, the viability curves are above the cytotoxicity index (IC50%)

line indicating no toxic effect. In the other hand the positive control presented cytotoxic

behavior with IC50% = 19 which means that the extract of positive control, in a dilution of

19% caused damage in 50% of the cell population in this assay. The ACP fibers by means of

the its cellular viability curve above IC50% line and are considered non-cytotoxic.

Figure 7. Viability curve of ACP fibers by the “in vitro” cytotoxicity assay.

Regarding graft done in alveoli of rats, the control group showed bone repair compatible

with the classic described in the literature, ie, centripetal way, showing presence of a central

or paracentral clot and blood vessels formation into the alveolus in all samples (Figure 8). The

cells typical of bone tissue, osteoblasts and osteoclasts were present. The surface of trabeculae

and abundance of immature fibroblasts in bone tissue occupying the spaces between the

trabecular bone was observed.

Figure 8. Alveoli of the Control group. HE. 20X:


Clot (CL), Alveolar bone (AB), Newly Formed Bone (NB) and Osteoid Tissue (OS).

In the experimental group the dispersion of blood vessels was more marked, with no clot

in most samples. Thus, there was the presence of neoformed tissue throughout the alveolar

space as can be seen in Figure 9. This group also showed good cell differentiation.

Figure 9. Alveoli of the Experimental group. HE. 20X:

Alveolar bone (AB), Newly Formed Bone (NB) and Osteoid Tissue (OS).

In general, 28 days after surgery, samples of the control group presented a higher amount of mature tissue. However the experimental samples showed a more organized tissue

arrangement filling the alveolar site as can be seen in Figure 10.

Figure 10. Collagen fibers in the alveolar bed after 28 days postoperatively. Picrosirius. 40x:

dark areas → mature bone; intensely stained → osteoid tissue.

In the human "in vivo" assay, he fibrotic region around the implant and the size of the

removed fibrous tissue can be seen in Figure 11a and 11b respectively.


Figure 11. Periapical radiographs showing fibrotic site (red dashed) around an abutment-

implant of 2 elements prosthesis and relative size of the removed fibrous tissue (black arrow).

After removal of the infected implant and curettage of existing fibrotic tissue around the

element as well as in the remaining hard tissue, the accommodation of the graft was

performed, as shown in the periapical radiograph of Figure 12a, and after 2 weeks of

postoperative the grafted site was evaluated showing a small exposed amount of fibers, but

without local gingival inflammation (Figure 12b).

Figure 12. Periapical radiographs showing the accommodation of the graft within the

curettage site (a) and after two weeks postoperatively (b) showing the delineation of the

grafted area (black arrows).

Throughout the process of tissue repair, the normal process of interaction between the

remaining bone and graft material were observed by weekly radiographs, as can be seen in

Figure 13.


Figure 13. Sequential periapical radiographs showing the morphological changes in the

grafted site over time of implantation.

The SEM images whose regions were subjected to chemical analysis by EDS showed

after 15 weeks of bone healing presence of the remaining fibers of the graft material in the central region of the insertion site, however in the vicinity and nearby with the remaining

alveolar bone were observed images where one can notice the new bone deposits interacting

with small residues of biomaterial (Figure 14).

Figure 14. Remaining fiber of the graft material (white arrow) and new bone deposit (blue

arrow).


Scaffold of fibers of the biomaterial anchoring deposits of new bone tissue it was

observed by light microscopy. Using Toluidine blue staining could be noticed the existence of

new bone matrix containing osteocytes (Figure 15). Images by PLM with staining in picro-

sirius demonstrate existence of type I and II collagen fibers by interacting with fibers of the

biomaterial. The existence of large amounts of type I collagen fibers represented by reddish

prove the presence of mature bone tissue (Figure 16).

Figure 15. Shaped neo-bone (star) containing residues of ACP fiber and fiber already in the

resorption process (arrow), conducting to new bone deposition.

Figure 16. View by PLM showing collagen type I (red) and type II (beige) fibers configuring

the neo-bone. The arrows point at fibers in the resorption process leading to new bone deposits.


4. CONCLUSIONS

a) ACP fibers proved be resorbable in "in vitro" and "in vivo" assays;

b) ACP fibers are considered non-cytotoxic ;

c) ACP fiber showed to stimulate a good vascular infiltration with further bone tissue

deposition and remodeling activities;

d) ACP fibers suggest be osteo-conductor when used as bone graft;

e) ACP fibers promises to be a solution as bone grafting material in cases of difficult

insertion and maintenance on the sites of defects such as fractures with tissue loss,

periodontal pocket and maxillary sinus lift surgery with rupture of the Schneiderian

membrane; f) ACP fiber, material of easy handling, enables its use in simple cases like filling in

alveoli after tooth extraction, filling cavities curetted due to fibrosis of the bone tissue, filling of cavitiy caused by apicoectomy, etc…

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calcium phosphate graft substitute: when the impact of innovation is in the form rather than content

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