Early weight bearing of porous HA/TCP (60/40) ceramics in vivo: a longitudinal study in a segmental bone defect model of rabbit

Porous interconnected hydroxyapatite (HA) and HA/tricalcium phosphate (TCP) (60/40) ceramics are promising materials for hard tissue repair. However, the mechanical properties of these materials have not been accurately determined under weight-bearing conditions. In this study, newly developed HA and HA/TCP (60/40) ceramics were used with intramedullary fixation in segmental bone defects of rabbits. Early radiological, histological, densitometric and biomechanical changes were evaluated. The mean radiological grade of healing and bonding to bone was higher in HA/TCP (60/40) ceramics than that of pure HA ceramics but the difference was not statistically significant. The densities of both implanted ceramics improved with time, supported by the histological evaluation of bone matrix ingrowth into ceramic pores, whereas the densities at the bone–ceramic interface decreased gradually. Flexural resonant frequencies and three-point bending strength increased, revealing an increase in mechanical stability during this early critical time interval where implant and/or bone–implant interface failures occur frequently. It can be concluded that both HA and HA/TCP (60/40) ceramics have a limited application in the treatment of load-bearing segmental bone defects but did not fail at the early stages of implantation.     

Calcium phosphates and glass composite coatings on zirconia for enhanced biocompatibility

Calcium phosphates (CaP) and phosphate-based glass (P-glass, xCaO-(0.55-x) Na(2)O-0.45P(2)O(5) composition) composite coatings were obtained on a strong ZrO(2) to improve biocompatibility, the mechanical strength and biological activity. Hydroxyapatite (HA) and P-glass mixed powder slurries were coated on the ZrO(2) substrate, and subsequently heat-treated to obtain CaP- and P-glass composite coatings. The effects of glass composition (x=0.3, 0.4, 0.5 mol), mixing ratio of glass to HA (30%, 40%, 50% wt/wt), and heat treatment temperature (800 degrees C, 900 degrees C, 1000 degrees C) on the coating properties were investigated. After heat treatment, additional calcium phosphates, i.e., dicalcium phosphate (DCP) and tricalcium phosphate (TCP), were crystallized, resulting in the formation of triphasic calcium phosphates (HA-TCP-DCP) surrounded by a glass phase. The relative amounts of the crystalline phases varied with coating variables. The higher heat treatment temperature and glass amount, and the lower CaO content in the glass composition rendered the composite coatings to retain the higher amounts of TCP and DCP while the initial HA decreased. These appearance of additional crystalline phases and reduction of HA amount were attributed to the combined effects, i.e., the melting-crystallization of P-glass and the reaction between glass liquid phase and HA powder during thermal treatment. As a result of the glass phase in the composite coatings, their microstructures became much denser when compared to the pure HA coating. In particular, a completely dense structure was obtained at coating conditions with large amount of glass addition (50 wt%) at the glass composition of lower CaO content (0.3 mol CaO), and the following heat treatment above 800 degrees C for 2h. As a result, the adhesion strengths of the composite coating layers were significantly improved when compared to the pure HA coating. The highest strength of the composite coating was approximately 40 MPa, an improvement of approximately 80% with respect to the pure HA coating. The composite coatings showed much higher dissolution rates than the pure HA coating due to the newly formed crystallines (TCP and DCP) and the remaining glass phase. The osteoblast-like cells grew and spread actively on the composite coating samples. The proliferation numbers and alkaline phosphate (ALP) activities of the cells on the composite coatings were improved by approximately 30-40% when compared to Thermanox control and ZrO(2) substrate, and were comparable to the pure HA coating. These findings suggested that the CaP and P-glass composites are potentially useful for hard tissue coating system, due to their morphological and mechanical integrity, enhanced bioactivity, and favorable responses to the osteoblast-like cells.     

