RESEARCH

TSMJ - Issue 10 - 2009 35

Comparative Evaluation of Three Different Collagen-Ceramic Composite Scaffolds for Use as Alternatives to Bone GraftsSultan Alqadiri*, Fergal O’Brien and John Gleeson

AbstractAim of StudyThis paper reports a comparative study conducted onnovel Collagen-Ceramic composite scaffolds. Three scaf-folds were compared based on pore size, porosity, andthickness of the collagen network. Characterisation ofthese properties will facilitate cell-specific optimisation ofthese scaffolds for use as successful clinical alternatives toauto- and allo-graft procedures.

MethodsCollagen-based scaffolds were fabricated using differentforms of calcium phosphate and freezedrying methods.Scaffold A, the control, was made with calcium phosphateR powder and lyophilised using a stainless steel freezingtray. Scaffold B was made with calcium phosphate S andlyophilised using a stainless steel freezing tray. Howeverscaffold C was made with calcium phosphate S andlyophilised using a polysulphone freezing tray.

ResultsThe results indicated that scaffold A had the minimumpore diameter for cell penetration and proper vascularisa-tion of the ingrown tissue, but is less than ideal for opti-mum bone growth. Scaffold B showed a pore diameterthat is almost three times larger and collagen networkstruts that were three times thicker than that of scaffold A.Scaffold C showed marked increase in porosity comparedwith scaffold B, but a thinner collagen frame.

Conclusionstudy has helped to compare and contrast some of the dif-ferent combinations of freezedrying treatments and cal-cium phosphate types, showing that numerous types ofcollagen scaffolds can be developed and customised tohelp in the treatment of various types of bone abnormali-ties. The large pore size and increased collagen framethickness of Scaffold B appear to make it the most prom-ising of the three scaffolds to be used in future bone graftprocedures.

IntroductionTissue engineering involves the application of principlesof materials science, cell biology, and engineering to thedevelopment of ideal tissue substitute materials that canmimic and maintain normal tissue function1. Large bonedefects often require reconstructive procedures for

anatomical restoration and to achieve stability. This can beachieved by many different means (ranging from the useof supportive casts to a series of invasive techniques) de-pending on the fracture pattern and relevant patient fac-tors.

Most clinical procedures involving large bone defects useautograft tissue transplantation (from the patient himself)as the source of the repair tissue. This autograft bone tis-sue is harvested from the patient and re-implanted at thesite of bone tissue damage. Other sources of bone tissueinclude allografts (from a different individual), xenografts(from a different species, mainly bovine) and syntheticmaterials2. At present, autografts are the clinically pre-ferred treatment since they allow earlier removal of exter-nal fixation devices, and improved anatomical andfunctional outcome3. However, there are a significantnumber of disadvantages with autograft tissue, specifi-cally limited supply of donor material (particularly inolder patients with degenerative bone diseases), donor-site morbidity, increased blood loss and postoperativepain4.

Allografts, although currently in clinical use, are oftencomplicated by fracture, resorption, and non-union be-tween the graft and recipient bone5. Synthetic materialsare slowly resorbed and replaced and therefore lead tolonger healing and recuperation times. Little informationis available regarding their long term effects and issueswith toxicity are also of major concern6,7.

However, the emergence of tissue engineering, throughthe development and application of scaffolds, promises toupgrade bone graft procedures and give physicians accessto numerous therapies that allow healing of fractures anddeformities. In 1970 W.T. Green, an orthopaedic surgeon,was responsible for conducting some of the first researchin the area of tissue engineering. He stipulated that by im-planting chondrocyte cells into the spicule of a bone (thearea of bone matrix where cell multiplication and bonegrowth occurs) he would be able to cause cartilage toform. Although he was unsuccessful, this opened thedoorway to tissue engineering. In the Mid-1980’s, Dr. Va-canti and Dr. Langer devised a method to generate func-tional tissue by using bio-degradable polymers, calledscaffolds, seeded with viable cells8.

Scaffolds are artificial structures capable of supportingthree-dimensional tissue formations, onto which cells canbe implanted. One approach is to implant scaffolds for tis-sue growth in vivo to direct tissue formation in situ. Bio-compatibility of the substrate materials is imperative – thematerial must not elicit an unresolved inflammatory re-sponse nor demonstrate immunogenicity or cytotoxicity9.The mechanical properties of the scaffold must be suffi-cient to facilitate the desired tissue healing and must notcollapse when handled by the surgeon during the proce-dure. Tissue scaffolds must be easy to sterilise to preventinfection. A requirement of particular importance in bonetissue engineering is a controllable interconnected poros-ity to allow cells to migrate into the scaffold centre and tofacilitate vascularisation of ingrown tissue. Porosity is a ‘

CLINICAL POINTS

Large bone defects often require reconstructive surgical procedures involvingtransplantation of tissue.

