CXCR4 Has the Potential to Enhance Bone Formation in Osteopenic Rats.

Osteoporosis is characterized by reduced bone mass and aberrant bone microarchitecture, thus increasing susceptibility to fracture due to reduced strength and quality. The aims of this study were to investigate the role of CXCR4 transfected on stem cell homing and osteogenic characteristics in osteopenic rats, particularly elucidating the effect on cell migration. METHODS: Mesenchymal stem cells (MSCs) were harvested from young, and ovariectomized animals and transfected with CXCR4; these cells were administered intravenously in ovariectomized rats. Micro CT and mechanical testing were completed after 12 weeks. RESULTS: Rats injected with young CXCR4 transfected cells had significantly higher bone mineral density (BMD) compared to placebo injected rats (p < 0.05). Rats injected with ovariectomized CXCR4 transfected cells had higher BMD compared to those injected with saline or nontransfected cells (p < 0.04). L4 vertebral stiffness was significantly higher in rats treated with young CXCR4 transfected cells compared to all other groups (p < 0.05). CONCLUSION: CXCR4 genetically modified cells from young and ovariectomized sources improve some aspects of bone formation in the ovariectomized model of osteoporosis and, thus, may play a role in patient treatment regimens.

Adsorption of Amorphous Silica Nanoparticles onto Hydroxyapatite Surfaces Differentially Alters Surfaces Properties and Adhesion of Human Osteoblast Cells.

Silicon (Si) is suggested to be an important/essential nutrient for bone and connective tissue health. Silicon-substituted hydroxyapatite (Si-HA) has silicate ions incorporated into its lattice structure and was developed to improve attachment to bone and increase new bone formation. Here we investigated the direct adsorption of silicate species onto an HA coated surface as a cost effective method of incorporating silicon on to HA surfaces for improved implant osseointegration, and determined changes in surface characteristics and osteoblast cell adhesion. Plasma-sprayed HA-coated stainless steel discs were incubated in silica dispersions of different concentrations (0-42 mM Si), at neutral pH for 12 h. Adsorbed Si was confirmed by XPS analysis and quantified by ICP-OES analysis following release from the HA surface. Changes in surface characteristics were determined by AFM and measurement of surface wettability. Osteoblast cell adhesion was determined by vinculin plaque staining. Maximum Si adsorption to the HA coated disc occurred after incubation in the 6 mM silica dispersion and decreased progressively with higher silica concentrations, while no adsorption was observed with dispersions below 6 mM Si. Comparison of the Si dispersions that produced the highest and lowest Si adsorption to the HA surface, by TEM-based analysis, revealed an abundance of small amorphous nanosilica species (NSP) of ~1.5 nm in diameter in the 6 mM Si dispersion, with much fewer and larger NSP in the 42 mM Si dispersions. 29Si-NMR confirmed that the NSPs in the 6 mM silica dispersion were polymeric and similar in composition to the larger NSPs in the 42 mM Si dispersion, suggesting that the latter were aggregates of the former. Amorphous NSP adsorbed from the 6 mM dispersion on to a HA-coated disc surface increased the surface's water contact angle by 53°, whereas that adsorbed from the 42 mM dispersion decreased the contact angle by 18°, indicating increased and decreased hydrophobicity, respectively. AFM showed an increase in surface roughness of the 6 mM Si treated surface, which correlated well with an increase in number of vinculin plaques. These findings suggest that NSP of the right size (relative to charge) adsorb readily to the HA surface, changing the surface characteristics and, thus, improving osteoblast cell adhesion. This treatment provides a simple way to modify plasma-coated HA surfaces that may enable improved osseointegration of bone implants.

Mesenchymal stem cells with increased stromal cell-derived factor 1 expression enhanced fracture healing.

Treatment of critical size bone defects pose a challenge in orthopedics. Stem cell therapy together with cytokines has the potential to improve bone repair as they cause the migration and homing of stem cells to the defect site. However, the engraftment, participation, and recruitment of other cells within the regenerating tissue are important. To enhance stem cell involvement, this study investigated overexpression of stem cells with stromal cell-derived factor 1 (SDF-1) using an adenovirus. We hypothesized that these engineered cells would effectively increase the migration of native cells to the site of fracture, enhancing bone repair. Before implantation, we showed that SDF-1 secreted by transfected cells increased the migration of nontransfected cells. In a rat defect bone model, bone marrow mesenchymal stem cells overexpressing SDF-1 showed significantly (p=0.003) more new bone formation within the gap and less bone mineral loss at the area adjacent to the defect site during the early bone healing stage. In conclusion, SDF-1 was shown to play an important role in accelerating fracture repair and contributing to bone repair in rat models, by recruiting more host stem cells to the defect site and encouraging osteogenic differentiation and production of bone.