Synthesis,characterization, and remodeling of weight-bearing allograft bone/polyurethane composites in the rabbit

The process of bone healing requires the restoration of both anatomy and physiology, and there is a recognized need for innovative biomaterials that facilitate remodeling throughout this complex process. While porous scaffolds with a high degree of interconnectivity are known to accelerate cellular infiltration and new bone formation, the presence of pores significantly diminishes the initial mechanical properties of the materials, rendering them largely unsuitable for load-bearing applications. In this study, a family of non-porous composites has been fabricated by reactive compression molding of mineralized allograft bone particles (MBPs) with a biodegradable polyurethane (PUR) binder, which is synthesized from a polyester polyol and a lysine-derived polyisocyanate. At volume fractions exceeding the random close-packing limit, the particulated allograft component presented a nearly continuous osteoconductive pathway for cells into the interior of the implant. By varying the molecular weight of the polyol and manipulating the surface chemistry of the MBP via surface demineralization, compressive modulus and strength values of 3–6 GPa and 107–172 MPa were achieved, respectively. When implanted in bilateral femoral condyle plug defects in New Zealand White rabbits, MBP/PUR composites exhibited resorption of the allograft and polymer components, extensive cellular infiltration deep into the interior of the implant, and new bone formation at 6 weeks. While later in vivo timepoints are necessary to determine the ultimate fate of the MBP/PUR composites, these observations suggest that allograft bone/polymer composites have potential for future development as weight-bearing devices for orthopedic applications.     

Impaction grafting with morsellised allograft and tricalcium phosphate-hydroxyapatite: incorporation within ovine metaphyseal bone defects

An ovine model was used to investigate the in vivo properties of impacted tricalcium phosphate-hydroxyapatite (TCP-HA) aggregates, varying in chemical composition (ratio of TCP to HA) and particle size distribution (8 versus 3 particle size ranges). All pellets were impacted to a standard compactive effort. Eight sheep underwent implantation of pellets in 4 metaphyseal defects in both rear limbs. Treatment groups consisted of: (1) allograft (clinical control). (2) 50/50 allograft/80% HA/20% TCP in 8 particle size ranges, (3) 50/50 allograft/80% TCP/20% HA in 8 sizes and (4) 50/50 allograft/80% HA/20% TCP in only 3 sizes of particles. Healing of defects was evaluated at 14 weeks with computed tomography, histology and histomorphometry. The computer tomography (CT) density measured in all defects containing synthetic agents was higher than in defects filled with allograft alone (p<0.01). Defects containing 8 sizes of 80% HA/ 20% TCP granules (group 2) achieved lower histological scores and contained less bone than the clinical control (p<0.05), whereas groups 3 and 4 did not differ from the control. Although all synthetic agents were osteoconductive, our results suggest that increasing the ratio of TCP over HA and limiting the number of particle size ranges to 3 instead of 8 improve the performance of impacted aggregates as graft expanders. Evaluation under loading conditions of morsellised allograft expanded with 80% TCP/20% HA (BoneSave) in 3 particle size ranges is warranted.     

Sodium citrate as an effective dispersant for the synthesis of inorganic–organic composites with a nanodispersed mineral phase

Although extensive efforts have been devoted to the development of polymer–ceramic composites for bone repair, those developed thus far were not able to mimic the nanostructure of bone, partly because of the aggregated, microscale organization of the mineral component. As a consequence, homogenization and intermixing of organic and inorganic components remain a major engineering challenge for the development of functional, biomimetic bone-substituting composites. In the current study, various dispersants were evaluated for their potential to be used as biocompatible dispersants in the synthesis of biomimetic composites with a nanodispersed mineral phase. Based on sedimentation experiments, tribasic sodium citrate was selected as the most effective dispersant for the stabilization of calcium phosphate (CaP) suspensions. Specific adsorption of citrate anions onto CaP nanocrystals was shown to result in a strong increase in the negative surface charge of the CaP particles and consequently increased repulsive interparticle forces that were able to overcome attractive van der Waals forces. Using sodium citrate as dispersant at a CaP/citrate ratio of 4.0, CaP–gelatin nanocomposites were fabricated which displayed a nanostructured mineral phase without occurrence of microscale CaP particles. Consequently, aggregation and sedimentation of CaP mineral phase was reduced considerably.     

Osteoblast adhesion on novel machinable calcium phosphate/lanthanum phosphate composites for orthopedic applications

Lanthanum phosphate (LaPO(4), LP) was combined with either hydroxyapatite (HA) or tricalcium phosphate (TCP) to form novel composites for orthopedic applications. In this study, these composites were prepared by wet chemistry synthesis and subsequent powder mixing. These HA/LP and TCP/LP composites were characterized in terms of phase stability and microstructure evolution during sintering using X-ray diffraction (XRD) and scanning electron microscopy (SEM). Their machinability was evaluated using a direct drilling test. For HA/LP composites, LP reacted with HA during sintering and formed a new phase, Ca(8)La(2)(PO(4))(6)O(2), as a reaction by-product. However, TCP/LP composites showed phase stability and the formation of a weak interface between TCP and LP machinability when sintered at 1100 degrees C, which is crucial for achieving desirable properties. Thus, these novel TCP/LP composites fulfilled the requirements for machinability, a key consideration for manufacturing orthopedic implants. Moreover, the biocompatibility of these novel LP composites was studied, for the first time, in this paper. In vitro cell culture tests demonstrated that the LP and its composites supported osteoblast (bone-forming cell) adhesion similar to natural bioceramics (such as HA and TCP). In conclusion, these novel LP composites should be further studied and developed for more effectively treating bone related diseases or injuries.     

