以工程化羊膜微粒为载体快速构建高增殖活性复合皮的实验研究

Construction of composite skin containing epidermal seed cells is the crux in skin tissue engineering research. The routine method usually employs the 2D culture model by two steps: first amplifying epidermal seed cells and then inoculating them to a dermal substitute for further culture. But as the conventional mehtod is time-consuming and cells are liable to differentiate.We hypothesize to use a 3D culture model simulating cell growth environment to construct high proliferative composite skin.Our pre-reasearch has been published on Biomaterials（IF:7.42）which demonstrated that human amniotic membrane (AM) could provide epidermal stem cells with an in vitro environment similar to the human physiological conditions. The aim of the present study was to develop engineered AM particles and use them as both a dermal substitute and a microcarrier to amplify large amounts of epidermal stems cells by using the rotating cell culture system (RCCS) so as to fasten the construction of epidermal stem cell-AM particle composite skin by one step.At the same time, we used ball-to-ball inoculation technique to transfer epidermal stem cells to the new AM particles thought contacts and collisions to amplify the composite skin automatically, thus shortening the in vitro culture time,avoiding injury to cells due to repeated digestions and improving proliferative activity. The result would abandon the routine composite skin construction model,provide a new way of thinking for improving the flexibility in using composite skin and survival of composite skin grafts.

构建含表皮种子细胞的复合皮是皮肤组织工程研究的重点。常规方法是在二维平面环境中先扩增表皮种子细胞,然后接种于真皮替代物表面再次培养，分二步完成，耗时长、反复消化后细胞增殖活性差。本项目设想：模拟细胞生长的体内微环境，一步法快速构建具有高增殖活性的复合皮，从而克服目前常规方法的不足。我们发表在Biomaterials（IF:7.42）的前期研究表明，人羊膜能为表皮干细胞提供更接近生理的体外培养微环境。本项目拟进一步制备工程化羊膜微粒，既能作为真皮替代物又能作为微载体，通过旋转细胞培养系统快速扩增表皮干细胞，同步构建表皮干细胞-羊膜微粒复合皮，从而缩短复合皮构建时间；并采用球传球接种技术，使表皮干细胞通过接触与碰撞转移到新的羊膜微粒，自动扩增复合皮，从而避免反复消化对细胞造成的损伤与消耗，提高增殖活性。将为摒弃常规的复合皮构建模式、提高其应用灵活性和移植存活率提供新思路。

构建含表皮种子细胞的复合皮是皮肤组织工程研究的重点。常规方法是在二维平面环境中先扩增表皮种子细胞,然后接种于真皮替代物表面再次培养，分二步完成，耗时长、反复消化后细胞增殖活性差。本项目成功制备了工程化羊膜微粒，具有良好的生物亲和性和高生物学活性，富含EGF、HGF等多种生长因子。进一步实验表明，羊膜微粒既能作为真皮替代物又能作为微载体，通过旋转细胞培养系统快速扩增表皮干细胞，表皮干细胞种植后半小时即可见粘附于羊膜微粒表面，培养3天时可见细胞呈三维立体生长，随培养天数增多，细胞密度逐渐增大，7天时见表皮干细胞密布于羊膜微粒表面。14d时表皮干细胞已形成2-3层。在RCCS培养过程中，采用球传球接种技术，即通过表皮干细胞—羊膜微粒复合皮与另一新的羊膜微粒间的接触与碰撞，完成表皮干细胞在羊膜微粒间的转移，从而自动扩增复合皮，避免了常规方法中反复消化对细胞造成的损伤与消耗，维持了表皮干细胞增殖活性。将上述复合皮移植于裸鼠全层皮肤缺损创面，3周左右形成了新的上皮，光滑平整。本研究可望为摒弃常规的复合皮构建模式、提高其应用灵活性和移植存活率提供新思路。