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.     

Bone response to radio frequency sputtered calcium phosphate implants and titanium implants in vivo.

The objective of this study was to evaluate the effect of radio frequency sputtered calcium phosphate (CaP) coatings of titanium (Ti) implants on the bond strength at the bone-implant interface and percent bone contact length. Cylindrical coated or noncoated implants (4.0-mm diameter by 8-mm long) were implanted for 3 and 12 weeks. At 3 weeks after implant placement, the ultimate interfacial strengths for as-deposited CaP-coated and heat-treated CaP-coated implants were 2.29 +/- 0.14 MPa and 1.28 +/- 0.04 MPa, respectively. These ultimate interfacial strength values at 3 weeks were statistically greater than the mean ultimate interfacial strength for control Ti implants (0.67 +/- 0.13 MPa). At 12 weeks after implant placement, no statistical differences in the mean ultimate interfacial strengths were observed between the as-deposited CaP-coated, heat-treated CaP-coated, and control Ti implants. Histomorphometric evaluation indicated greater percent bone contact lengths for the as-deposited CaP-coated implants compared with the heat-treated CaP-coated and control Ti implants 3 and 12 weeks after implant placement.     

In vitro responses to electrosprayed alkaline phosphatase/calcium phosphate composite coatings

Surface modification of titanium implants to improve their fixation in bone tissue is of great interest. We present a novel approach to enhance implant performance by applying important principles of bone mineralization to biomedical coatings. As an attempt to mimic the biphasic biomineralization process, both the enzyme alkaline phosphatase (ALP) and calcium phosphate (CaP) were immobilized onto Ti discs, thereby triggering enzymatically and physicochemically controlled biomineralization pathways. ALP, CaP and ALP–CaP composite coatings with preserved functionality of ALP were successfully deposited using electrospray deposition. In vitro soaking studies in cell culture medium revealed that crystal growth initially proceeded at a faster rate on CaP-coated Ti than on ALP-containing coatings, but mineral deposition onto ALP-coated Ti caught up with the calcification behaviour of CaP coatings upon long-term soaking. Cell culture experiments with osteoblast-like cells, however, demonstrated the opposite effect in mineral deposition on the electrosprayed CaP and ALP coatings. The ALP–CaP composite coatings showed delayed proliferation as well as accelerated mineralization in comparison to cells cultured on the CaP-coated and uncoated Ti. In conclusion, these in vitro results showed that the osteogenic potential of Ti can be stimulated by ALP-containing coatings.     

Ectopic bone formation in rats: the importance of the carrier

Much research has been done to develop the ideal bone graft substitute (BGS). One approach to develop this ideal BGS is the use of growth factors, but for this approach osteoprogenitor cells are needed at the site of reconstruction. An alternative is a cell-based approach, where enough cells are provided to form bone in a carrier material. In previous studies of our group, titanium (Ti) carriers have been used, because of the excellent mechanical properties and the bone-compatibility of this material. On the other hand, calcium phosphate (CaP) ceramics are known for their excellent osteoconductivity. The aim of this study is to investigate the influence of the carrier in a cell-based bone regeneration approach, whereby we hypothesize that CaP-ceramic implants will induce more bone formation than Ti-fiber implants, in the same animal model as our previous experiment. Ti-fiber mesh implants and ceramic implants were seeded with rat bone marrow cells (RBM) and implanted subcutaneously. Histological analysis after one, three and six weeks showed differences in the way of bone formation in the two groups: bone appeared to grow from the center to the periphery of the implant in the titanium group, while bone formation in the ceramic group occurred through the whole implant. Histomorphometrical analysis after one week showed very limited bone formation for both the titanium and ceramic group. At three weeks, the amount of bone formation was increased till about 10% for the titanium group and 18% for the ceramic group. No significant difference between the two groups could be observed. In the six week group, the bone formation was 6% (Ti) and 23% (CaP), respectively (P < 0.001). Further, bone formation started earlier in the CaP-ceramic scaffolds than in the Ti scaffolds. Our hypothesis could be confirmed: ceramic implants induce more bone formation than titanium implants.     