The emergence of tissue engineering and the development of scaffolds is likely to circumventsome of the problems associated with traditional transplantation methods.

Biocompatibility, ease of sterilization and mechanical strength are important considerationsin determining the suitability of a scaffold for a particular site.

Interconnected porosity is important also, as it affects the extent of vascularisation and themigration of proliferating cells within the scaffold.

Scaffold properties can be influenced by the properties of the materials it is composed ofand the methods used in its preparation.

*4th Year Medicine, TCD

RESEARCH

TSMJ - Issue 10 - 2009 36

measure of void spaces in a material, where the void maycontain gas or liquid. It is defined by the ratio:

and can be expressed as a percentage. A typical porosityof 90% as well as a pore diameter of at least 100 µm isknown to be a prerequisite for cell penetration and com-plete vascularisation of the implanted scaffold in bone tis-sue engineering6.

This paper reports a study conducted on novel collagen-ceramic composite scaffolds and discusses their potentialuse in the treatment of various ailments involving bonedeformities and abnormalities.

BackgroundIn previous experimental work, calcium phosphate andcollagen have been combined to make bone graft scaf-folds, as they mimic the natural composition of bone. Col-lagen is the most abundant and ubiquitous structuralprotein in the body, and may be readily purified from bothanimal and human tissues10. Around 60% of bone weightis made of calcium phosphate (Ca10(PO4)6(OH)2), thusexplaining why calcium phosphates have been intensivelyinvestigated as a major component of scaffold materialsfor bone tissue engineering6. Calcium phosphates have anexcellent biocompatibility due to their close chemical andcrystal resemblance to bone mineral. They also have os-teoconductive properties and can bind directly to boneunder specific conditions6.

While the excellent biological performance of calciumphosphates has been well documented, their relativelyslow biodegradation and their low mechanical strengthlimit their application in engineering of new bone tissue,especially at load bearing sites. The properties of syntheticcalcium phosphates vary significantly with their crys-tallinity, pore diameter, porosity and composition. Moreextensive interconnected porosity of a substance permitsfaster bone ingrowth but weakens the material; the idealpore size is thought to be between 150-500 µm11. Increasedcrystallinity, low porosity and small pore diameter tend togive higher stiffness, higher compressive and tensilestrengths, and greater fracture toughness6.

This study investigated three different types of scaffoldsmade with different forms of calcium phosphate (CP) andtreated with different freezedrying methods. Scaffold A,the control, was made with calcium phosphate R powder(CP R) and lyophilised using a stainless steel freeze dryingtray. This was chosen as the control because previous test-ing found it to have the qualities needed for sufficientbone growth12. Scaffold B was made with calcium phos-phate S (CP S) and lyophilised using a stainless steel freezedrying tray. Scaffold C was also made with CP S but waslyophilised using a polysulphone freeze drying tray. Theexact compositions of the two different types of calciumphosphate preparations can not be disclosed in this paper,as they are awaiting patency for future distribution andmarketing. However, it can be mentioned that the molec-ular make up of CP S is subjectively weaker than CP R asit has a lower crystallinity and density.

The three scaffolds were compared based on the thicknessof the collagen frame, porosity and pore size. To achieve amore complete comparison of the three scaffolds, mechan-ical testing should be carried out and their tensile andcompressive strengths should be compared with various

types of bones within the human body. Use of Calciumphosphate S rather than Calcium phosphate R andfreezedrying with the polysulphone tray rather than thestainless steel tray are two areas that are expected to havean important role in the future of tissue engineering prac-tices. Characterisation of these properties will facilitatecell-specific optimisation of these scaffolds for use as suc-cessful clinical alternatives to auto- and allo-graft proce-dures.

MethodsScaffolds are produced using a lyophilisation (Freeze-dry-ing) technique. Firstly, a collagen slurry is fabricated byblending the collagen in a mild acetic acid solution. Main-taining a constant low temperature during this step en-sures that the collagen protein does not denature andgelatinise. The suspension is then Freeze-dried usingrigidly controlled temperature and pressure parametersresults in a highly porous collagen-calcium phosphatecomposite scaffold. Briefly freezing the suspension resultsin the nucleation of ice crystals, which are surrounded bythe collagen-calcium phosphate composite material. Es-sentially, the collagen-calcium phoshpate co-precipitate isforced into the spaces between the growing ice crystals toform a continuous interpenetrating network of ice and co-precipitate. By sublimating the ice crystals out over a longperiod of time, a sponge like three dimensional construct,comprised of a biocompatible collagen-CP composite, isproduced. The use of a freezedryer allows extremely pre-cise control of the shelf (and consequently the slurry), tem-perature. This controls the size of the resulting pores inthe fabricated collagen scaffold.