Histological findings of long-term healing of the experimental defects by application of a synthetic biphasic ceramic in rats

Calcium phosphate ceramics are generally biocompatible and can develop interactions with human recipient bone. Therefore, they can be widely used in the field of periodontology and dentistry. The purpose of this investigation was to assess the long-term histological bone healing results of experimentally created critical size parietal bone defects in rats. Twelve Wistar rats were used in this investigation. Two 6-mm wide, symmetrical, and circular critical size defects were created in each parietal bone of the animals. While the right defects filled with granular implant (Ceraform), the symmetrical defects were taken as controls. Eighteen months after implantation, rats were killed and defects including the biomaterial with surrounding bone was taken for histological examination. Serial histological sections were cut across the defects and stained for the histological analysis. Both control and Ceraform implanted regions contained dense collagenous tissue. In the implantation site, multinuclear giant cells were observed around the material. On the other hand, there were no necrosis, tumour, and infection in the implantation region. There was no statistical difference between the control and ceraform implanted groups when the bone formation results were compared (p > 0.05). In conclusion, the results revealed that this material is biocompatible and does enhance the new bone building despite the long-term observation period. Although this biphasic ceramic shows within the limits of the study as a less resorptive and not osteoconductive properties, it can be considered as a biocompatible bone defect filling material having a limited application alternative in dentistry and medicine.     

The influence of genetic factors on the osteoinductive potential of calcium phosphate ceramics in mice

The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, not as high as that of autologous bone grafting. Recently, we reported that CaP ceramics with osteoinductive properties were as efficient in healing an ilium defect of a sheep as autologous bone graft was, which makes this subclass of CaP ceramics a powerful alternative for bone regeneration. Although osteoinduction by CaP ceramics has been shown in several large animal models it is sporadically reported in mice. Because the lack of a robust mouse model has delayed understanding of the mechanism, we screened mice from 11 different inbred mouse strains for their responsiveness to subcutaneous implantation of osteoinductive tricalcium phosphate (TCP). In only two strains (FVB and 129S2) the ceramic induced bone formation, and in particularly, in FVB mice, bone was found in all the tested mice. We also demonstrated that other CaP ceramics induced bone formation at the same magnitude as that observed in other animal models. Furthermore, VEGF did not significantly increase TCP induced bone formation. The mouse model here described can accelerate research of osteoinductive mechanisms triggered by CaP ceramics and potentially the development of therapies for bone regeneration.     

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.     

Phenotypic expression of bone-related genes in osteoblasts grown on calcium phosphate ceramics with different phase compositions

Calcium phosphate ceramics with different hydroxyapatite (HA) and tricalcium phosphate (TCP) ratios have different chemical properties. Does the difference in phase composition affect osteoblast behavior? In this study, osteoblasts were cultured on 4 kinds of calcium phosphate ceramics, i.e. pure (HA), HT1 (HA/TCP, 70/30), HT2 (HA/TCP, 35/65), and pure TCP. Cell proliferation of SaOS-2 cells together with bone-related genes' mRNA expression and protein production in osteoblasts cultured on different calcium phosphate ceramics were detected at different time points. Data suggested that cell proliferation rate on TCP ceramics was lower than that on the other substrates tested. Generally, mRNA expressions for osteonectin and osteocalcin were similar among the four kinds of ceramics in most circumstances, whereas at six days, alkaline phosphatase mRNA expression was higher on HA and HT1 surfaces than on the other two materials. Collagen I mRNA expression was also affected by the phase composition of substrates. Osteocalcin and bone sialoprotein production in SaOS-2 cells was very similar no matter which ceramic surface the cells were grown upon. This study revealed that calcium phosphate ceramics substrate could support osteoblast growth and bone-related gene expression and its gene expression pattern explained the basis of the biocompatibility and bioactivity for calcium phosphate ceramics.     

Silicon substitution in the calcium phosphate bioceramics

Silicon (Si) substitution in the crystal structures of calcium phosphate (CaP) ceramics such as hydroxyapatite (HA) and tricalcium phosphate (TCP) generates materials with superior biological performance to stoichiometric counterparts. Si, an essential trace element required for healthy bone and connective tissues, influences the biological activity of CaP materials by modifying material properties and by direct effects on the physiological processes in skeletal tissue. The synthesis of Si substituted HA (Si-HA), Si substituted alpha-TCP (Si-alpha-TCP), and multiphase systems are reviewed. The biological performance of these Si substituted CaP materials in comparison to stoichiometric counterparts is discussed. Si substitution promotes biological activity by the transformation of the material surface to a biologically equivalent apatite by increasing the solubility of the material, by generating a more electronegative surface and by creating a finer microstructure. When Si is included in the TCP structure, recrystallization to a carbonated HA is mediated by serum proteins and osteoblast-like cells. Release of Si complexes to the extracellular media and the presence of Si at the material surface may induce additional dose-dependent stimulatory effects on cells of the bone and cartilage tissue systems.     

On the feasibility of phosphate glass and hydroxyapatite engineered coating on titanium

In this report, bioactive calcium phosphate (CaP) coatings were produced on titanium (Ti) by using phosphate-based glass (P-glass) and hydroxyapatite (HA), and their feasibility for hard tissue applications was addressed in vitro. P-glass and HA composite slurries were coated on Ti under mild heat treatment conditions to form a porous thick layer, and then the micropores were filled in with an HA sol-gel precursor to produce a dense layer. The resultant coating product was composed of HA and calcium phosphate glass ceramics, such as tricalcium phosphate (TCP) and calcium pyrophosphate (CPP). The coating layer had a thickness of approximately 30-40 microm and adhered to the Ti substrate tightly. The adhesion strength of the coating layer on Ti was as high as 30-33 MPa. The human osteoblastic cells cultured on the coatings produced by the combined method attached and proliferated favorably. Moreover, the cells on the coatings expressed significantly higher alkaline phosphatase activity than those on pure Ti, suggesting the stimulation of the osteoblastic activity on the coatings. On the basis of these observations, the engineered CaP coating layer is considered to be potentially applicable as a hard tissue-coating system on Ti-based implants.     

Osteoconductive properties of poly(96L/4D-lactide)/beta-tricalcium phosphate in long term animal model

The objective of this study was to determine the effect of calcium phosphate mineral content on the bone in-growth at the expense of composite of co-polylactide polymer charged with 2 different ratios of β-TCP granules (10 and 24 w-% of β-TCP). The evaluation was realized in a long term rabbit bone model. After 24, 48 and 76 weeks, the implants were examined by micro CT, scanning electron microscopy (SEM) using backscattered electron (BSE) and light microscopy (polarized and blue light microscopy). No foreign body reaction was detected during the 76 weeks follow-up in any of the test samples. Polymer hydrolysis began at approximately 24 weeks, by 76 weeks, the pure polymer implant had begun to release P(96L/4D)LA particles and show signs of peripheral localized bone resorption. A decrease in the amount of CaP was noticed between 24 and 76 weeks in both 10?wt-% and 24?wt-% β-TCP/P(96L/4D)LA composites. The study showed that the highest bone in-growth was with 24 wt-% β-TCP/P(96L/4D)LA composite. Bone in-growth and mineralization were evident for the composites associated with specific peripheral bone architecture. Fluorescent labelling demonstrated high bone in-growth and remodeling at the interface, while for pure co-polymer no bone remodeling or bone activity was maintained after 48 weeks. The study demonstrated the positive effect of calcium phosphate content into P(96L/4D)LA. This kind of composite is a suitable resorbable osteoconductive matrix, which provides long term stability required for ligament fixation device.     

