Marrow cell induced osteogenesis in porous hydroxyapatite and tricalcium phosphate: a comparative histomorphometric study of ectopic bone formation

To investigate the bone formation ability of porous hydroxyapatite (HA) and tricalcium phosphate (TCP), ceramic discs were implanted with or without rat marrow cells into subcutaneous sites in syngeneic rats. The discs of HA and TCP had identical microstructures: pore size was 190-230 microns, porosity was 50-60%, and they were fully interconnected. Implants without marrow cells (discs themselves) did not show bone formation, whereas implants with marrow cells showed bone formation in the pores of the ceramics. The bone formation of both HA and TCP occurred initially on the surface of the ceramic and progressed towards the center of the pore. The de novo bone was quantitated from decalcified serial sections of the implants. One month after implantation with marrow cells, the percentage fractions of the pore area filled with bone for implanted HA and TCP were 16.9 and 15.1, respectively. At 2 months after implantation with marrow cells, the fractions of bone were 34.3 and 30.9, respectively. These results indicate that both HA and TCP ceramics can show comparable osteogenic ability in the presence of marrow cells.     

Material-dependent bone induction by calcium phosphate ceramics: a 2.5-year study in dog

Bone induction by different calcium phosphate biomaterials has been reported previously. With regard to (1) whether the induced bone would disappear with time due to the absence of mechanical stresses and (2) whether this heterotopically formed bone would give rise to uncontrolled growth, a long-time investigation of porous hydroxyapatite ceramic (HA), porous biphasic calcium phosphate ceramic (TCP/HA, BCP), porous alpha-tricalcium phosphate ceramic (alpha-TCP) and porous beta-tricalcium phosphate ceramic (beta-TCP) was performed in dorsal muscles of dog, for 2.5 years. Histological observation, backscattered scanning electron microscopy observation and histomorphometric analysis were made on thin un-decalcified sections of retrieved samples. Normal compact bone with bone marrow was found in all HA implants (n = 4) and in all BCP implants (n = 4), 48 +/- 4% pore area was filled with bone in HA implants and 41 +/- 2% in BCP implants. Bone-like tissue, which was a mineralised bone matrix with osteocytes but lacked osteoblasts and bone marrow, was found in all beta-TCP implants (n = 4) and in one of the four alpha-TCP implants. Both normal bone and bone-like tissues were confined inside the pores of the implants. The results show that calcium phosphate ceramics are osteoinductive in muscles of dogs. Although the quality and quantity varied among different ceramics, the induced bone in both HA and BCP ceramics did neither disappear nor grow uncontrollably during the period as long as 2.5 years.     

Osteoinduction of hydroxyapatite/β-tricalcium phosphate bioceramics in mice with a fractured fibula

Many studies have shown that calcium phosphate ceramics can induce bone formation in non-osseous sites without the application of any osteoinductive biomolecules, but the mechanisms of this phenomenon (intrinsic osteoinduction of bioceramics) remain unclear. In this study, we compared the intrinsic osteoinduction of porous hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) implanted in mice at different sites. In 30 mice the left fibula was fractured and the right fibula was kept intact. A porous HA/β-TCP cylinder was implanted into both the left (group 1) and right (group 2) leg muscles of each animal. In addition, two HA/β-TCP cylinders were bilaterally implanted into leg subcutaneous pockets (group 3) in each of the remaining 15 mice. New bone formation was studied in the three groups by histology, histomorphometry and immunostaining. In group 1 new bone was observed at week 6 and bone marrow appeared at week 12. In group 2 new bone was observed at week 8 and bone marrow appeared at week 12. The new bone area percentage in group 1 was significantly higher than in group 2 at both weeks 8 and 12. In contrast, group 3 did not show any new bone within the period studied. These differences were explained based on the location of the implants and thus their proximity to the osteogenic environment of fracture healing. The results support the hypothesis that intrinsic osteoinduction by calcium phosphate ceramics is the result of adsorption of osteoinductive substances on the surface.     

