Osteogenic efficacy of bone marrow concentrate in rabbit maxillary sinus grafting.

Maxillary sinus grafting is required to increase bone volume in the atrophic posterior maxilla to facilitate dental implant placement. Grafting with autogenous bone (AB) is ideal, but additional bone harvesting surgery is unpleasant. Alternatively, bone substitutes have been used but they limit new bone formation. The strategy of single-visit clinical stem cell therapy using bone marrow aspirate concentrate (BMAC) to facilitate new bone formation has been proposed. This study aimed to assess bone regeneration capacity of autologous BMAC mixed with bovine bone mineral (BBM) in maxillary sinus grafting. Twenty-four white New Zealand rabbits were used and their maxillary sinuses were randomly assigned for grafting with 4 different materials. Rates of new bone apposition in augmented sinuses were measured and bone histomorphometry were examined. Significant increase in the quantity of nucleated cells and colony forming unit-fibroblasts were confirmed in BMAC. Mesenchymal stem cells in BMAC retained their in vitro multi-differentiation capability. Higher rates of mineral appositions in the early period were detected in BBM + BMAC and AB than BBM alone, though they are not significantly different. Graft volume/tissue volumes in BBM and BBM + BMAC were found to be higher than those in AB and sham.

Alendronate-induced atypical bone fracture: evidence that the drug inhibits osteogenesis.

WHAT IS KNOWN AND OBJECTIVE: Alendronate (ALN) is used for the treatment of post-menopausal osteoporosis. By reducing bone turnover, it increases bone mineral density. However, recent reports suggest an increased risk of atypical bone fractures after long-term ALN administration. Despite its well-known anti-osteoclastic activity, it is unclear whether ALN also suppresses human mesenchymal stem cell (hMSC)-mediated osteogenesis, thus possibly resulting in atypical bone fragility. We hypothesized that ALN does this and we look at its in vitro effects on osteogenesis. METHODS: Morphological analysis, reverse transcriptase polymerase chain reaction, cell viability, alkaline phosphatase (ALP) activity and mineralization assays were investigated in hMSCs treated with a wide range of ALN. RESULTS AND DISCUSSION: After treatment with high concentrations of ALN for 3 and 7 days, cell viability was significantly reduced and cell morphology was altered. Osteogenic differentiation of hMSCs was also substantially suppressed as demonstrated by decreased ALP activity although ALN did not affect osteogenic-related genes tested. Furthermore, ALN at all concentrations tested drastically inhibited alizarin red S-positive mineralized matrix. WHAT IS NEW AND CONCLUSION: ALN has a strong inhibitory effect on hMSC-mediated osteogenesis by suppressing cell proliferation, osteoblast differentiation and function. The insight gained may help in the development of safer alternatives.

RNA interference of the BMPR-IB gene blocks BMP-2-induced osteogenic gene expression in human bone cells.

We have previously shown that human bone cells express bone morphogenetic protein receptor-IB (BMPR-IB). However, little is known about the precise role of this receptor in the response of osteoblastic genes to the BMP in these cells. To determine BMPR-IB-dependent osteoblastic gene expression, the present study examined the effects of BMPR-IB knockdown on BMP-induced osteoblast-associated genes. BMPR-IB mRNA and protein were markedly suppressed by transfection of cells with BMPR-IB siRNA. Using three different bone cell samples, BMP-2 stimulation of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox-5 (Dlx5) and core binding factor alpha-1 (Cbfa1) was found to be specifically and significantly reduced in the BMPR-IB siRNA-transfected cultures compared with that of control cultures. Our study has provided evidence that BMPR-IB-dependent signaling plays a crucial role in BMP-2 up-regulation of the ALP, OC, Dlx5 and Cbfa1 genes in bone cells, suggesting a pivotal role of this receptor in BMP-2-induced osteoblast differentiation in vitro. These findings thus suggest the possibility that BMPR-IB could be a therapeutic target for enhancing bone regeneration in vivo.

Bone morphogenetic protein receptors and bone morphogenetic protein signaling are controlled by tumor necrosis factor-alpha in human bone cells.

Bone morphogenetic proteins (BMP) stimulate osteoblast differentiation by signal transduction via three BMP receptors (BMPR-IA, -IB and -II), whereas the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) has been shown to suppress osteoblast differentiation. Although the mechanisms which regulate the BMPR are not yet known, it is possible that they may be negatively controlled by TNF-alpha, thereby inhibiting BMP-induced osteoblast differentiation. To test this hypothesis, we have examined the effects of TNF-alpha on BMPR-IA, -IB and -II expression and the functional consequences of this cytokine on BMPR-mediated functions in human bone cells. The results showed that although TNF-alpha down-regulated BMPR-IA and -II transcripts, it increased the level of BMPR-IB mRNA via a MAPK-dependent pathway. In marked contrast, however, TNF-alpha nevertheless caused marked down-regulation of the expression of the BMPR-IB surface antigen specifically. Moreover, the cytokine-induced decrease in BMPR-IB expression was found to be associated with the concurrent presence of a 'soluble' form of this antigen in supernatants of TNF-alpha-treated cultures. Furthermore, the TNF-alpha-induced loss of BMPR-IB was found to ablate BMP-2-stimulated bone cell functions, including phosphorylation of Smad1/5/8, alkaline phosphatase activity and osteocalcin expression. In conclusion, our study has provided evidence, for the first time, that BMPR can be differentially modulated by TNF-alpha at both the post-transcriptional and post-translational levels, with the TNF-alpha-induced shedding of the BMPR-IB antigen associated with a significantly diminished response to BMP-2 in vitro.

Shedding of a soluble form of BMP receptor-IB controls bone cell responses to BMP.

Bone morphogenetic proteins (BMP) are members of the transforming growth factor beta (TGF-beta) superfamily and are involved in a wide variety of biological processes, including osteoblast differentiation and bone healing. The activities of the BMP are mediated by signal transduction via three BMP receptors (BMPR-IA, -IB and -II), which are thus essential for the biological actions of the BMP. Although the precise mechanisms which control the BMPR are not yet known, it is possible that post-translational regulation of these cell surface antigens by shedding could modulate their expression and thereby at least partly determine the response of the cells to the BMP. To test this possibility, the present study has examined whether soluble forms of the BMPR are produced by shedding from primary human bone cells in vitro. The results showed that human bone cells expressed both mRNA transcripts and antigens corresponding to BMPR-IA, -IB and -II. Incubation of the cells with phorbol 12-myristate 13-acetate (PMA), a potent inducer of proteolytic shedding, resulted in a pronounced decrease in cell surface expression of all three BMPR and, concurrently, the presence of "soluble" forms of these antigens in culture supernatants. Moreover, PMA treatment significantly reduced the level of BMP-2-induced Smad1/5 phosphorylation, a major early activation step in signal transduction initiated by BMP/BMPR interaction. It is notable that, while treatment of bone cells with interleukin-1beta (IL-1beta) also reduced the level of surface BMPR-IB, this inflammatory cytokine had no effect on BMPR-IA or -II levels, hence only the soluble form of BMPR-IB was detected. Furthermore, in addition to down-regulating BMP-2-induced Smad1/5 phosphorylation, IL-1beta also caused a reduction in the level of BMP-2-induced alkaline phosphatase activity and osteocalcin expression, both closely associated with bone cell differentiation. In conclusion, our study has provided evidence, for the first time, that BMPR can be modulated at the cell surface by the shedding of a soluble form of the antigen, resulting in a markedly diminished response to BMP-2 in vitro.