Tissue-Engineered Bone Regeneration

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Massive bone defects are a great challenge to reconstructivesurgery. The preferred treatment is an autologous bone graft. Butthe supply of suitable bone is limited and its collection is painful,with a risk of infection, hemorrhage, cosmetic disability, nervedamage, and a loss of function1. Surgeons can overcome theseproblems by using scaffolds of synthetic or natural biomaterialsthat promote the migration, proliferation, and differentiation of bone cells1,2. However, the success of these materials in repairinglarge bone defects is limited. They lack the osteogenic and osteoin-ductive properties of bone autografts. It has been postulated thatgreater regeneration could be obtained by supplementing aresorbable scaffold with regeneration-competent cells such as mar-row stromal cells (MSC) to recreate an embryonic environment inthe injured adult tissue, and thus improve clinical outcome 3,4. Thepropensity of MSC to adhere to tissue culture plastic permits theirisolation from other marrow cells. They can differentiate intoosteoblasts, chondrocytes, adipocytes, and myoblasts, and are pop-ularly referred to as mesenchymal stem cells5,6. A small bone hybridmade of a ceramic combined with MSC has been prepared inrats7,8. But scaleup to a bone hybrid that can be used to reconstructbone defects of clinically relevant volume has given suboptimalresults9. The form of scaffold (hydroxyapatite–tricalcium phos-phate ceramic) used may not have been ideal for applications

involving large skeletal defects under predominantly compressiveloads9. The scaffold was too brittle and fractures frequent. The lackof completely interconnected pores and the slow resorption of theceramic made it difficult for new bone to invade the defect. The useof synthetic calcium-based ceramics such as hydroxyapatite orhydroxyapatite-tricalcium phosphate ceramics are at present limit-ed because they do not combine good mechanical properties withan open porosity and are prone to fragile failure10,11. By contrast,naturally produced ceramic organic composites can combine goodmechanical properties with an open porosity, which makes themcandidates for use as delivery vehicles for MSC. One such materialis the natural coral exoskeleton, where an inorganic calcium car-bonate phase grows onto a charged, organized organic template.This natural ceramic has the best mechanical properties of the

porous calcium-based ceramics, given that its interconnectedporous architecture is similar to that of spongy bone 12,13.Transcortical bony defects implanted with coral become vascular-ized and invaded by newly formed bone, whereas the coral isresorbed at a rate commensurate with bone formation14. Coral hasbeen used in various clinical applications for more than adecade15–18. These properties make the coral exoskeleton a suitablecandidate for delivering bone marrow cells. We therefore exploredits use as a delivery vehicle for MSC or fresh bone marrow (FBM)aspirate in a large segmental defect model. This study was done todetermine whether the combination of coral scaffold loaded withMSC or FBM improved bone formation in a bone defect of clini-cally relevant volume and whether coral acts as a template to guidebone morphogenesis and allow epimorphic-like bone regenera-tion. We prepared large bone defects in sheep. Bone remodeling inthese animals is comparable to that in humans. Successful recon-struction of large bone defects was obtained using a combinationof MSC and coral.

Results

Establishment of a critical-size defect model in the sheep metatar-sus. Defects 6, 12, 15, and 25 mm long were created in sheepmetatarsals. Healing was followed radiographically for 16 weeks,

after which all animals were killed. The defects then underwenthigh-definition radiographic examination and histomorphologicalanalysis. Osteogenesis was always minimal in untreated defectswhatever their size, and bone union never occurred (Fig. 1A, B). Thebone-regenerating ability of the implants was subsequently evaluat-ed using bone defects 25 mm long to obtain an environment that isvery hostile to bone regeneration.

Preparation of the biohybrids. An average of 7 × 106 ± 1 × 106

autologous nucleated cells per milliliter (range 6 × 106 to 9 × 106

cells/ml) was collected from the iliac crests of sheep. The aspiratecontained cells from the erythroid (immature and mature pro-erythroblasts) and myeloid lineages, as well as a few macrophagesand fibroblastic cells. We estimated that there was 1 MSC per3,000 nucleated cells. No mature osteoblastic or osteoclastic cells

Tissue-engineered bone regenerationHerve Petite*, Veronique Viateau, Wassila Bensaïd, Alain Meunier, Cindy de Pollak, Marianne Bourguignon,

Karim Oudina, Laurent Sedel, and Genevieve Guillemin

Laboratoire de Recherches Orthopédiques, CNRS UPRES A 7052, Université D. Diderot, Faculté de Médecine Lariboisière Saint-Louis, 10 avenue de Verdun,75010 Paris, France. *Corresponding author ([email protected]).