多孔碳酸钙陶瓷与干细胞源性成骨细胞的相容性

背景：国内外的研究证实普通碳酸钙陶瓷作为骨替代材料时具有细胞支架作用。 目的：观察多孔碳酸钙陶瓷与成骨细胞的相容性,及作为骨组织工程支架的可能性。 方法：SD大鼠骨髓基质干细胞经矿化诱导培养、扩增并检测证实其已具成骨细胞表型后,分别与多孔碳酸钙陶瓷支架、普通羟基磷灰石陶瓷支架体外复合培养。 结果与结论：骨髓基质干细胞经体外诱导形成成骨细胞,钙结节、Ⅰ型胶原和碱性磷酸酶免疫染色结果阳性。多孔碳酸钙陶瓷支架材料与羟基磷灰石陶瓷材料皆有细胞附着生长,但多孔碳酸钙陶瓷支架材料细胞的黏附能力、增殖活力及成骨活性均强于羟基磷灰石陶瓷材料。提示多孔碳酸钙陶瓷支架材料与SD大鼠骨髓基质干细胞源性成骨细胞有良好相容性。     

Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.     

The effect of the microstructure of beta-tricalcium phosphate on the metabolism of subsequently formed bone tissue

The response of bone cells to a newly developed porous beta-tricalcium phosphate composed of rod-shaped particles (RSbeta-TCP), beta-TCP composed of conventional non-rod-shaped particles (Cbeta-TCP), and hydroxyapatite (HA) was analyzed using in vivo implantation and in vitro osteoclastogenesis systems. Implantation of the materials into the rabbit femur showed that RSbeta-TCP and Cbeta-TCP were bioresorbable, but HA was not. Up to 12 weeks after the implantation, bioresorption of RSbeta-TCP and Cbeta-TCP accompanied by the formation of new bone occurred satisfactorily. At 24 weeks post-implantation, most of the RSbeta-TCP had been absorbed, and active osteogenesis was preserved in the region. However, in the specimens implanted with Cbeta-TCP, the amount of not only the implanted Cbeta-TCP but also the newly formed bone tissue decreased, and bone marrow dominated the region. The implanted HA was unbioresorbable throughout the experimental period. When osteoclasts were generated on RSbeta-TCP, Cbeta-TCP, or HA disks, apparent resorption lacunae were formed on the RSbeta-TCP and Cbeta-TCP, but not HA disks. Quantitation of the calcium concentration in the culture media showed an earlier and more constant release of calcium from RSbeta-TCP than Cbeta-TCP. These results showed that the microstructure of beta-TCP affects the activity of bone cells and subsequent bone replacement.     

The induction of bone formation by coral-derived calcium carbonate/hydroxyapatite constructs

The spontaneous induction of bone formation in heterotopic rectus abdominis and orthotopic calvarial sites by coral-derived biomimetic matrices of different chemical compositions was investigated in a long-term study in the non-human primate Papio ursinus. Coral-derived calcium carbonate constructs were converted to hydroxyapatite by hydrothermal exchange. Limited conversion produced hydroxyapatite/calcium carbonate (HA/CC) constructs of 5% and 13% hydroxyapatite. Rods of 20 mm in length and 7 mm in diameter were implanted in heterotopic rectus abdominis sites; discs 25 mm in diameter were implanted in orthotopic calvarial defects of six adult non-human primates P. ursinus. Heterotopic samples also included fully converted hydroxyapatite replicas sintered at 1100 °C. To further enhance spontaneous osteoinductive activity, fully converted hydroxyapatite replicas were coated with the synthetic peptide P15 known to increase the adhesion of fibroblasts to anorganic bovine mineral. Bone induction was assessed at 60, 90 and 365 days by histological examination, alkaline phosphatase and osteocalcin expression, as well as by the expression of BMP-7, GDF-10 and collagen type IV mRNAs. Induction of bone occurred in the concavities of the matrices at all time points. At 365 days, bone marrow was evident in the P15-coated and uncoated implants. Resorption of partially converted calcium carbonate/hydroxyapatite was apparent, as well as remodeling of the newly formed bone. Northern blot analyses of samples from heterotopic specimens showed high levels of expression of BMP-7 and collagen type IV mRNA in all specimen types at 60 days, correlating with the induction of the osteoblastic phenotype in invading fibrovascular cells. Orthotopic specimens showed prominent bone formation across the different implanted constructs. The concavities of the matrices biomimetize the remodeling cycle of the osteonic primate cortico-cancellous bone and promote the ripple-like cascade of the induction of bone formation. This study demonstrates for the first time that partially converted HA/CC constructs also induce spontaneous differentiation of bone, albeit only seen one year post-implantation.     