In vitro response of human osteoblasts to multi-step sol-gel derived bioactive glass nanoparticles for bone tissue engineering.

A multi-step sol-gel process was employed to synthesize bioactive glass (BG) nanoparticles. Transmission electron microscopy (TEM) revealed that the BG nanoparticles were spherical and ranged from 30 to 60 nm in diameter. In vitro reactivity of the BG nanoparticles was tested in phosphate buffer saline (PBS), Tris-buffer (TRIS), simulated body fluid (SBF), and Dulbecco's modified Eagle's medium (DMEM), in comparison with similar sized hydroxyapatite (HA) and silicon substituted HA (SiHA) nanoparticles. Bioactivity of the BG nanoparticles was confirmed through Fourier transform infrared spectroscopy (FTIR) analysis. It was found that bone-like apatite was formed after immersion in SBF at 7 days. Solutions containing BG nanoparticles were slightly more alkaline than HA and SiHA, suggesting that a more rapid apatite formation on BG was related to solution-mediated dissolution. Primary human osteoblast (HOB) cell model was used to evaluate biological responses to BG nanoparticles. Lactate dehydrogenase (LDH) cytotoxicity assay showed that HOB cells were not adversely affected by the BG nanoparticles throughout the 7day test period. Interestingly, MTS assay results showed an enhancement in cell proliferation in the presence of BG when compared to HA and SiHA nanoparticles. Particularly, statistically significant (p<0.05) alkaline phosphatase (ALP) activity of HOB cells was found on the culture containing BG nanoparticles, suggesting that the cell differentiation might be promoted by BG. Real-time quantitative PCR analysis (qPCR) further confirmed this finding, as a significantly higher level of RUNX2 gene expression was recorded on the cells cultured in the presence of BG nanoparticles when compared to those with HA and SiHA.

Nanohydroxyapatite shape and its potential role in bone formation: an analytical study.

Bone cells (osteoblasts) produce a collagen-rich matrix called osteoid, which is mineralized extracellularly by nanosized calcium phosphate (CaP). Synthetically produced CaP nanoparticles (NPs) have great potential for clinical application. However few studies have compared the effect of CaP NPs with different properties, such as shape and aspect ratio, on the survival and behaviour of active bone-producing cells, such as primary human osteoblasts (HOBs). This study aimed to investigate the biocompatibility and ultrastructural effects of two differently shaped hydroxyapatite [Ca10(PO4)6(OH)2] nanoparticles (HA NPs), round- (aspect ratio 2.12, AR2) and rice-shaped (aspect ratio 3.79, AR4). The ultrastructural response and initial extracellular matrix (ECM) formation of HOBs to HA NPs were observed, as well as matrix vesicle release. A transmission electron microscopy (TEM)-based X-ray microanalytical technique was used to measure cytoplasmic ion levels, including calcium (Ca), phosphorus (P), sodium (Na) and potassium (K). K/Na ratios were used as a measure of cell viability. Following HA NP stimulation, all measured cytoplasmic ion levels increased. AR2 NPs had a greater osteogenic effect on osteoblasts compared with AR4 NPs, including alkaline phosphatase activity and matrix vesicle release. However, they produced only a moderate increase in intracellular Ca and P levels compared with AR4. This suggests that particular Ca and P concentrations may be required for, or indicative of, optimal osteoblast activity. Cell viability, as measured by Na and K microanalysis, was best maintained in AR2. Initial formation of osteoblast ECM was altered in the presence of either HA NP, and immuno-TEM identified fibronectin and matrilin-3 as two ECM proteins affected. Matrilin-3 is here described for the first time as being expressed by cultured osteoblasts. In summary, this novel and in-depth study has demonstrated that HA NP shape can influence a range of different parameters related to osteoblast viability and activity.

A comparison of allogeneic and autologous mesenchymal stromal cells and osteoprogenitor cells in augmenting bone formation around massive bone tumor prostheses.

Spraying autologous mesenchymal stromal cells (MSCs) onto hydroxyapatite (HA)-coated ingrowth collars, located at the shoulder of massive bone tumor implants, significantly increased extracortical bone-bridging and osteointegration in an ovine model. In this study, we investigated the hypothesis that allogeneic MSCs and osteoprogenitor cells (OPCs) will augment bone growth equally when compared with autologous BMSCs. All collars were HA coated. In group i, the HA collar was coated with fibrin glue only. Cells were combined with fibrin glue and implants received (ii) 2 × 10(6) autologous MSCs, (iii) 10 × 10(6) autologous MSCs, (iv) 2 × 10(6) OPCs, (v) 10 × 10(6) OPCs, or (vi) 10 × 10(6) allogeneic MSCs. In group vii, collars were HA coated only. New bone area and bone-implant contact onto the ingrowth collar was quantified radiographically and using histological techniques. Results showed that no extracortical bone formed adjacent to any collars sprayed with allogeneic MSCs and significantly more new bone was measured when all other experimental groups were compared (p < 0.05 in all cases). Most bone growth and bone-implant contact occurred in the 10 × 10(6) OPC group. Spraying MSCs or OPCs onto the implant surface may be used in patients; however, further work is needed to determine the role of allogeneic cells in bone augmentation in vivo.

