Julius Wolff Institute, Charité - Universitätsmedizin, Berlin, Germany.
Bone. 2010 Aug;47(2):438-44. doi: 10.1016/j.bone.2010.05.026. Epub 2010 May 24.
Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72 h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20% compression, 1 Hz) over 3 days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24 h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24 h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical stimulation; this was significant in haematomas from younger, but not from older patients. Other GFs were not mechanically regulated. In conclusion, the paracrine pro-angiogenic capacity of early human haematomas is enhanced by mechanical stimulation. This effect lasts even after removing the mechanical stimulus and appears to be VEGFR2-dependent.
受损的血管生成似乎是各种骨愈合不良情况下的主要限制因素。适当的机械刺激支持血管形成，并改善愈合效果。然而，负责这种关联的机制尚不清楚。为了解决这个问题，研究了早期人骨折血肿的旁分泌血管生成潜力及其对机械加载的反应性，以及涉及的血管生成生长因子。从健康患者在骨折后 72 小时内接受手术时收集人血肿。将血肿嵌入纤维蛋白基质中，并在类似于骨愈合早期（20%压缩，1 Hz）的生物反应器中培养 3 天。然后分析生物反应器中的条件培养基（CM）。还将基质在新鲜培养基中再孵育 24 小时，以评估效果的持久性。通过 ELISA 测量 CM 中的生长因子（GF）浓度。用 HMEC-1 细胞系在 Matrigel 上进行体外管形成试验，有或没有血管内皮生长因子受体 2（VEGFR2）的抑制。使用 MTS 试验定量细胞数量。与纤维蛋白对照相比，来自血肿的 CM 增强了体外内皮管形成。血管生成调节剂血管内皮生长因子（VEGF）和转化生长因子β1（TGF-β1）被释放到血肿 CM 中，但血管生成素 1 或 2（Ang1，2）、碱性成纤维细胞生长因子（bFGF）或血小板衍生生长因子（PDGF）未被释放。血肿的机械刺激，而不是纤维蛋白对照，进一步增加了其 CM 诱导管形成的能力。机械刺激的血肿基质保留了其升高的促血管生成能力 24 小时。VEGFR2 信号通路的抑制取消了促血管生成作用。CM 中的 VEGF 浓度倾向于通过机械刺激升高；在较年轻的患者的血肿中这是显著的，但在较年长的患者中则不然。其他 GF 不受机械调节。总之，早期人血肿的旁分泌促血管生成能力通过机械刺激增强。这种效应甚至在去除机械刺激后仍然存在，并且似乎依赖于 VEGFR2。