Synthesis of Scaffolds3.6 grams of type 1 microfibrillar bovine tendon collagenwas added to a 0.05M acetic acid solution. This suspensionwas blended in a cooled reaction vessel at 4°C for 90 min-utes. Fifty percent by weight of calcium phosphate pow-der was added to the slurry and the slurry was blendedfor a further 90 minutes. The slurry was then degassed.Once degassed, 67 ml of slurry were placed in a suitablefreeze drying tray (stainless steel or polysulphone), andplaced in the freezedryer. Once the freeze drying processhad been completed, samples of the scaffold were re-moved using a sharp circular punch (9.5 mm diameter) foranalysis of pore structure.

Preparation of scaffolds for microscopyThe samples were embedded in JB-4 glycol methacrylatein order to make them suitable for slicing, staining and ob-servation under a light microscope. Firstly, the collagen-CP samples were fixed using 10% neutral buffered

formalin for 24 hours at room temperature to ensure sam-ple morphology was not affected by the embeddingprocess. Samples were then dehydrated in a series of al-cohol solutions over 24 hours: The distilled H20 and the100% EtOH steps were repeated multiple times to allow‘

volume of void space

total volume of the object

Solution Time Temperaturedistilled H20 30 mins - 1 hour room temp.distilled H20 30 mins - 1 hour room temp.50% EtOH 30 mins - 1 hour room temp.70% EtOH 30 mins - 1 hour room temp.80% EtOH 30 mins - 1 hour room temp.95% EtOH 30 mins - 1 hour room temp.95% EtOH 30 mins - 1 hour room temp.100% EtOH 30 mins - 1 hour room temp.100% EtOH 30 mins - 1 hour room temp.100% EtOH 30 mins - 1 hour room temp.

RESEARCH

TSMJ - Issue 10 - 2009 37

extended time for proper hydration and de-hydration respectively. Samples were equili-brated for 12 hours at 4°C in 1:1ethanol:catalysed JB4 solution A. Sampleswere then infiltrated with 100% JB4 A and thesolution was catalysed using JB4 B to poly-merise the embedding solution. The sampleswere sliced 10 micrometers thick using themicrotome and every fifth slice was stainedusing either Alizarin Red or Toluidine Blueand mounted upon a slide for microscopy.

Comparative analysis of scaffoldsA quantitative pore analysis was then carriedout in which stained samples were imagedusing light microscopy at a magnification of10 times (10X) and representative images ofthe pore structure were digitally captured.The definitive porosity and collagen thick-ness were not calculated, but a subjectivecomparison of the images obtained was doneto get an indication of the differences betweenthe scaffold samples. To measure strengthand rigidity, mechanical testing on the differ-ent scaffolds was scheduled. However, due totime constraints, the results are not availablefor inclusion in this paper.

ResultsScaffold A (the control) was made with cal-cium phosphate “R” powder and placed withthe standard metal tray in the freezedryer. Itdisplayed a pore size of approximately 100µm in diameter. Figure 1 shows a Scaffold Asample stained with Alizarin Red.

Scaffold B was made with calcium phos-phate “S” powder and placed with the stan-dard metal tray in the freezedryer. The porediameter was approximately 250- 300 µmwith a thick collagen frame. Figure 2 shows aScaffold B sample stained with ToluidineBlue.

Scaffold C was made with calcium phos-phate “S” powder and placed with the poly-sulphone plastic tray. The pore diameter wasapproximately 200 µm. Using subjective com-parison, a marked increase in porosity com-

pared with scaffold B was noted, with a markedly thinnercollagen frame. Figure 3 shows a Scaffold C samplestained with Toluidine Blue.

DiscussionFor the successful transfer of extracorporal bone tissue en-gineering in clinical practice, clear and precise definitionof the clinical demand or problem should be addressed.The severity and the location of the bone defect needs tobe precisely assessed before planning tissue engineering.Clinical aspects that should be considered in bone tissueengineering include the evaluation of the biomechanicalaspects of the skeletal implantation site, the immunologi-cal reactions towards the scaffold construct and thebiodegradability of the scaffolds13.

One can infer from comparison of Scaffolds A, B and Cthat their different porosities and pore sizes may result indifferent bone healing responses and thus could be used

to develop different types of bone grafts for use in variousbody sites. Scaffold A displayed the minimum pore diam-eter (100 µm) for cell penetration and sufficient vasculari-sation of the ingrown tissue, but it was less than ideal(150-500 µm) for optimum bone growth6. In scaffold B, thefreezedrying method remained the same, but the form ofcalcium phosphate used was switched from CP R to thesubjectively inferior CP S. This led to a pore diameter thatwas almost three times larger and a collagen frame thatwas markedly thicker due to the increase in amount of col-lagen available per pore. Although the results from me-chanical testing are not available at present, it can bepostulated that scaffold B would be weaker than scaffoldA due to the greater pore diameter of scaffold B.

From a clinical perspective, bone grafts made with CP Sscaffolds would be reserved for areas requiring weaker,non weight bearing bones such as those in the maxillaryfacial areas. Considering the fact that materials of low heatconductance allowed greater nucleation of ice crystals inthe freezedryer, the study expected that treating ScaffoldC with the polysulphone plastic trays should result in alarger pore diameter compared to those treated with thestainless steel tray (i.e. Scaffolds A and B). Its reason forhaving a smaller pore diameter than scaffold B remainsunclear and should be studied extensively.

ConclusionThe current objective in tissue engineering is to design re-producible bioactive and bio-resorbable three dimensionalscaffolds with customized porosity and pore structure.These should also retain their structure and integrity forpredetermined times in the presence or absence of loadbearing conditions(6). Use of Calcium phosphate S ratherthan Calcium phosphate R and freezedrying with thepolysulphone tray rather than the stainless steel tray aretwo areas that are expected to have an important role inthe future of tissue engineering practices.

Although all three scaffolds had the minimum pore diam-eter for cell penetration and proper vascularisation of theingrown tissue, scaffold B seemed to have struck the bestbalance in terms of large pore size for fast vascularisationand thick collagen frames for support. Given these char-acteristics, scaffold B seems to be the most promising scaf-fold for use in future bone graft procedures. Furtherevaluation of its strength, toxicity and impact on humanhealing must be carried out before recommending its usein a clinical setting.

This study has helped to compare and contrast some ofthe different combinations of freezedrying treatments andcalcium phosphate types, showing that numerous typesof collagen scaffolds can be developed and customised tohelp in the treatment of various types of bone abnormali-ties. However, before being deemed safe and clinically ap-plicable, in vitro and in vivo studies are imperative inorder to integrate this technology into the human biolog-ical system and to reduce unwanted side effects and toxi-city.

AcknowledgementsI would like to thank Prof. Fergal O’Brien and Dr. John P.Gleeson for allowing me to take part in his project and fortheir technical support throughout the project. I wouldalso like to acknowledge Enterprise Ireland and the Com-mercialisation Fund Proof of Concept (PC/2005/226)funding that allowed us to carry out this work. n

s Fig. 1. Scaffold A (control).at 10X magnification.

s Fig. 2. Scaffold B.at 10X magnification. Note enlarged pore diameter.

s Fig. 3. Scaffold C.at 10X magnification. Note increased porosity.

RESEARCH

References1. Williams, D. Benefit and risk in tissue engineering. Mater Today 2004; 7:p.24-29.2. Koh, C. & Atala, A. Tissue engineering, stem cells, and cloning: opportunities forregenerative medicine. J Am Soc Nephrol 2004; 15: p.1113-1125.3. Einhorn, T. et al. The healing of segmental bone defects induced by demineralisedbone matrix: a radiographic and biomechanical study. J Bone Joint Surg 1984; 66:p.274-279.4. Heiple, K. & Kendrick, R. & Herndon, C. & Chase, S. A critical evaluation ofprocessed calf bone. J Bone Joint Surg 1967; 49: p.1119-11275. Jarcho, M. Calcium Phosphate Ceramics as Hard Tissue Prosthetics. Clin OrthopRelat Res 1981; 157: p.259-278.6. Rezwan, K. & Chen, Q. & Blaker, J. & Boccaccini, A. Biodegradable and bioactiveporous polymer/ inorganic composite scaffolds for bone tissue engineering. Bioma-terials 2006; 27: p.3413-3431.7. Chaikof, I. et al. Biomaterial and scaffolds in reparative medicine. Ann NY AcadSci 2002; 96: p.96-105.8. Charles, V. The history of tissue engineering. J Cell Mol Med 2006; 10: p.569-76.9. Antoniou, G. & Mikos, A. & Temenoff, J. Formation of highly porous biodegrad-able scaffolds for tissue engineering. Electron. J. Biotechnol 2000; 3.10. Ramachandran, G. & Redid, A. Biochemistry of Collagen. New York: plenumpress 1976.11. Hinz, P. et al. A new resorbable bone void filler in trauma: early clinical expeienceand histologic evaluation. Orthopedics 2002; 25: p.597-600.12. Al-Munajjed, A. & O’Brien F. Influence of a novel calcium-phosphate coatingon the mechanical properties of highly porous collagen scaffolds for bone repair. MechBehav Biomed Mater 2009; 2: 138-146.13. Meyer, U. & Joos, H. & Weismann, P. Biological and biophysical principles inextracorporal bone tissue engineering Part III. Int J Oral Maxillofac Surg 2004; 33:p.635-641.