基于外泌体精准调控的“核-壳”（core-shell）同步血管化骨组织工程策略的应用与机制探讨

组织工程骨（TEBG）移植体内后血管化只发生于外周而非内核区域导致修复能力差，限制骨组织工程（BTE）临床转化。我们前期通过内皮祖细胞（EPC）与间充质干细胞（MSC）共培养构建“核-壳”结构TEBG，可实现核和壳区域同步血管化，促进成骨，但共培养制备复杂、细胞特性易丢失。因此，本项目创新性提出一种基于外泌体精确调控的“核-壳”同步血管化BTE策略，并研究其机理：①利用我们建立的外泌体规模化提取技术，分别获得EPC和MSC的外泌体，3D打印成“核-壳”结构的功能支架，具有血管化“核”、成骨化“壳”的区域niche微环境；②将单独培养的EPC和MSC分别注入到“核”和“壳”中，移植体内，在外泌体niche调控下实现核-壳同步血管化和骨再生；③与传统BTE比较，从组织、细胞和分子水平探索作用机制。该策略简化原有核-壳同步血管化技术，有利于临床转化，对骨和其他组织修复与血管化研究有积极借鉴意义。

The in vivo vascularization of tissue-engineered bone grafts (TEBG) after transplantation is limited to the peripheral area (“Shell”), and the lack of vascularization in the core area (“Core”) results in limited bone regeneration capacity, which hinders the clinical translation of the bone tissue engineering (BTE) technology. In our preliminary research, we cocultured the endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) to construct the “core-shell” structured TEBG, which can achieve a synchronized vascularization in both the core and shell area, leading to enhanced bone regeneration. Nevertheless, the complexity of the coculture system and the losing of the cell phenotype make it difficult for clinical application. To boost its clinical translation, a novel bone tissue engineering strategy based on the exosome-regulated “core-shell” synchronous vascularization is proposed in this research, and the mechanism would be comprehensively investigated. ① EPC-derived exosome (serves as the vascularized microenvironment niche in the “core” area) and MSC-derived exosome (serves as the ossified microenvironment niche in the “shell” area) are extracted from our custom-designed continuous cell culture system, respectively, which are then been fabricated into a “core-shell” functional scaffold via the 3D printing; ② Inject the separately cultured EPC and MSC into the "core" and "shell", respectively, before the transplantation, so as to achieve the synchronous vascularization and bone regeneration under the modulation of the different microenvironment niches. ③ The mechanism is explored on a histologic, cellular, and molecular level, compared to the traditional prevascularized bone tissue engineering (BTE). This strategy simplifies the original core-shell vascularization technique, thus bestows value on further clinical translation. More importantly, this is an important supplement to the vascularization in BTE and can be used as a reference for other tissue regeneration.