Osteogenic potential of culture-expanded rat marrow cells as assayed in vivo with porous calcium phosphate ceramic

It has been established that, when whole marrow is introduced into porous calcium phosphate ceramic, bone forms on the walls of the pores. To extend earlier studies, bone marrow cells derived from the femora of inbred rats were introduced into tissue culture and the adherent cells cultivated, mitotically expanded, passaged, harvested, placed in small cubes of porous calcium phosphate ceramics and grafted into subcutaneous sites of syngeneic rats. Marrow-derived, cultured mesenchymal cells introduced into ceramics showed strong osteogenic potential, with bone forming in the pore regions of ceramics as early as 2 wk after implantation. Osteogenesis could be observed after the eighteenth passage. With increasing passage number, the initiation of osteogenesis and the apparent rate of bone formation declined and the course of osteogenesis was delayed. In the future, it may be possible to culture marrow cells as a source for reparative cells for implantation back into autologous in vivo sites.     

Bone formation in calcium-phosphate-coated titanium mesh

The osteogenic activity of porous titanium fiber mesh and calcium phosphate (Ca-P)-coated titanium fiber mesh loaded with cultured syngeneic osteogenic cells was compared in a syngeneic rat ectopic assay model. In 30 syngeneic rats, (Ca-P)-coated and non-coated porous titanium implants were subcutaneously placed either without or loaded with cultured rat bone marrow (RBM) cells. Fluorochrome bone markers were injected at 2, 4, and 6 weeks. The rats were sacrificed, and the implants were retrieved at 2, 4, and 8 weeks post-operatively. Histological analysis demonstrated that none of the (Ca-P)-coated and non-coated meshes alone supported bone formation at any time period. In RBM-loaded implants, bone formation started at 2 weeks. At 4 weeks, bone formation increased. However, at 8 weeks bone formation was absent in the non-coated titanium implants, while it had remained in the (Ca-P)-coated titanium implants. Also, in (Ca-P)-coated implants more bone was formed than in non-coated samples. In general, osteogenesis was characterized by the occurrence of multiple spheres in the porosity of the mesh. The accumulation sequence of the fluorochrome markers showed that the newly formed bone was deposited in a centrifugal manner starting at the center of a pore. Our results show that the combination of Ti-mesh with RBM cells can indeed generate bone formation. Further, our results confirm that a thin Ca-P coating can have a beneficial effect on the bone-generating properties of a scaffold material.     

Osteoconduction at porous hydroxyapatite with various pore configurations

To assess the histological response and the reinforcing effects of bone ingrowth within porous hydroxyapatite (HA) implants depending on pore geometry, four kinds of cylindrical-type with parallel linear pores phi50, 100, 300, 500 microm), one kind of sponge-type with irregular interconnecting pores (phi250 microm) and one cross-type with crossing linear pores (phi100 x 120 microm) of porous HA were prepared. Eighty-four rabbits were divided into six groups, and a 5 x 5 x 7 mm sized porous HA block was inserted through the medial cortical window of the proximal tibia. Histomorphological changes were examined using light and scanning electron microscopy. A biomechanical compression test was performed using material test machines. After implantation, the implants showed different histological changes depending on pore geometry. Active osteoconduction was also found in the phi50 microm sized cylindrical-type porous HA. Evidence of remodeling of new bone and bone marrow formation within porous HA was found in the larger cylindrical-types (phi300, 500 microm), and the sponge- and cross-types. The biomechanical test showed that the ultimate compressive strength increased significantly in the phi300 microm sized cylindrical-type, and in the sponge- and cross-types eight weeks after implantation. Porous HA with cylindrical pores could be a useful graft material due to its strength, osteoconductivity and the ease with which its pore geometry can be controlled.     