Histological characterization of the early stages of bone morphogenetic protein-induced osteogenesis

On the basis of currently available knowledge, we hypothesize that the initial bone formation, as induced by bone morphogenetic protein (BMP), is influenced by the chemical composition and three-dimensional spatial configuration of the used carrier material. Therefore, in the current study, the osteoinductive properties of porous titanium (Ti) fiber mesh with a calcium phosphate (Ca-P) coating (Ti-CaP), insoluble bone matrix (IBM), fibrous glass membrane (FGM), and porous particles of hydroxy apatite (PPHAP) loaded with rhBMP-2 were compared in a rat ectopic assay model at short implantation periods. Twelve Ti-CaP, 12 IBM, 12 FGM, and 12 PPHAP implants, loaded with rhBMP-2, were subcutaneously placed in 16 Wistar King rats. The rats were sacrificed at 3, 5, 7, and 9 days post-operative, and the implants were retrieved. Histological analysis demonstrated that IBM and Ti-CaP had induced ectopic cartilage and bone formation by 5 and 7 days, respectively. However, in PPHAP, bone formation and cartilage formation were seen together at 7 days. At 9 days, in Ti-CaP, IBM, and PPHAP, cartilage was seen together with trabecular bone. At 9 days, in FGM, only cartilage was observed. Quantitative rating of the tissue response, using a scoring system, demonstrated that the observed differences were statistically significant (Wilcoxon rank sum test, p < 0.05). We conclude that IBM, CaP-coated Ti mesh, FGM, and PPHAP provided with rhBMP-2 can indeed induce ectopic bone formation with a cartilaginous phase in a rat model at short implantation periods. Considering the different chemical composition and three-dimensional spatial configuration of the carrier materials used, these findings even suggest that endochondral ossification is present in rhBMP-2-induced osteogenesis, even though the amount of cartilage may differ.     

Long-term evaluation of porous poly(epsilon-caprolactone-co-L-lactide) as a bone-filling material

Porous poly(epsilon-caprolactone-co-L-lactide) (P(CL-co-LA, wt % ca. 5/95) sponges were prepared, coated biomimetically with CaP/apatite, and implanted with noncoated control sponges into rat femur cortical defects and dorsal subcutaneous space. The implants were inspected histologically at 2, 4, and 33 weeks after the operation. All implants were filled with fibrovascular tissue within 4 weeks. The femur implants were partially ossified with compact bone, which in the CaP-coated sponges was less mature and more fragmented. Approximately equal amounts of bone were observed in both types of implants. The polymer induced a mild inflammatory reaction with foreign body giant cells but no accumulation of fluid. Degradation of the polymer was slow; most of it was found intact at 33 weeks in histological samples. Nondegraded polymer seems to prevent complete ossification. Cultured osteoblasts proliferated well on apatite-coated material, whereas only a few cells were seen on noncoated material. Thus CaP/apatite coating helped the attachment of osteoblasts in cell cultures but did not offer any advantage in bone formation over noncoated material in vivo. We conclude that a shorter degradation time of P(CL-co-LA) is needed to create an optimal implant. Furthermore, in vivo experiments seem to be necessary for the estimation of osteopromotive properties of a biomaterial.     

Drug release and bone growth studies of antimicrobial peptide-loaded calcium phosphate coating on titanium

Preventing infection is one of the major challenges in total hip and joint arthroplasty. The main concerns of local drug delivery as a solution have been the evolution of antibiotic-resistant bacteria and the potential inhibition of osseointegration caused by the delivery systems. This work investigated the in vitro drug release, antimicrobial performance, and cytotoxicity, as well as the in vivo bone growth of an antimicrobial peptide loaded into calcium phosphate coated Ti implants in a rabbit model. Two potent AMP candidates (HHC36: KRWWKWWRR, Tet213: KRWWKWWRRC) were first investigated through an in vitro cytotoxicity assay. MTT absorbance values revealed that HHC36 showed much lower cytotoxicity (minimal cytotoxic concentration 200 μg/mL) than Tet 213 (50 μg/mL). The AMP HHC36 loaded onto CaP (34.7 ± 4.2 μg/cm(2)) had a burst release during the first few hours followed by a slow and steady release for 7 days as measured spectrophotometrically. The CaP-AMP coatings were antimicrobial against Staphylococcus aureus and Pseudomonas aeruginosa strains in colony-forming units (CFU) in vitro assays. No cytotoxicity was observed on CaP-AMP samples against MG-63 osteoblast-like cells after 5 days in vitro. In a trabecular bone growth in vivo study using cylindrical implants, loading of AMP HHC36 did not impair bone growth onto the implants. Significant bone on-growth was observed on CaP-coated Ti with or without HHC36 loading, as compared with Ti alone. The current AMP-CaP coating thus offers in vivo osteoconductivity to orthopedic implants. It also offers in vitro antimicrobial property, with its in vivo performance to be confirmed in future animal infection models.     