Received 17 February 2000; accepted 19 May 2000

Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We

have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with

regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus

improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow

stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the

scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large

segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis

leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical

bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled

with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs whenthe defects were filled with the tissue-engineered bone.

Keywords: tissue engineering, mesenchymal stem cells, bone repair, marrow stromal cells, coral

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were observed. These cells were either loaded within coralimplants or cultured for three weeks before implantation. MSCgrew with a population doubling time of 48 h and were passagedtwice before implantation. In a separate experiment, they were

allowed to reach confluency and differentiate. Cultures had alka-line phosphatase activity and von Kossa-positive bone nodules,colonies of adipocytes (oil red O-positive) as well as numerousfibroblastic cells. We made no attempt to demonstrate thepluripotentiality of these cells.

Additional implants were loaded with FBM or MSC andprocessed for histology or scanning electron microscopy to deter-mine the distribution of MSC in coral implants. They showed cellsthroughout the implant, with a concentration toward the periph-eral areas.

In vivo evaluation of the biohybrids. Coral scaffolds and biohy-brids were implanted in 25 mm defects in sheep metatarsals, andhealing was followed as described earlier.

Bone regeneration and implant resorption were monitored by taking X-rays every four weeks. Radiographs were taken in theanteroposterior and mediolateral planes when the sheep were killed.Clinical union was defined as full continuity of both cortices on bothviews. Control defects never healed spontaneously (Fig. 1A, B).Osteogenesis spread slowly through the coral medullary canal indefects filled with coral alone, confirming its osteoconductivity (Fig. 1F). However, osteogenesis never led to clinical union(Fig. 1E, F). Defects filled with coral plus FBM were radiolucent inall animals at eight weeks, indicating massive coral resorption andpoor bone apposition. Bone formation was minimal in this group at16 weeks, and no sheep showed bridging of any cortices (Fig. 2C, D).In contrast, defects filled with implants of coral loaded with MSC

maintained their diameter in five out of seven sheep, suggesting thatbone formation occurred at about the same rate as implant resorp-tion. All but one of the sheep had at least one healed cortex at16 weeks (Fig. 2G, H). Clinical bone healing occurred in three sheep,

whereas at least one cortex was healed in three sheep. There was nounion in one animal.

Implants were collected at 16 weeks and processed, undecalcified,for histology . Defects filled with coral alone contained newly formedbone chiefly within the medullary area, and cortical continuity wasnever restored (Fig. 3C, D). Cavities filled with a coral scaffoldloaded with FBM were invaded by a dense, almost avascular, fibroustissue after 16 weeks, indicating that scarring had occurred, ratherthan bone regeneration (Fig. 3E, F). Cavities loaded with a coralscaffold plus MSC resulted in new bone formation with a tubularpattern. This pattern reflected normal bone macrostructure, with awell-differentiated marrow cavity and cortices (Fig. 3G, H). Thecoral had almost completely disappeared after four months in allgroups. Only a few scattered fragments remained embedded in boneor fibrous tissue.

The bone-regenerating ability of all implants was assessed by measuring cortical and medullary bone surface areas by imageanalysis (Table 1). The metatarsal diaphyses are a thick ring of cor-tical bone without any medullary bone. Therefore, we expectedregenerated bone to contain very little medullary bone comparedwith cortical bone. Adding FBM to coral did not significantly increase cortical or medullary bone surface area compared withcontrol or coral alone. In contrast, coral plus MSC produced asignificant increase in cortical bone surface area (54 ± 11%)compared with coral alone (13 ± 14%, p < 0.0001) or coral loadedwith FBM (18 ± 3%, p < 0.001), confirming the radiological and

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Figure 1. Radiographic follow-ups. (A, B)Control: X-rays of the metatarsus taken 16weeks after a 25 mm resection. Note theabsence of bone formation within the defect.(C–F) Coral alone: X-rays taken (C)immediately post-operatively, and at (D) 4and (E, F) 16 weeks following a coral implant.

Animals treated with coral were not healed

after 16 weeks (E, F), but a minimal bonyingrowth in the medullary area wasoccasionally observed (F).

Figure 2. Radiographic follow-ups.

(A–D) Coral–FBM: X-rays taken (A)immediately post-operatively (B) 4 and(C, D) 16 weeks following an implant ofcoral–FBM. Note the almost completedisappearance of coral by 4 weeks andthe absence of newly formed bone after16 weeks. (E–H) Coral–MSC: X-raystaken (E) immediately post-operatively(F) 4 and (G, H) 16 weeks following animplant of coral–MSC. Bone formationwas sufficient for union of the defectafter 16 weeks.

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histological data. The coral scaffold had almost completely disap-peared four months after implantation.

Discussion

One of the most extraordinary properties of bone is its ability to healwith practically no scarring. However, perturbations of the fracturesite may disrupt the repair process when bone defects reach a criticalsize, resulting in nonunion. The principal bone defects created dur-ing the resection of neoplasms are clinical examples of this situation.Because of the multiple limitations associated with the use of auto-grafts or bank bones for bone reconstruction, investigators havesought alternative solutions. Recent tissue engineering approacheshave attempted to create new bone based on MSC seeded ontoporous ceramic scaffolds. These attempts have given suboptimalresults that are due to the slow resorption rate of the hydroxyapatite-based ceramics7–9,19 producing a bony ingrowth onto a porous surfacerather than a true bone regeneration. We have used a combination of coral, a natural calcium carbonate–based ceramic, and MSC andfound a progressive, complete resorption of the scaffold leaving rela-tively mature remodeled bone. In the most favorable cases, MSC pro-duced complete recorticalization and formed a medullary canal withmature lamellar bone within four months. To our knowledge, thealmost complete disappearance of a biomaterial when used in con- junction with MSC and its replacement by mature bone with the

appropriate architecture, hence true bone regeneration, has not beendemonstrated previously.

We evaluated the capacity of the tissue-engineered bone torepair defects in a large-animal model of clinically relevantvolume. The sheep metatarsus is surrounded by a poorly vas-cularized environment with minimal amounts of soft tissue.The resections in untreated animals never healed, whatevertheir size, thereby confirming the critical size of this defect.

We found that the extent of healing differed signifi-cantly, depending on the source of cells. Filling a defectwith a coral scaffold alone allowed osteogenesis to occurin the medullary areas. This could be due to bone marrowcells migrating from the adjacent host bone marrow cavity onto the osteoconductive coral surfaces. The coral scaffold

was resorbed too rapidly in animals treated with the combina-tion of coral and FBM, probably because there were too many myeloid cells loaded within the coral scaffold. These myeloidcells include the progenitors of multinucleated giant cells, whichresorb coral14,20.

The tissue-engineered bone performed better than all othercombinations tested, confirming the positive influence of MSC sup-plements on biomaterials. Of the seven animals treated with coralscaffolds loaded with 107 cells, three produced clinical unions. Weplan to carry out additional studies with increased cell numbers toobtain more satisfactory results, because bone formation is directly related to the number of MSC used21. The MSC were also cultured inthe absence of dexamethasone, an osteogenic inducer, to transplantundifferentiated MSC with a high proliferative capacity 5. Furtherinvestigations are required to assess the effect of transplanting differ-entiated MSC.

The design of a scaffold is based on sound biological principlesthat have evolved from understanding how autografts function andhow they are remodeled after transplantation22. Scaffolds for MSC,regardless of the material from which they are formed, should encour-age MSC adhesion, proliferation, and differentiation to elicit boneformation. The implanted scaffold should become vascularized,because osteogenesis requires a well-developed vascular supply 23. Theporosity, pore size distribution, and continuity of the scaffold are also

critical for vascular invasion. These parameters dictate the interactionof scaffolds and transplanted cells with the host environment.

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Figure 3. Micro-X-rays and photomicrographs at 16 weeks.Histological sections of defects (A) left empty or filled with(C) coral (E) coral–FBM, and (G) coral–MSC. Note theinvasion of the defect with fibrous tissue (FT) in (A) and (E).