Coralline hydroxyapatite granules inferior to morselized allograft around uncemented porous Ti implants: unchanged fixation by addition of concentrated autologous bone marrow aspirate

We compared early fixation of titanium implants grafted with impacted allograft bone or coralline hydroxyapatite (HA) granules (Pro Osteon 200) with and without the addition of concentrated bone marrow cells (BMC). Autologous bone marrow aspirate was centrifuged to increase the BMC concentration. Four nonloaded cylindrical, porous coated titanium implants with a circumferential gap of 2.3 mm were inserted in the proximal humeri of eight dogs. Coralline HA granules +/- BMC were impacted around the two implants on one side, and allograft +/- BMC was impacted around the contra lateral implants. Observation time was 4 weeks. The implants surrounded by allograft bone had a three-fold better fixation than the HA-grafted implants. The concentration of BMC after centrifugation was increased with a factor 2.1. The addition of BMC to either of the bone graft materials had no statistically significant effects on implant fixation. The allografted implants were well osseointegrated, whereas the HA-grafted implants were largely encapsulated in fibrous tissue. The addition of concentrated autologous BMCs to the graft material had no effect on implant fixation. The HA-grafted implants were poorly anchored compared with allografted implants, suggesting that coralline HA granules should be considered a bone graft extender rather than a bone graft substitute.     

Long-term durability of porous hydroxyapatite with low-pressure system to support osteogenesis of mesenchymal stem cells

In this study, we hypothesize that loading more marrow-derived mesenchymal stem cells (MSCs) into porous material by using a low-pressure system during subculture, creating a composite which combines MSCs and a novel mechanical reinforced porous hydroxyapatite, can result in more bone tissue formation in vivo. Within 26 weeks postimplantation, we examined in vivo bone formation of the experimental group with 100 mmHg pressure applied to porous HA blocks loaded with MSCs. For in vivo testing, the 2-week subcultured HA/MSC composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested at 13 and 26 weeks after implantation. SEM showed that the pore surface is covered by osteoblasts as well as collagenous extracellular matrix at 13 weeks. Light microscopy revealed the quantity of bone at 26 weeks was greater than at 13 weeks. These results showed that the novel mechanical reinforced porous HA combined with MSC has more potential for bone formation at 100 mmHg, making this method very efficient for bone reconstruction.     

Viable osteogenic cells are obligatory for tissue-engineered ectopic bone formation in goats

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.     

Transforming growth factor beta released from natural coral implant enhances bone growth at calvarium of mature rat.

Earlier studies have shown that transforming growth factor beta (TGF-beta) has the capability of enhancing bone formation after a single application to an orthotopic site. We investigated whether 1, 5, or 25 microg of recombinant human TGF-beta1 added to porous natural coral (NC) blocks could promote bone ingrowth in a critical size defect (CSD) model in nongrowing rats. A 6-mm CSD in the parietal bone of Wistar rats was filled with NC disks, which were retrieved at 3 and 8 weeks. We prepared undecalcified sections for microscopy and histomorphometry to study bone formation in the implants. The differences in the means of the measured variables were compared with a one-way analysis of variance and Tukey's Student range test, and p values smaller than 0.05 were considered statistically significant. Bone formation was enhanced in all the TGF-beta1-treated implants at 8 weeks in comparison with the controls, but none of the implants showed complete bridging across the defect. The number of macrophages and giant cells was reduced in the TGF-beta1 implants, which showed less resorption and more intact structure than the coral controls. Void areas without any fibrous tissue ingrowth were found only in the TGF-beta1-treated implants, which may partly explain the reduced resorption. The data suggested that TGF-beta1 induced enhanced but limited bone formation in mature rats and prevented resorption of the coral calcium carbonate matrix, possibly by hindering reactive cell formation and fibrous tissue ingrowth.     

Nanoindentation study of interfaces between calcium phosphate and bone in an animal spinal fusion model.

Intertransverse process spinal fusion is a common surgical procedure for the treatment of spinal disorders. In the present study, a porous hydroxyapatite (HA)/beta-tricalcium phosphate (beta-TCP) ceramic was tested as graft material using a rabbit lumbar transverse process (L5-L6) fusion model. The porous ceramic blocks were implanted onto the dorsal decorticated surface of the lumbar transverse processes. The specimens were harvested at the seventh week after implantation. Histomorphological observation revealed that the integration of HA/beta-TCP with the host bone of the transverse process occurred by both cancellous bone formation and cartilage formation. Scanning electron microscopy-wavelength dispersive X-ray spectrometry examinations showed significant differences in calcium, phosphorus, and sulfur contents in the newly formed tissues and the porous HA/TCP implants. Nanoindentations were used to evaluate the intrinsic mechanical properties of the implants and the newly formed tissues. The Young's moduli of the newly formed cartilage, new cancellous bone, and HA/TCP, were 0.66 +/- 0.02 GPa, 2.36 +/- 0.50 GPa, and 10.2 +/- 1.21 GPa, respectively. Nanoindentation results revealed degradation of the porous ceramics and incomplete calcification of the new cancellous bone at the seventh week after implantation. Nanoindentation appeared to be a useful technique for assessing the mechanical status of spinal fusion in animal models.     

Mechanical and biological properties of hydroxyapatite/tricalcium phosphate scaffolds coated with poly(lactic-co-glycolic acid)

Regeneration of bone, cartilage and osteochondral tissues by tissue engineering has attracted intense attention due to its potential advantages over the traditional replacement of tissues with synthetic implants. Nevertheless, there is still a dearth of ideal or suitable scaffolds based on porous biomaterials, and the present study was undertaken to develop and evaluate a useful porous composite scaffold system. Here, hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds (average pore size: 500 μm; porosity: 87%) were prepared by a polyurethane foam replica method, followed by modification with infiltration and coating of poly(lactic-co-glycolic acid) (PLGA). The thermal shock resistance of the composite scaffolds was evaluated by measuring the compressive strength before and after quenching or freezing treatment. The porous structure (in terms of pore size, porosity and pore interconnectivity) of the composite scaffolds was examined. The penetration of the bone marrow stromal stem cells into the scaffolds and the attachment of the cells onto the scaffolds were also investigated. It was shown that the PLGA incorporation in the HA/TCP scaffolds significantly increased the compressive strength up to 660 kPa and the residual compressive strength after the freezing treatment decreased to 160 kPa, which was, however, sufficient for the scaffolds to withstand subsequent cell culture procedures and a freeze–drying process. On the other hand, the PLGA coating on the strut surfaces of the scaffolds was rather thin (<5 μm) and apparently porous, maintaining the high open porosity of the HA/TCP scaffolds, resulting in desirable migration and attachment of the bone marrow stromal stem cells, although a thicker PLGA coating would have imparted a higher compressive strength of the PLGA-coated porous HA/TCP composite scaffolds.     

Biological performance in goats of a porous titanium alloy-biphasic calcium phosphate composite

In this study, porous 3D fiber deposition titanium (3DFT) and 3DFT combined with porous biphasic calcium phosphate ceramic (3DFT+BCP) implants, both bare and 1 week cultured with autologous bone marrow stromal cells (BMSCs), were implanted intramuscularly and orthotopically in 10 goats. To assess the dynamics of bone formation over time, fluorochrome markers were administered at 3, 6 and 9 weeks and the animals were sacrificed at 12 weeks after implantation. New bone in the implants was investigated by histology and histomorphometry of non-decalcified sections. Intramuscularly, no bone formation was found in any of the 3DFT implants, while a very limited amount of bone was observed in 2 BMSC 3DFT implants. 3DFT+BCP and BMSC 3DFT+BCP implants showed ectopic bone formation, in 8 and 10 animals, respectively. The amount of formed bone was significantly higher in BMSC 3DFT+BCP as compared to 3DFT+BCP implants. Implantation on transverse processes resulted in significantly more bone formation in composite structure as compared to titanium alloy alone, both with and without cells. Unlike intramuscularly, the presence of BMSC did not have a significant effect on the amount of new bone either in metallic or in composite structure. Although the 3DFT is inferior to BCP for bone growth, the reinforcement of the brittle BCP with a 3DFT cage did not negatively influence osteogenesis, osteoinduction and osteoconduction as previously shown for the BCP alone. The positive effect of BMSCs was observed ectopically, while it was not significant orthotopically.     

Osteogenicity of biphasic calcium phosphate ceramics and bone autograft in a goat model

The aim of this work was to compare the osteogenicity of calcium phosphate ceramic granules with autologous bone graft in ectopic and orthotopic sites. Biphasic calcium phosphate (BCP) granules composed of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) in a 60/40 ratio were sintered at 1050, 1125 and 1200 degrees C, producing different microporosities. Either BCP ceramic granules or autologous bone chips (n=7) were implanted into paraspinal muscles. Osteoinduction was not observed in either the BCP implants or autologous bone chips after 6 or 12 weeks in the ectopic sites. Hollow and bored polytetrafluoroethylene (PTFE) cylinders were filled with autologous bone, BCP granules or left empty, then implanted into critical-sized defects in femoral epiphyses. The PTFE cylinders left empty contained marrow and blood vessels but not mineralized bone, indicating that this model prevented bone ingrowth (0.56+/-0.43% at 12 weeks). Bone formation was observed in contact with the BCP1050 and BCP1125 granules in the femoral sites after 6 weeks. The amount of bone after 12 weeks was 5.6+/-7.3 and 9.6+/-6.6% for BCP1050 and BCP1125, respectively. Very little bone formation was observed with the BCP1200 implants (1.5+/-1.3% at 12 weeks). In both the ectopic and orthotopic sites, autologous bone chips were drastically resorbed (from 19.4+/-3.7% initially to 1.7+/-1.2% at 12 weeks). This study shows that synthetic bone substitutes may have superior stability and osteogenic properties than autologous bone grafts in critical-sized bone defects.     

Recombinant growth/differentiation factor-5 (GDF-5) stimulates osteogenic differentiation of marrow mesenchymal stem cells in porous hydroxyapatite ceramic

To evaluate the growth/differentiation factor-5 (GDF-5) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), we subcutaneously implanted five different kinds of hydroxyapatite (HA) ceramic implants: HA alone, GDF-5/HA composites (GDF/HA), MSCs/HA composites, the MSCs/HA composites supplemented with GDF-5 (GDF/MSCs/HA), and recombinant bone morphogenetic protein-2 (BMP/MSCs/HA). Neither the HA alone nor the GDF/HA composites exhibited any bone formation at any time after implantation. At 4 weeks, the MSCs/HA composites exhibited a certain amount of bone formation in some pore areas. In contrast, at 2 weeks, the GDF/MSCs/HA composites exhibited histologically obvious de novo bone formation together with active osteoblasts in many pore areas and additional bone formation at 4 weeks. In the de novo formed bone, neither chondrocytes nor endochondral bone was detected. The GDF/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels and the high level of expression was well maintained even at 4 weeks. Compared with GDF/MSCs/HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. The results indicate that GDF-5 synergistically enhances de novo bone formation capability of MSCs/HA composite and suggest that tissue-engineered GDF/MSCs/HA composites could be used as bone graft substitutes.     

Ectopic osteogenesis with biphasic ceramics of hydroxyapatite and tricalcium phosphate in rabbits.

Porous calcium phosphate ceramics consisting of hydroxyapatite (HA) and tricalcium phosphate (TCP) with different HA to TCP ratio were implanted intramuscularly in rabbits for six months in order to carry out a comparative study on osteogenic activity of the ceramics. Bone formation was detected only in HT73 (HA to TCP ratio, 7-3) specimens. Other implants, HT28 (2-8) and HT010 (0-10), could not induce bone. After a six-month period of implantation, HT28 and HT010 implants showed obvious degradation of the implants changing their shape and size macro and microscopically. Microscopically, they showed aggregates of fine particles and appearance of multinucleated cells. However, HT73 implants was less degraded and could maintain their original structure macro and microscopically. This study showed that HT73 ceramics can induce bone in rabbit muscle tissue and it is considered that maintenance of porous structure, that is, degradation rate of the materials may be one of the affecting factors in ceramic-induced osteogenesis.