3D打印猪源性真皮支架复合低氧BM-MSC膜片构建新型真皮替代物的实验研究

Treatment for dermal defects is difficult in clinics, and a challenge for tissue engineers. The structure and vascularization of current dermal substitutes cannot meet clinical standards of care. As for this project, we will exploit porcine derived ECM nano-fiber powder to build bioprint scaffold that simulates human dermis. Then we will combine it with hypoxic bone marrow derived mesenchymal stem cell (BM-MSC) sheets to make novel artificial dermis. This could act as a substitute of micro- nanoscale topological structure, stem cell therapy and rapid angiogenesis. To evaluate the interaction between scaffold structure and cells, we will measure the cell viability, genomic stability, colony- forming efficiency, differentiation potential, morphological structure，protein differential expression, immunogenicity, and physical properties of the substitute in vitro. Then, we will trace BM-MSCs differentiation, inspect the synergistic effect of the 3D printed matrix and the cell sheets, as well as explore a new method to realize rapid neovascularization in vivo. This new dermal substitute may provide a novel and superior clinical treatment for skin wound healing.

真皮缺损的修复是临床治疗的难点和组织工程学研究的重点，现有真皮替代物在结构、血管化速度等方面尚不能满足临床需要。课题组拟以猪源性ECM纳米纤维微粉为原料，按人真皮结构特点打印支架，将其与低氧骨髓间充质干细胞（Bone marrow derived mesenchymal stem cell，BM-MSC）膜片结合，构建集高精度微米级打印孔隙、纳米级ECM孔隙、干细胞疗法及快速血管化于一体的新型真皮替代物。我们将就细胞增殖活力、基因稳定性、集落形成能力、分化潜能、形态结构、蛋白表达差异、免疫原性及物理性能对新型真皮替代物进行体外检测，考察支架拓扑结构与细胞之间的相互作用，然后行动物实验检测BM-MSCs分化，研究支架与低氧BM-MSC膜片对创面修复的作用，探索实现组织工程真皮快速血管化的新方法。本研究结果将为提高皮肤创面修复质量带来新的手段和思路。

真皮缺损的修复是临床治疗的难点和组织工程学研究的重点，现有真皮替代物在结构、血管化速度等方面尚不能满足临床需要。课题组在项目经费的资助下进行了系列研究：① 利用低氧培养条件和自研的纳米蚀刻细胞模板优化制备出低氧骨髓间充质干细胞(Bone marrow derived mesenchymal stem cell，BM-MSC)膜片(HCS)及人脐静脉内皮细胞预血管化的HCS(PHCS)膜片；② 以“贴近临床、解决临床实际问题”为目标，建立了小鼠超长皮瓣模型及大鼠复合植皮模型，可为真皮替代物/促血管元件的体内检测提供生物学工具；③ 证实了优化的PHCS膜片既能解决超长带蒂皮瓣远端缺血坏死问题，又可有效提高自体断层皮片对全层皮肤缺损创面的修复质量；④ 通过3D扫描获得创面数据，按照 “对称对照”的原则，通过计算机处理获得契合于缺损组织的3D重建立体结构，经过再处理获得创面再生材料的结构数据。此数据处理过程将通过“机器学习”不断优化，为制作与创面完全契合的“个体化”组织工程填充材料打下基础，以期在未来实现高质量创面修复的目标；⑤ 以猪源性ECM纳米纤维微粉为原料，快速制备出孔径在130μm左右的3D打印真皮基质，通过体内外检测筛选出最佳的制备条件，证实其在创面高修复中的作用，并寻找到进一步改良靶点。课题组的前述研究成果将为皮肤损伤修复提供新的思路与方法，具有转化应用潜能。