In defects filled with coral alone (C), osteogenesisoccurred within the medullary canal (MB). Defects filledwith coral plus MSC show cortical-like bone formationperipherally (CB). Cortical continuity was achievedbetween the edges of the defect. Micro X-rays confirmedthe histological observations. There was no boneformation in defects (B) left empty or (F) filled withcoral–FBM. Bone formation occurred in the medullary areain defects (D) filled with coral alone, but was insufficient forbone union. In contrast, defects filled with (H) coral–MSCshow osteogenesis chiefly at the periphery of the defect,leading to cortical bone union of the defect.

Table 1. Histomorphometry dataa

Control Coral Coral + FBM Coral + MSC

Medullary BSA 0.77 ± 0.6 23 ± 13 1.5 ± 1 17 ± 6.8

Cortical BSA 9.4 ± 7.8 13 ± 14 18 ± 3 54 ± 11CSA – 1.44 ± 1.4 0 1.17 ± 1.28

aThe defect surface area was split into medullary and cortical locations. Newly formedbone surface areas (BSA) and residual coral surface area (CSA) are expressed as the per-centage of total available surface areas in both locations. The metatarsus diaphysis is athick ring of cortical bone. Regenerated bone should therefore contain mainly corticalbone. Results showed that adding MSC significantly increased cortical BSA. CSA neverexceeded 2%.

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Pioneering studies showed that pore sizes less than 15–50 µm result infibrovascular ingrowth, pore sizes of 50–150 µm encourage osteoidformation, and pore sizes greater than 150 µm encourage theingrowth of mineralized bone24. A Porites coral scaffold, with an aver-age pore size of 250 µm and an interconnected structure with nodead-end pockets, should facilitate vascular invasion and bonedevelopment25. Ideally, the scaffold should be resorbed at a rate com-

mensurate with new bone formation: Porites scaffolding is dissolvedby multinucleated giant cells. These cells are presumably osteoclasts 14.Carbonic anhydrase, an enzyme present in osteoclasts26, is believed toplay a key role in the resorption of coral14,27, however, other possiblemechanisms cannot be excluded. The Porites scaffold is resorbedrapidly, within a few weeks25. This makes it very different from mosthydroxyapatite ceramics that are virtually undegraded during the firstfew weeks of implantation8,9,19,28. Presumably, the disappearance of thecoral scaffold left in place only bone tissue that then self-organizedaccording to the mechanical environment. In this respect, scaffoldsmade from the new generation of coral-like hydroxyapatite, with anarchitecture similar to Porites coral and an adjustable rate of resorp-tion28, tricalcium phosphate–hydroxyapatite (β-TCP-HA) ceramicswith a different ratio of β-TCP to HA (ref. 29), and biodegradablepolymers30, such as polylactic and polyglycolic acids may all be inter-

esting alternatives to coral.This study has left open the question of whether the coral

becomes vascularized in concert with expansion of the MSC mass.The average rate of vascularization in the rabbit ear chamber is esti-mated at 0.09–0.25 mm/day 31. If one assumes a similar rate of vascu-larization in the sheep, blood vessels should reach the center of theimplant within at least 10 days. Although the results obtained withthe tissue-engineered bone suggest good cell viability and the directparticipation of MSC in osteogenesis, it is still possible that there ismassive cell death within the core of the implant due to a lack of vas-cularization.

The choice of the associated osteosynthesis is critical to the per-formance of a tissue-engineered bone. Mechanical stresses arebelieved to be responsible for determining the architecture of bone,and changes in the pattern of mechanical loading result in a newmechanically optimized bone architecture. We found that remodel-ing of the tissue-engineered bone led to the formation of new corti-cal bone matching the architecture of the adjacent metatarsus. Thissuggests that the osteosynthesis elicited an appropriate stress trans-fer along the tissue-engineered bone, resulting in a normal bonearchitecture.

In summary, previous evidence indicated that MSC within aporous ceramic implant can lead to osteogenesis7–9. Our results areconsistent with this interpretation. Our findings also demonstratethat MSC-loaded implants can elicit true bone regeneration with thecomplete disappearance of the biomaterial and formation of corticalbone in a bone defect of clinically relevant volume. We believe thatsetting conditions for uniform loading and retention of MSC shouldensure systematic bone union.

