Autologous bone marrow cells implantation induces bone formation in a sheep spinal fusion model

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Abstracts / Bone 44 (2009) S142–S161

inhibitor, did not promote the osteoblastic differentiation in MC3T3E1 cells. In contrast, TPA, which activated not only PKCα but other isoforms, suppressed osteoblastic differentiation in a dose-dependent fashion. Mineralization of extracellular matrix was correlated with the result of ALP activity and mRNA expression. In the transcriptional factors, Runx2 was not influenced for PKCs inhibitors and activator. Discussion: The osteoblastic differentiation was accelerated by Go6976, PKCα and PKCβI inhibitor, but not PKCβ inhibitor. Additionally, TPA, PKC stimulator, which also promoted PKCα activity, inhibited the differentiation. These results indicated that PKCα was involved in the osteoblastic differentiation and that its inhibition positively affected the differentiation. Conclusion: PKCα might regulate the osteoblastic differentiation. The inhibition of its kinase could promote the differentiation. We are going to study signal transduction pathways of these phenomena. doi:10.1016/j.bone.2009.01.338

432 Regulation of osteoblast function and mineralisation by extracellular nucleotides: The role of P2Y and P2X receptors I.R. Orrissa, G. Burnstockb, A. Gartlandc, T.R. Arnetta a Cell and Developmental Biology, University College London, London, UK b Autonomic Neuroscience Centre, Royal Free and University College Medical School, London, UK c Academic Unit of Bone Biology, University of Sheffield, Sheffield, UK Extracellular nucleotides, signalling through P2 receptors, play a significant role in bone biology, modulating both osteoblast and osteoclast function. To date we have demonstrated P2X2, P2X5, P2X7, P2Y1, P2Y2, P2Y4 and P2Y6 receptor expression by osteoblasts. We previously showed that ATP/UTP, potently inhibit alkaline phosphatase (ALP) activity and bone mineralisation in vitro, an effect which could be mediated, at least in part, via the P2Y2 receptor. MicroCT analysis of 2month-old P2Y2 receptor deficient mice revealed increased femoral (43%, p < 0.01) and tibial (21%) trabecular bone volume. Increases in femoral trabecular thickness (17%, p < 0.01), trabecular number (33%, p < 0.05) and cortical bone volume (25%, p < 0.01) were also observed. Using qPCR and immunofluorescence we have now extended our investigation of P2 receptor expression by primary osteoblasts derived from rat calvariae. Osteoblasts were found also to express mRNA and protein for P2X1, P2X3, P2X4, P2X6, P2Y12, P2Y13 and P2Y14 receptors. Receptor expression changed with cellular differentiation; for example, P2X4 receptor mRNA levels were 5-fold higher in mature bone-forming osteoblasts relative to immature, proliferating cells. To investigate whether receptors other than P2Y2 might influence osteoblast function, osteoblasts were cultured for 14 days with the P2 agonists α,β-meATP, β,γ-meATP, Bz-ATP and 2-MeSATP (1–100 μM). Mineralised bone nodule formation was measured by image analysis of alizarin redstained cell layers. The P2X1 and P2X3 receptor agonists, α,β-meATP and β,γ-meATP (1 μM) inhibited bone mineralisation by 70% and 90%, respectively, with complete abolition at ≥25 μM. Bz-ATP, a potent P2X7 receptor agonist, reduced bone mineralisation by 65% and 90% at 1 μM and 100 μM, respectively; this inhibition was abolished by the selective P2X7 antagonist, A438079. The P2 agonist 2-MeSATP, which is not active at P2Y2 receptors, also reduced bone mineralisation by > 50% at 10 μM. Osteoblast ALP activity was similarly decreased in each case by these agonists. These responses are consistent pharmacologically with involvement of the P2X1, P2X3, P2X5 and/or P2X7 receptors. These data highlight the expression of multiple P2 receptor subtypes by osteoblasts and indicate that extracellular nucleotides could function as local signaling agents that “switch off” bone mineralisation. doi:10.1016/j.bone.2009.01.339

433 Autologous bone marrow cells implantation induces bone formation in a sheep spinal fusion model Y. Qiana, Z. Lina, J. Chena, Y. Fanb, T. Daveya, J. Xua, M.H. Zhenga a Centre for Orthopaedic Research, University of Western Australia, Perth, WA, Australia b Perth Bone and Tissue Bank, Perth, WA, Australia Bone marrow cells (BMCs), containing mesenchymal stem cells, have pluripotent potential to differentiate into multiple mesenchymal lineages, and presents as a potential cell source for bone tissue engineering, such as spinal fusion. The purpose of this study was to evaluate the efficiency of osteogenesis of sheep bone marrow cells in natural bone collagen scaffold (NBCS) in vitro, and the ability of these cells combined with NBCS to induced new bone formation in a sheep interbody lumbar fusion model. BMCs were co-cultured with NBCS for 1, 2, 3 and 4 weeks to investigate the proliferation, and differentiation of MSCs in NBCS by histological examination, scanning electron microscopy (SEM), immunohistochemistry and semi-quantitative RTPCR. In sheep interbody fusion model, BMCs combined with NBCS were implanted in disc space, and was compared with autograft, NBCS alone and BMCs alone at 6 and 10 weeks postoperativity. In vivo results showed that sheep BMCs exhibited good proliferation 3dimentionally in NBCS, which was evidenced by histology and SEM. Osteogenesis differentiation was induced in NBCS as well. Collagen type I matrix was also detected by immunohistochemistry, mineralization was demonstrated by von Kossa staining, and osteogenesis gene were investigated by RT-PCR. In vivo study showed that BMCs combined with NBCS yielded spinal fusion with higher fusion rate, greater biomechanical stiffness and larger bone volume than control groups. Histological finding also revealed that more mature new bone formation was induced by BMCs combined with NBCS, and integrated well with host bone tissue. We conclude that BMCs is a potential cell source for bone tissue engineering, and combined with NBCS, can be used as bone formation inducer for spinal fusion. doi:10.1016/j.bone.2009.01.340

434 Inhibition of bone loss using dynamic muscle stimulation in a disuse osteopenia model Y. Qina, H. Lama, M. Malbaria, M. Shihb, W. Carrollb a Biomedical Engineering, Stony Brook University, SUNY, Stony Brook, New York, USA b RS Medical, Vancouver, Washington, USA Musculoskeletal adaptations to aging and disuse environment have significant physiological effects on skeletal health, i.e., osteopenia. Mechanical loading through muscle stimulation (MS) has been shown to promote interstitial and blood flow to bone, possibly by creating a pressure gradient within the microvasculature and tissue [[1] Lam and Qin: Bone, 2008;43(6):1093–100]. The hypothesis for this study is that dynamic MS can enhance anabolic activity in bone, and mitigate bone loss in a functional disuse condition. Using a hindlimb suspension (HLS) rat model, dynamic MS was applied as replacement of the normal weight-bearing activity of the hindlimb with two skin patch electrodes at the right quadriceps muscles for total of 30 animals. The stimulus was applied at 115 mA, 71 Hz, 0.2 ms pulse with 3 s on and 8 s rest. The animals were divided into six groups (5 per group), including 1) baseline control, 2) age-matched control, 3) hindlimb suspended (HLS) sham control, 4) HLS + 10 min MS, 5) HLS + 30 min MS, 6) HLS + 60 min MS, 5 days per week, for a total of 4 weeks. Left and right femurs were harvested for micro-computed tomography analysis. Distal metaphyseal regions and one epiphyseal region of the femurs