Guided bone regeneration (GBR) often utilizes a combination of autologous bone grafts, deproteinized bovine bone mineral (DBBM), and collagen membranes. DBBM and collagen membranes pre-coated with bone-conditioned medium (BCM) extracted from locally harvested autologous bone chips have shown great regenerative potential in GBR. However, the underlying molecular mechanism remains largely unknown. Here, we investigated the composition of BCM and its activity on the osteogenic potential of mesenchymal stromal cells. We detected a fast and significant (P < 0.001) release of transforming growth factor-β1 (TGF-β1) from autologous bone within 10 min versus a delayed bone morphogenetic protein-2 (BMP-2) release from 40 min onwards. BCMs harvested within short time periods (10, 20, or 40 min), corresponding to the time of a typical surgical procedure, significantly increased the proliferative activity and collagen matrix production of BCM-treated cells. Long-term (1, 3, or 6 days)-extracted BCMs promoted the later stages of osteoblast differentiation and maturation. Short-term-extracted BCMs, in which TGF-β1 but no BMP-2 was detected, reduced the expression of the late differentiation marker osteocalcin. However, when both growth factors were present simultaneously in the BCM, no inhibitory effects on osteoblast differentiation were observed, suggesting a synergistic TGF-β1/BMP-2 activity. Consequently, in cells that were co-stimulated with recombinant TGF-β1 and BMP-2, we showed a significant stimulatory and dose-dependent effect of TGF-β1 on BMP-2-induced osteoblast differentiation due to prolonged BMP signaling and reduced expression of the BMP-2 antagonist noggin. Altogether, our data provide new insights into the molecular mechanisms underlying the favorable outcome from GBR procedures using BCM, derived from autologous bone grafts.

引导性骨再生（GBR）通常利用自体骨移植物，脱蛋白的牛骨矿物质（DBBM）和胶原膜的组合。从局部收获的自体骨碎屑中提取的预涂有骨条件培养基（BCM）的DBBM和胶原膜在GBR中显示出巨大的再生潜力。但是，基本的分子机制仍然是未知的。在这里，我们调查了BCM的组成及其对间充质基质细胞成骨潜力的活性。我们检测到了快速而重要的（P <0.001）在10分钟内从自体骨释放了转化生长因子-β1（TGF-β1），而从40分钟开始则释放了延迟的骨形态发生蛋白2（BMP-2）。在短时间段（10、20或40分钟）内收获的BCM（对应于典型的外科手术时间）显着增加了BCM处理的细胞的增殖活性和胶原基质的产生。长期（1、3或6天）提取的BCM促进了成骨细胞分化和成熟的后期阶段。短期提取的BCMs，其中检测到TGF-β1，但未检测到BMP-2，降低了晚期分化标记骨钙素的表达。但是，当两种生长因子同时存在于BCM中时，未观察到对成骨细胞分化的抑制作用，表明TGF-β1/ BMP-2活性具有协同作用。因此，在重组TGF-β1和BMP-2共同刺激的细胞中，我们发现TGF-β1对BMP-2诱导的成骨细胞分化具有显着的刺激和剂量依赖性作用，这是由于BMP信号延长和BMP-2表达降低所致。 BMP-2拮抗剂头蛋白。总之，我们的数据提供了对使用自体骨移植物进行BCM的GBR程序产生良好结果的分子机制的新见解。