Experimental protocolEstablishment of a critical size defect model in sheep metatarsus. Twenty-four month-old Pré-Alpes sheep weighing an average of 60 kg were obtainedfrom a licensed vendor (Institut National de la Recherche Agronomique,Jouy en Josas, France) and cared for according to the European guidelines forthe care and use of laboratory animals (Directive 24.11.1986, 86/609/CEE).The sheep were anesthetized and an osteotomy of the metatarsal diaphysiswas performed by a standard lateral approach. An eight-hole 3.5 mm narrowDC plate was provisionally attached to the shaft using the three most distaland proximal screws. The plate was removed and, under permanent salineirrigation, a 6 mm (three sheep), 12 mm (three sheep), or 25 mm (five sheep)long mid-diaphyseal osteotomy was performed with a 1 mm thick Gigli wire.The defect was stabilized with the anchor plate. The subcutaneous and skinlayers were closed in a routine fashion. A full cylinder cast (Vetcast Plus, 3M

Animal Care Products, St. Paul, MN) extending from beneath the knee to theend of the limb was applied with the animal in a standing position. Eachsheep was housed in a 16 m2 pen and fed a maintenance ration of hay plusfree access to water for the duration of the study.

Coral implant preparation. The pieces of coral exoskeleton from Poritessp. were 14 mm in diameter and 25 mm long, with a 5 mm diameter centralcanal. The coral consists of calcium carbonate (98–99%), as aragonite, traceelements (0.5–1%), and amino acids (0.07 ± 0.02%). Volume porosity was

49 ± 2%, and mean pore diameter was 250 µm (range 150–400). All poresintercommunicated (open porosity). Coral implants were sterilized by auto-claving, which does not affect the coral composition32.

Coral-FBM implant preparation. A 2 ± 0.5 ml sample of autologous bonemarrow was obtained from the left dorsal iliac crest and mixed with100 IU/ml heparin. A single-cell suspension was prepared by repeatedly aspi-rating the cells through 18- and then 21-gauge needles. The number of nucle-ated cells was determined by counting an aliquot in 3% acetic acid using anhemocytometer. The suspension was placed in a 4.8 mm diameter stainless-steel mold specially designed in the laboratory. The bone marrow was thenpushed into the defect using a plunger. Bone marrow smears were routinely prepared and examined by a qualified hematologist. Cell phenotype wasdetermined using morphological criteria.

Coral–MSC implant preparation. Samples (14 ± 3 ml) of autologous bonemarrow were obtained from the left dorsal iliac crest. A single-cell suspensionwas prepared as above. The cell suspension was cultured in α-minimal essen-

tial medium (α-MEM) plus 15% fetal calf serum supplemented with0.28 mM ascorbate with no dexamethasone addition33 and grown for 21 days.Cells (3.25 ± 0.25 × 107) from the first or second passage were used to inocu-late the coral implants. The implant was placed in the cell suspension for 2 h.Then, 4 ml of α-MEM were added and the hybrid implant was allowed toincubate overnight at 37°C before surgery. Four additional implants wereseeded with MSC and processed for histology and scanning electronmicroscopy to qualitatively evaluate cell adhesion and cell penetration.

The frequency of MSC was determined by counting under the microscopethe number of fibroblasts after three days of culture and expressed as thenumber of MSC per initially nucleated cells.

In a separate experiment, MSC were grown to confluence and became dif-ferentiated in the presence of dexamethasone to assess their osteogenicpotential. Alkaline phosphatase/von Kossa staining was performed asdescribed34, except that toluidine blue staining was omitted. Adipocytes werecharactarized by oil red O staining35. Fibroblastic cells were characterizedmorphologically by hematoxylin staining.

Transplantation. The 25 mm osteoperiosteal defects stabilized by ananchor plate were filled with a coral implant (seven sheep), with coral plusFBM (seven sheep), or with coral plus MSC (seven sheep). The follow-up of all the animals was identical to the follow-up of control animals.