Initial interaction of U2OS cells with noncoated and calcium phosphate coated titanium substrates

From previous studies, we know that calcium phosphate (CaP) coated implants stimulate bone formation compared to uncoated implants. Nevertheless, the mechanism by which substrate surface characteristics affect cell function is unclear. In this study, we examined the initial interaction (30 min to 24 h) of U2OS cells with titanium substrates with or without a CaP coating. The effect of substrate roughness was also studied. When cell attachment was studied, we found that cells attached more readily to rough than to smooth surfaces. Also, more cells attached to the uncoated than to the CaP coated surface. After 24 h, cell numbers were similar for all substrate surfaces. Further, cells spread to a larger area on noncoated titanium than on the CaP coated substrates. At 24 h, the sequence of cell size was smooth titanium > rough titanium > CaP coated titanium. Shape measurements showed differences in cell shape between the cells on the different materials only at 7 h, not at different culture times. Cells expressed alpha2, alpha3, alpha5, alpha6, alphav, and beta1 subunits. Expression of alpha1, alpha4, alphavbeta3, beta3, beta4, and beta7 was extremely low or was not found.The beta1 integrin expression was higher on the coated than on the noncoated titanium at 3 h, but not on the other studied times. Expression of alpha2, alpha5, alpha6, and alphav expression was found to be upregulated at 24 h compared to earlier culture times on coated titanium, but not on uncoated titanium substrates. From this we conclude that the surface characteristics of a material (roughness and composition) can affect the initial interaction of cells with the material.     

Biological performance of uncoated and octacalcium phosphate-coated Ti6Al4V

The in vivo behavior of a porous Ti6Al4V material that was produced by a positive replica technique, with and without an octacalcium phosphate (OCP) coating, has been studied both in the back muscle and femur of goats. Macro- and microporous biphasic calcium phosphate (BCP) ceramic, known to be both osteoconductive and able to induce ectopic bone formation, was used for comparison purpose. The three groups of materials (Ti6Al4V, OCP Ti6Al4V and BCP) were implanted transcortically and intramuscularly for 6 and 12 weeks in 10 adult Dutch milk goats in order to study their osteointegration and osteoinductive potential. In femoral defects, both OCP Ti6Al4V and BCP were performing better than the uncoated Ti6Al4V, at both time points. BCP showed a higher bone amount than OCP Ti6Al4V after 6 weeks of implantation, while after 12 weeks, this difference was no longer significant. Ectopic bone formation was found in both OCP Ti6Al4V and BCP implants after 6 and 12 weeks. The quantity of ectopically formed bone was limited as was the amount of animals in which the bone was observed. Ectopic bone formation was not found in uncoated titanium alloy implants, suggesting that the presence of calcium phosphate (CaP) is important for bone induction. This study showed that CaPs in the form of coating on metal implants or in the form of bulk ceramic have a significantly positive effect on the bone healing process.     

Modulation of integrin expression on rat bone marrow cells by substrates with different surface characteristics

Biomaterials have been shown to be able to influence the growth and differentiation of osteogenic cells cultured on the surface. Although the precise mechanisms by which the materials influence osteogenic cells are unclear, it is possible that the materials manipulate the expression of integrins by the cells. We therefore studied the expression of a number of integrins by rat bone marrow (RBM) cells, after culture on culture polystyrene, on machined and grit-blasted titanium, and on calcium phosphate-coated titanium. Integrin expression was studied by FACS analysis. We found a large variation in the expression of integrins by cells in replicate experiments. After culture on polystyrene for 7 days, cells expressed alpha1, alpha2, alpha3, alpha5, alpha6, beta1, and beta3, although some of the subunits were expressed only occasionally. The cells did not express the alpha4 subunit. After culture of RBM cells for 8 days on coated and noncoated titanium substrates, cells always expressed alpha3, alpha5, alpha6, and beta1. The alpha1 and beta3 subunits were only expressed in some of the experiments. Frequently, the expression of alpha5, alpha6, and beta1 was higher on the coated than on the noncoated titanium substrates. Based on our results, we conclude that the studied materials are capable of influencing the expression of integrins by RBM cells cultured on relevant implant materials.     

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.     