Direct inhibition of osteoclast formation and activity by the vitamin E isomer gamma-tocotrienol.

Vitamin E homologues, specifically tocotrienols, have been shown to have favorable effects on bone. They possess properties that are indicative of anti-resorptive activity, suggesting the potential for vitamin E in preventing bone loss. To investigate the anti-resorptive activity of the various vitamin E homologues, we cultured human osteoclasts from blood-derived CD14+ cells on collagen, dentin, and calcium phosphate substrates, with some samples supplemented with vitamin E homologues in their cell culture medium. These were compared to the clinically used bisphosphonate, pamidronate. Compounds were either added at the start of culture to study effects on osteoclast formation, or at the start of osteoclastic resorption to determine their effects on activity. The alpha- and gamma-tocotrienol isomers inhibited osteoclast formation without consequent reduction in total cell number. Only gamma-tocotrienol inhibited osteoclast activity without toxicity. Gamma-tocotrienol was the most potent inhibitor of both osteoclast formation and activity and requires further investigation into its anti-resorptive effects on bone.

Augmentation of bone growth onto the acetabular cup surface using bone marrow stromal cells in total hip replacement surgery.

Aseptic loosening of acetabular components in total hip arthroplasty is the major cause of implant failure. Our hypothesis was that spraying autologous bone marrow-derived stromal cells (BMSCs) in fibrin glue onto the surface of hydroxyapatite-coated uncemented acetabular components would increase bone formation and contact in a caprine model. Ten million BMSCs were sprayed onto the acetabular cup at the time of surgery. Animals in the control group received fibrin glue only. Ground reaction force measurements were taken preoperatively and at 6 and 12 weeks postsurgery. After retrieval at 12 weeks new bone formation, bone-implant contact and fibrous tissue thickness adjacent to the cup were quantified. Viability and proliferation assays showed that the majority of the BMSCs survived spraying in fibrin glue at pressures of up to 1.5 atm. New bone growth adjacent to the bone implant interface in the BMSC-treated group (71.42 +/- 8.97%) was 30% greater than in control (54.22 +/- 16.56%) although this difference was not statistically significant. However, significantly increased new bone formation was measured at the periphery of the cup (zone 5) in the BMSC-treated group (71.97 +/- 10.91%) when compared with control (23.85 +/- 15.13%, p = 0.028). Bone-implant contact was significantly greater in the BMSC-treated group (20.03 +/- 4.64%) (control: 13.71 +/- 8.32%, p = 0.027); correspondingly, the average thickness of the fibrous tissue membrane where present was significantly reduced at the periphery of the cups in the BMSC-treated group (327.49 +/- 20.38 mum) when compared with control (887.21 +/- 158.89 mum) (p = 0.02). This study has clinical applications as greater bone contact at the cup surface will improve fixation and may decrease longer-term aseptic loosening by preventing wear debris-induced bone loss at the implant interface.

Do autologous mesenchymal stem cells augment bone growth and contact to massive bone tumor implants?

This study investigated the hypothesis that spraying autologous mesenchymal stem cells (MSCs) onto grooved hydroxyapatite (HA) coated collars of segmental bone tumor implants would increase bone growth and contact to the implant surface in an ovine model. Autologous MSCs were isolated from bone marrow, grown in culture and during surgery implants in group 1 were sprayed with MSCs, suspended within fibrin glue. Implants in group 2 received no MSC therapy and acted as control. Implants remained in vivo for 6 months. New bone area and contact to the implant was quantified on radiographs and histologically. Radiographic analysis demonstrated greater total bone area in the MSC treated group in both ML (MSC = 79.738 mm2 +/- 22.964; control = 30.135 mm2 +/- 6.717) (p = 0.018) and AP (MSC = 90.338 +/- 19.361 mm2; control = 57.384 +/- 9.035 mm2 (p = 0.074) radiographs at 6 months. Results demonstrated significantly increased bone growth in the MSC group at 2 (p = 0.03) and 3 months (p < 0.05). Histological analysis demonstrated significantly greater bone area adjacent to the collars in the treated group (53.994 +/- 10.641 mm2) when compared with the control group 21.069 +/- 7.339 mm2 (p = 0.020). Increased bone contact in the MSC group (19.833 +/- 8.729 %) was observed when compared with controls (8.667 +/- 8.667%). This novel application of spraying MSCs onto the implant surface has significant implications for the future of successful implant fixation.