Implant analysis. Radiographs were taken on the day of surgery and atmonthly intervals. At sacrifice, all metatarsals were clinically evaluated forimplant loosening, fixed in 10% formalin solution, and embedded inmethylmethacrylate25. All metatarsals were cut lengthwise using a circularwater-cooled diamond saw (200–300 µm). Sections closest to the longitudi-nal mid-sagittal plane and including the entire length of the implant and thetwo nearest screws were selected. These sections were ground down to athickness of 100 µm, polished, microradiographed, and surface-stained withStevenel blue and van Gieson picro-fuchsin25. Histomorphometry was car-ried out using a microscope linked through a 3-CCD video camera(DXC-930P, Sony) to an image processing system (Quantimet Q500, Leica,France). Coral surface area (CSA), total bone surface area (total BSA),medullary BSA, and cortical BSA were determined and converted to a per-centage of bone and coral per total surface area36. The data were analyzed by one-way analysis of variance (ANOVA). Between-group differences wereassessed using Bonferroni/Dunn post hoc tests ( p < 0.05).

AcknowledgmentsWe thank Inoteb (France) for donating the coral implants (Biocoral), Mrs M.Vallot for animal care and the Fondation pour l’Avenir ET8-263, AssistancePublique des Hopitaux de Paris AP-HP 97-002, CNRS and INSERM for finan-cial support.

1. Damien, C. & Parsons, R. Bone graft and bone graft substitutes: a review ofcurrent technology and applications. J. Appl. Biomat. 2, 187–208 (1991).

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2. Langer, R. & Vacanti, J.P. Tissue engineering. Science 260, 920–926 (1993).3. Caplan, A.I. Mesenchymal stem cells. J. Orthop. Res. 9, 641–650 (1991).4. Caplan, A.I. & Bruder, S.P. Cell and molecular engineering of bone regeneration.

In Principles of tissue engineering. (eds Lanza, R.P., Langer, R. & Chick, W.L.)603–619 (Landes, Georgetown, TX; 1997).

5. Prockop, D.J. Marrow stromal cells as stem cells for nonhematopoietic tissues.Science 276, 71–74 (1997).

6. Triffitt, J.T. The stem cell of the osteoblast. In Principles of bone biology . (edsBilezikian, J.P. & Raisz, L.G.) 39–50 (Academic, San Diego, CA; 1996).

7. Ohgushi, H., Goldberg, V.M. & Caplan, A.I. Repair of bone defects with marrowcells and porous ceramic. Experiments in rats. Acta Orthop. Scand. 60, 334–339

(1989).8. Bruder, S.P et al. Bone regeneration by implantation of purified, culture-expand-

ed human mesenchymal stem cells. J. Orthop. Res. 16, 155–162 (1998).9. Bruder, S.P., Kraus, K.H., Goldberg, V.M. & Kadiyala, S. The effect of implants

loaded with autologous mesenchymal stem cells on the healing of canine seg-mental bone defects. J. Bone Joint Surg. Am. 80, 985–996 (1998).

10. Moore, D.C., Chapman, M.W. & Manske, D. The evaluation of a biphasic calciumphosphate ceramic for use in grafting long-bone diaphyseal defects. J. Orthop.Res. 5, 356–365 (1987).

11. Grundel, R.E., Chapman, M.W., Yee, T., and Moore, D.C. Autogeneic bone mar-row and porous biphasic calcium phosphate ceramic for segmental bonedefects in the canine ulna. Clin. Orthop. 266, 244–258 (1991).

12. Guillemin, G., Patat, J.L. & Meunier, A. Natural corals used as bone graft substi-tutes. Bulletin de l’institut océanographique Monaco 14, 67–77 (1995).

13. Piecuch, J.F., Goldberg, A.J., Shastry, C.V. & Chrzanowski, R.B. Compressivestrength of implanted porous replamineform hydroxyapatite. J. Biomed. Mater.Res. 18, 39–45 (1984).

14. Guillemin, G., Patat, J.L., Fournié, J. & Chétail M. The use of coral as a bone graftsubstitute. J. Biomed. Mater. Res. 21, 557–567 (1987).

15. Yukna, R.A., & Yukna C.N. A 5-year follow-up of 16 patients treated with corallinecalcium carbonate (Biocoral) bone replacement grafts in infrabony defects. J.Clin. Periodontol. 25, 1036–1040 (1998).