Analysis of the osteoinductive capacity and angiogenicity of an in vitro generated extracellular matrix

In this study, the osteoinductive potential of an in vitro generated extracellular matrix (ECM) deposited by marrow stromal cells seeded onto titanium fiber mesh scaffolds and cultured in a flow perfusion bioreactor was investigated. Culture periods of 8, 12, and 16 days were selected to allow for different amounts of ECM deposition by the cells as well as ECM with varying degrees of maturity (Ti/ECM/d8, Ti/ECM/d12, and Ti/ECM/d16, respectively). These ECM-containing constructs were implanted intramuscularly in a rat animal model. After 56 days, histologic analysis of retrieved constructs revealed no bone formation in any of the implants. Surrounding many of the implants was a fibrous capsule, which was often interspersed with fat cells. Within the pore spaces, the predominant tissue response was the presence of blood vessels and young fibroblasts or fat cells. The number of blood vessels on a per area basis calculated from a histomorphometric analysis increased as a function of the amount of ECM within the implanted constructs, with a significant difference between Ti/ECM/d16 and plain Ti constructs. These results indicate that although an in vitro generated ECM alone may not induce bone formation at an ectopic site, its use may enhance the vascularization of implanted constructs.     

Effect of calcium phosphate coating crystallinity and implant surface roughness on differentiation of rat bone marrow cells

In this study, we examined the effect of calcium phosphate (Ca-P) coating crystallinity and of surface roughness on growth and differentiation of osteogenic cells. Grit-blasted titanium substrates were provided with Ca-P coatings of different crystallinities. Rat bone marrow (RBM) cells were cultured on these substrates and on noncoated rough and smooth titanium substrates. After specific culture times, expression of osteogenic markers by the cells was studied. Cells cultured on crystalline coatings and on titanium substrates proliferate, express alkaline phosphatase, osteocalcin (OC), and show mineralization of the extracellular matrix. Rough titanium substrates only express low OC levels. Significantly higher OC levels were expressed on smooth titanium, and even higher levels on the crystalline Ca-P coating. No difference was found in calcification between smooth and rough titanium. The crystalline coating showed more calcification than the titanium substrates. When substrates without cells were incubated in medium, precipitation of calcium was found. On the titanium substrates, this precipitate disappeared after prolonged incubation. The precipitate on the crystalline coating was stable and increased with longer incubation times. On the amorphous coatings, no proliferation and differentiation of RBM cells were found. After longer culture periods, substrates showed extensive dissolution. Cells on the amorphous coatings did express high levels of prostaglandin E2. In contrast, prostaglandin E2 expression was low for the other substrates. We conclude that crystalline Ca-P coatings stimulate differentiation of RBM cells, to a higher extent than titanium substrates. Surface roughness only has a limited effect on phenotype expression of the cells. In contrast, thin amorphous coatings show negative effects on the growth and differentiation of cultured RBM cells.     

Osteointegration of femoral stem prostheses with a bilayered calcium phosphate coating

Our purpose was to evaluate the osteointegration of bilayered calcium phosphate (CaP)-coated femoral hip stems in a canine model. A first layer of hydroxyapatite (HA) 20 microm thick and a superficial layer of Biphasic Calcium Phosphate (BCP) 30 microm thick were plasma-sprayed on to the proximal region of sandblasted Ti6Al4V prostheses. Bilayered CaP-coated and non-coated canine femoral stems were implanted bilaterally under general anesthesia in 6 adult female Beagle dogs. After 6 and 12 months, a significant degradation of the bilayered coating occurred with a remainder of 33.1+/-12.4 and 23.6+/-9.2 microm in thickness, respectively. Lamellar bone apposition was observed on bilayered coated implants while fibrous tissue encapsulation was observed on non-coated femoral stems. The bone-implant contacts (BIC) were 91+/-3% and 81+/-8% for coated and 7+/-8% and 8+/-12% for non-coated implants, at 6 and 12 months, respectively. Our study supports the concept of a direct relationship between the biodegradation of CaP coating and the enhanced osteointegration of titanium prostheses. A bilayered CaP coating might therefore enhance bone apposition in the early stages because of the superior bioactivity of the BCP layer while the more stable HA layer might sustain bone bonding over long periods.     

Designing optimal calcium phosphate scaffold-cell combinations using an integrative model-based approach

Bone formation is a very complex physiological process, involving the participation of many different cell types and regulated by countless biochemical, physical and mechanical factors, including naturally occurring or synthetic biomaterials. For the latter, calcium phosphate (CaP)-based scaffolds have proven to stimulate bone formation, but at present still result in a wide range of in vivo outcomes, which is partly related to the suboptimal use and combination with osteogenic cells. To optimize CaP scaffold selection and make their use in combination with cells more clinically relevant, this study uses an integrative approach in which mathematical modeling is combined with experimental research. This paper describes the development and implementation of an experimentally informed bioregulatory model of the effect of calcium ions released from CaP-based biomaterials on the activity of osteogenic cells and mesenchymal stem cell driven ectopic bone formation.The amount of bone formation predicted by the mathematical model corresponds to the amount measured experimentally under similar conditions. Moreover, the model is also able to qualitatively predict the experimentally observed impaired bone formation under conditions such as insufficient cell seeding and scaffold decalcification. A strategy was designed in silico to overcome the negative influence of a low initial cell density on the bone formation process. Finally, the model was applied to design optimal combinations of calcium-based biomaterials and cell culture conditions with the aim of maximizing the amount of bone formation. This work illustrates the potential of mathematical models as research tools to design more efficient and cell-customized CaP scaffolds for bone tissue engineering applications.     