Bone response and mechanical strength of rabbit femoral defects filled with injectable CaP cements containing TGF-beta 1 loaded gelatin microparticles

This study focused at the potential of transforming growth factor beta 1 (TGF-beta 1) loaded gelatin microparticles to enhance the bone response and mechanical strength of rabbit femoral defects filled with injectable calcium phosphate (CaP)/gelatin microparticle composites. Therefore, TGF-beta1 loaded composites and non-loaded controls were injected in circular defects as created in the femoral condyles of rabbits and were left in place for 4, 8 and 12 weeks. The specimens were evaluated mechanically (push-out test), and morphologically (scanning electron microscopy (SEM), histology, and histomorphometry). The results showed a gradual increase in mechanical strength with increasing implantation periods. Histological and histomorphometrical evaluation showed similar results for both composite formulations regarding histological aspect, new bone formation and bone/implant contact. However, TGF-beta1 loading of the composites demonstrated a significant effect on composite degradation after twelve weeks of implantation. The results of this study showed that CaP/gelatin composites show excellent osteogenic properties and a rapid increase in mechanical strength. The addition of TGF-beta1 significantly enhances the bone remodeling process.     

HA/PDLLA复合材料的体内成骨过程研究

目的研究HA／PDLLA复合材料植入体内后与细胞、组织的相互作用，探讨HA／PDLLA复合材料在体内的成骨过程，为其临床应用及设计具有生物功能的人工骨替换材料和骨组织工程支架材料提供依据。方法采用液相吸附法制备了HA／PDLLA复合材料，以纯PDLLA和空白组进行对照，进行体内植入实验，通过组织学观察和四环素标记考察其成骨过程。结果HA／PDLLA复合材料植入机体后，体内的无菌性炎症轻微，新骨形成速率高于PDLLA材料。HA微粒的存在，加强了复合材料的机械强度，使之可以避免过早的丧失力学强度。第24w时，材料被组织分隔包裹，新生骨组织长入材料，骨愈合情况良好。结论HA／PDLLA复合材料具有良好的生物相容性、生物降解性能、生物学活性和骨传导性能。     

Production and evaluation of biodegradable composites based on PHB-PHV copolymer

In recent years, emphasis in biomaterials engineering has moved from materials that remain stable in the biological environment to materials that can degrade in the human body. Biodegradable materials are designed to degrade gradually and be replaced eventually by newly formed tissue in the body. In this investigation, two particulate bioactive ceramics, i.e., hydroxyapatite (HA) and tricalcium phosphate (TCP), were incorporated into polyhydroxybutyrate-polyhydroxyvalerate (PHB-PHV), which is a biodegradable copolymer. to produce new biomaterials for potential medical applications. All raw materials were commercially available and they were characterised prior to composite production. HA/PHB-PHV and TCP/PHB-PHV composites containiing up to 30 vol% of the bioceramics were produced through an established procedure. Compounded and compression moulded materials were evaluated using various techniques including thermogravimatric analysis, scanning electron microscopy, differential scanning calorimetry and dynamic mechanical analysis. The results showed that intended compositions of composites had been achieved and bioceramic particles were well distributed in the polymer. The degradation temperature of PHB-PHV was significantly reduced by the incorporation of bioceramics, while the melting temperature was slightly affected by the addition of bioceramics. The crystallinity of PHB-PHV was also varied with the presence of HA or TCP particles. The storage modulus and loss modulus of the composites increased with the increase in HA or TCP content. Composites containing the highest percentage of bioceramics exhibited the highest stiffness. Preliminary in vitro study indicated enhanced ability of the composites to induce the formation of bone-like apatite on their surfaces.     

A new in vivo screening model for posterior spinal bone formation: comparison of ten calcium phosphate ceramic material treatments

This study presents a new screening model for evaluating the influence of multiple conditions on the initial process of bone formation in the posterior lumbar spine of a large animal. This model uses cages designed for placement on the decorticated transverse process of the goat lumbar spine. Five conduction channels per cage, each be defined by a different material treatment, are open to both the underlying bone and overlying soft tissue. The model was validated in ten adult Dutch milk goats, with each animal implanted with two cages containing a total of ten calcium phosphate material treatments according to a randomized complete block design. The ten calcium phosphate ceramic materials were created through a combination of material chemistry (BCP, TCP, HA), sintering temperature (low, medium, high), calcination and surface roughness treatments. To monitor the bone formation over time, fluorochrome markers were administered at 3, 5 and 7 weeks and the animals were sacrificed at 9 weeks after implantation. Bone formation in the conduction channels was investigated by histology and histomorphometry of non-decalcified sections using traditional light and epifluorescent microscopy. According to both observed and measured bone formation parameters, materials were ranked in order of increasing magnitude as follows: low sintering temperature BCP (rough and smooth) approximately medium sintering temperature BCP approximately = TCP > calcined low sintering temperature HA > non-calcined low sintering temperature HA > high sintering temperature BCP (rough and smooth) > high sintering temperature HA (calcined and non-calcined). These results agree closely with those obtained in previous studies of osteoconduction and bioactivity of ceramics thereby validating the screening model presented in this study.     

Evaluation of the biocompatibility of calcium phosphate cement/PLGA microparticle composites

In this study, the biocompatibility of a calcium phosphate (CaP) cement incorporating poly (D,L-lactic-co-glycolic acid) (PLGA) microparticles was evaluated in a subcutaneous implantation model in rats. Short-term biocompatibility was assessed using pure CaP discs and CaP discs incorporating PLGA microparticles (20% w/w) with and without preincubation in water. Long-term biocompatibility was assessed using CaP discs incorporating varying amounts (5, 10, or 20% w/w) and diameter sizes (small, 0-50 mum; medium, 51-100 mum, or large, 101-200 mum) of PLGA microparticles. The short-term biocompatibility results showed a mild tissue response for all implant formulations, irrespective of disc preincubation, during the early implantation periods up to 12 days. Quantitative histological evaluation revealed that the different implant formulations induced the formation of similar fibrous tissue capsules and interfaces. The results concerning long-term biocompatibility showed that all implants were surrounded by a thin connective tissue capsule (<10 layers of fibroblasts). Additionally, no significant differences in capsule and interface scores were observed between the different implant formulations. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth throughout the implants, indicating PLGA degradation and interconnectivity of the pores. The results demonstrate that CaP/PLGA composites evoke a minimal inflammatory response. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth after 12- and 24-weeks indicating PLGA degradation and interconnectivity of the pores. Therefore, CaP/PLGA composites can be regarded as biocompatible biomaterials with potential for bone tissue engineering and advantageous possibilities of the microparticles regarding material porosity.     

A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation

For the repair of bone defects, a tissue engineering approach would be to combine cells capable of osteogenic (i.e. bone-forming) activity with an appropriate scaffolding material to stimulate bone regeneration and repair. Human mesenchymal stem cells (hMSCs), when combined with hydroxyapatite/beta-tricalcium phosphate (HA/TCP) ceramic scaffolds of the composition 60% HA/40% TCP (in weight %), have been shown to induce bone formation in large, long bone defects. However, full repair or function of the long bone could be limited due to the poor remodeling of the HA/TCP material. We conducted a study designed to determine the optimum ratio of HA to TCP that promoted hMSC induced bone formation yet be fully degradable. In a mouse ectopic model, by altering the composition of HA/TCP to 20% HA/80% TCP, hMSC bone induction occurred at the fastest rate in vivo over the other formulations of the more stable 100% HA, HA/TCP (76/24, 63/37, 56/44), and the fully degradable, 100% TCP. In vitro studies also demonstrated that 20/80 HA/TCP stimulated the osteogenic differentiation of hMSCs as determined by the expression of osteocalcin.     

Establishment of efficacy and safety assessment of human adipose tissue-derived mesenchymal stem cells (hATMSCs) in a nude rat femoral segmental defect model

Human adipose tissue-derived mesenchymal stem cell (hATMSC) have emerged as a potentially powerful tool for bone repair, but an appropriate evaluation system has not been established. The purpose of this study was to establish a preclinical assessment system to evaluate the efficacy and safety of cell therapies in a nude rat bone defect model. Segmental defects (5 mm) were created in the femoral diaphyses and transplanted with cell media (control), hydroxyapatite/tricalcium phosphate scaffolds (HA/TCP, Group I), hATMSCs (Group II), or three cell-loading density of hATMSC-loaded HA/TCP (Group III-V). Healing response was evaluated by serial radiography, micro-computed tomography and histology at 16 weeks. To address safety-concerns, we conducted a GLP-compliant toxicity study. Scanning electron microscopy studies showed that hATMSCs filled the pores/surfaces of scaffolds in a cell-loading density-dependent manner. We detected significant increases in bone formation in the hATMSC-loaded HA/TCP groups compared with other groups. The amount of new bone formation increased with increases in loaded cell number. In a toxicity study, no significant hATMSC-related changes were found in body weights, clinical signs, hematological/biochemical values, organ weights, or histopathological findings. In conclusion, hATMSCs loaded on HA/TCP enhance the repair of bone defects and was found to be safe under our preclinical efficacy/safety hybrid assessment system.