16. Yukna, R.A. Clinical evaluation of coralline calcium carbonate as a bone replace-ment graft material in human periodontal osseous defects. J. Periodontol. 65,

177–185 (1994).17. Roux, F.X., Brasnu, D., Loty, B., Georges, B. & Guillemin, G. Madreporic coral: a

new bone graft substitute for cranial surgery. J. Neuro. Surg. 69, 510–513 (1988).18. Pouliquen, J.C., Noat, M., Verneret, C., Guillemin, G. & Patat J.L. Coral as a sub-

stitute for bone graft in posterior spine fusion in childhood. Fr. J. Orthop. Surg. 3,272–280 (1989).

19. Kadiyala, S., Jaiswal, N. & Bruder, S. Culture-expanded bone marrow-derivedmesenchymal stem cells can regenerate a critical-sized segmental bone defect .Tissue Engineering 3, 173–185 (1997).

20. Gross, U., Müller-Mai, C. and Voigt, C. Comparative morphology of the boneinterface with glass ceramics, hydroxyapatite and natural coral. In The bone–bio-

material interface. (ed. Davies, J.E.). 308–320 (University of Toronto Press,Toronto, ON; 1991).

21. Goshima, J., Goldberg, V.M. & Caplan, A.I. Osteogenic potential of culture-expanded rat marrow cells as assayed in vivo with porous calcium phosphateceramic. Biomaterials 12, 253–258 (1991).

22. Ostrum, R.F. et al. Bone injury, regeneration and repair. In Orthopaedic basic sci-ence. (ed. Simon, S.R.) 277–323 (American Academy of Orthopaedic Surgeons,Rosemont, IL; 1994).

23. Marks, S.C., & Hermey, D.C. The structure and development of bone. In

Principles of bone biology. (eds Bilezikian, J.P. & Raisz, L.G.) 3–14 (AcademicPress, San Diego, CA ; 1996).

24. Hulbert, S.F et al. Potential of ceramic materials as permanently implantableskeletal prostheses. J. Biomed. Mater. Res. 4, 433–456 (1970).

25. Guillemin, G. et al. Comparison of coral resorption and bone apposition with twonatural corals of different porosities. J. Biomed. Mater. Res. 23, 765–779 (1989).

26. Gay, C.V. & Mueller, W.J. Carbonic anhydrase and osteoclasts: localization bylabeled inhibitor autoradiography. Science 183, 432–434 (1974).

27. Guillemin, G., Hunter, S.J. & Gay, C.V. Resorption of natural calcium carbonateby avian osteoclasts in vitro. Cells and Material s5, 157–165 (1995).

28. Shors, E.C. Coralline bone graft substitutes. Orthop. Clin. North Am. 30,599–613 (1999).

29. Daculsi, G., Bouler, J.M. & LeGeros, R.Z. Adaptative crystal formation in normaland pathological calcifications in synthetic calcium phosphate and related bio-materials. Int. Rev. Cytol. 172, 129–191 (1997).

30. Pachence, J.M. & Kohn, J. Biodegradable polymers for tissue engineering. InPrinciples of tissue engineering. (eds Lanza, R.P., Langer, R. & Chick, W.L.)274–193 (R.G. Landes, Georgetown, TX 1997).

31. Zawicki, D.F., Jain, R.K., Schmid-Schoenbein, G.W. & Chien, S. Dynamics ofneovascularization in normal tissue. Microvasc. Res. 21, 27–47 (1981).

32. Irigaray, J.L. et al. Effet de la température sur la structure cristalline d’unBiocoral. J. Thermal Analysis 39, 3–14 (1993).

33. Petite, H., Kacem, K. & Triffitt, J.T. Adhesion, growth and differentiation of humanbone marrow cells on non porous calcium carbonate and pastic substrata:effects of dexamethasone and 1,25 dihydroxyvitamin d3. Mater. Med. 7,

665–671 (1996).34. Herbertson, A. & Aubin, J.E. Dexamethasone alters the subpopulation make-up

of rat bone marrow stromal cell cultures. J. Bone Miner. Res. 10, 285–294(1995).

35. Gamou, S., Shimizy, Y. & Shimizu, N. In Animal cell culture. (ed. Pollard, J. &Walker, J.) 197–207 (Humana, Clifton, NJ; 1990).

36. Louisia, S., Stromboni, M., Meunier, A., Sedel, L. & Petite, H. Coral grafting sup-plemented with bone marrow. J. Bone Joint Surg. Br. 81, 719–724 (1999).

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