Use of injectable calcium-phosphate cement for the fixation of titanium implants: an experimental study in goats

This in vivo study evaluated the fixation of two types of titanium implants with the use of an injectable calcium-phosphate (CaP) cement. The cement was either used to create a cement mantle (Type A implant) or as an additive to press-fit placed titanium plasma sprayed implants (Type B implant). The implants were placed in trabecular bone of the medial femoral condyle of goats and left in place for 2 and 10 weeks. Mechanical evaluation of the implant fixation was done by torque testing. This showed that for the Type A implants the calcium-phosphate cement's performance was significantly inferior (P < 0.05) to that of polymethylmethacrylate cement fixation. For the two-week Type B implants a significant increase (P < 0.05) in failure load was found for calcium-phosphate cemented implants compared with just press-fitted Type B implants. Histological evaluation revealed that for Type A implants, failure during torque testing occurred at the implant-cement interface. In contrast, for Type B implants, failure occurred in the bone-implant interface for press-fit-placed devices and in the cement layer for CaP-cemented devices. Further, the CaP cement was found to be overgrown with new formed bone already after 2 weeks of implantation. The cement showed resorption due to regular bone remodeling. On the basis of these observations, it was concluded that the use of injectable CaP cement might facilitate earlier loading of press-fit inserted titanium implants. Nevertheless, the results have to be confirmed in dynamical mechanical as well as loaded in vivo studies.     

Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering

High strength porous scaffolds and mesenchymal stem cells are required for bone tissue engineering applications. Porous titanium scaffolds (TiS) with a regular array of interconnected pores of 1000 microm in diameter and a porosity of 50% were produced using a rapid prototyping technique. A calcium phosphate (CaP) coating was applied to these titanium (Ti) scaffolds with an electrodeposition method. Raman spectroscopy and energy dispersive X-ray analysis showed that the coating consisted of carbonated hydroxyapatite. Cross-sectioned observations by scanning electron microscopy indicated that the coating evenly covered the entire structure with a thickness of approximately 25 microm. The bonding strength of the coating to the substrate was evaluated to be around 25 MPa. Rat bone marrow cells (RBMC) were seeded and cultured on the Ti scaffolds with or without coating. The Alamar Blue assay provided a low initial cell attachment (40%) and cell numbers were similar on both the uncoated and coated Ti scaffolds after 3 days. The Ti scaffolds were subsequently implanted subcutaneously for 4 weeks in syngenic rats. Histology revealed the presence of a mineralized collagen tissue in contact with the implants, but no bone formation. This study demonstrated that porous Ti scaffolds with high strength and defined geometry may be evenly coated with CaP layers and cultured mesenchymal stem cells for bone tissue engineering.     

Titania and titania-silica coatings for titanium: comparison of ectopic bone formation within cell-seeded scaffolds

The aim of this study was to compare titania (TiO(2))-coated, titania-silica (TiSi)-coated, and uncoated (cpTi) titanium fiber meshes as scaffolds for bone engineering. The scaffolds were loaded with bone marrow stromal cells and implanted subcutaneously in rats. Ectopic bone formation after 1, 4, and 12 weeks of implantation was evaluated using histology and histomorphometry. After 1 week of implantation, multiple patches of unorganized mineralizing tissue were seen in all implants. The amount of this bone-like tissue clearly increased from 1 to 4 weeks. Bone apposition occurred in direct contact with coated meshes, while a thin layer of unmineralized fibrous tissue was often observed surrounding cpTi mesh fibers. After 12 weeks, the structure of bone, with bone marrow-like tissue, was further matured and mesh fibers were embedded in lamellar bone. No statistical differences in the amount of mineralized bone were observed between scaffold types at any point of time. Only TiSi scaffolds showed further increase in bone area from 4 to 12 weeks (p < 0.01). A notable difference was that the sol-gel coatings resulted in enhanced initial bone contact and distribution of bone tissue, whereas uncoated implants showed bone formation mainly in the center of the scaffolds. In conclusion, TiO(2)-based sol-gel coatings may be used in tissue engineering to gain more uniform distribution of bone throughout titanium fiber mesh scaffolds.     

Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (μCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials.