施万细胞通过骨髓间充质干细胞来源的内皮细胞促进组织工程骨的血管生成和成骨。

BACKGROUND: Tissue-engineered bone grafts (TEBGs) that undergo vascularization and neurotization evolve into functioning bone tissue. Previously, we verified that implanting sensory nerve tracts into TEBGs promoted osteogenesis. However, the precise mechanisms and interaction between seed cells were not explored. In this study, we hypothesized that neurotization may influence the osteogenesis of TEBGs through vascularization. METHODS: We cultured rat Schwann cells (SCs), aortic endothelial cells (AECs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) and then obtained BM-MSC-derived induced endothelial cells (IECs) and induced osteoblasts (IOBs). IECs and AECs were cultured in an SC-conditioned medium (SC-CM) to assess proliferation, migration, capillary-like tube formation, and angiogenesis, and the vascular endothelial growth factor (VEGF) levels in the supernatants were detected. We established an indirect coculture model to detect the expression of nestin and VEGF receptors in IECs and tissue inhibitor of metalloproteinase (TIMP)-2 in SCs. Then, SCs, IECs, and IOBs were labeled and loaded into a β-tricalcium phosphate scaffold to induce prevascularization, and the scaffold was implanted into a 6-mm-long defect of rat femurs. Three groups were set up according to the loaded cells: I, SCs, and IECs (coculture for 3 days) plus IOBs; II, IECs (culture for 3 days) plus IOBs; III, IOBs. Nestin and TIMP-2 expression and osteogenesis of TEBGs were evaluated at 12 weeks post-implantation through histological and radiological assessments. RESULTS: We found that SC-CM promoted IEC proliferation, migration, capillary-like tube formation, and angiogenesis, but no similar effects were observed for AECs. IECs expressed nestin extensively, while AECs barely expressed nestin, and SC-CM promoted the VEGF secretion of IECs. In the coculture model, SCs promoted nestin and VEGF receptor expression in IECs, and IECs inhibited TIMP-2 expression in SCs. The promotion of prevascularized TEBGs by SCs and IECs in group I augmented new bone formation at 6 and 12 weeks. Nestin expression was higher in group I than in the other groups, while TIMP-2 expression was lower at 12 weeks. CONCLUSIONS: This study demonstrated that SCs can promote TEBG osteogenesis via IECs and further revealed the related specific characteristics of IECs, providing preliminary cytological evidence for neurotization of TEBGs.

背景：经过血管化和神经化的组织工程化骨移植物（TEBG）演变成具有功能的骨组织。以前，我们已经验证了将感觉神经束植入 TEBG 中可以促进成骨。然而，种子细胞之间的确切机制和相互作用尚未得到探索。在这项研究中，我们假设神经化可能通过血管化影响 TEBG 的成骨作用。

方法：我们培养大鼠雪旺细胞（SCs）、主动脉内皮细胞（AECs）和骨髓间充质干细胞（BM-MSCs），然后获得 BM-MSC 衍生的诱导内皮细胞（IECs）和诱导成骨细胞（IOBs）。将 IECs 和 AECs 在雪旺细胞条件培养基（SC-CM）中培养，以评估增殖、迁移、毛细血管样管形成和血管生成，并检测上清液中的血管内皮生长因子（VEGF）水平。我们建立了间接共培养模型，以检测 IECs 中的巢蛋白和血管内皮生长因子受体的表达以及 SCs 中的组织金属蛋白酶抑制剂（TIMP）-2 的表达。然后，将 SCs、IECs 和 IOBs 标记并加载到 β-磷酸三钙支架中以诱导预血管化，并将支架植入大鼠股骨 6mm 长的缺损中。根据加载的细胞，我们设置了三组：I 组，SCs 和 IECs（共培养 3 天）加 IOBs；II 组，IECs（培养 3 天）加 IOBs；III 组，IOBs。通过组织学和影像学评估，在植入后 12 周评估 TEBG 的巢蛋白和 TIMP-2 表达和骨生成。

结果：我们发现 SC-CM 促进了 IEC 的增殖、迁移、毛细血管样管形成和血管生成，但 AEC 则没有类似的作用。IECs 广泛表达巢蛋白，而 AECs 几乎不表达巢蛋白，SC-CM 促进了 IECs 的 VEGF 分泌。在共培养模型中，SCs 促进了 IECs 中巢蛋白和血管内皮生长因子受体的表达，而 IECs 抑制了 SCs 中 TIMP-2 的表达。第 I 组中 SCs 和 IECs 对预血管化 TEBG 的促进作用增强了第 6 周和第 12 周的新骨形成。第 I 组的巢蛋白表达高于其他组，而第 12 周的 TIMP-2 表达较低。

结论：本研究表明，SCs 可以通过 IEC 促进 TEBG 的成骨作用，进一步揭示了 IEC 的相关特征，为 TEBG 的神经化提供了初步的细胞学证据。