基于可降解微载体-生物反应器组织工程皮肤再生单元构建的研究

Traditional tissue-engineered skin is hard to circumvent dermal scaffold revascularization in the clinical application while epidermal cell suspension, the lack of dermal structure. Based on this, we propose a new concept of the tissue-engineered skin regenerative units (TESRUs): a smallest unit has a complete full-thickness skin regeneration capacity. In this study, we use the biodegradable macroporous microcarriers as scaffold and hierarchically assembled the fibroblasts, epidermal melanocytes layer by layer. The different cellular components can be amplified by the hydrodynamic response of the cells in the bioreactor. The formation of the micro-organizational-structured TESRUs may achieved by suspended culture in the bioreactor. The association of different cellular components with flow field in the bioreactor can be derived and visualized by computational fluid dynamics method, and then, the suitable operation conditions are determined. Experiments are planned to set up to observe the vitality of the cells in the TESRUs, the growth curves of the cells, chromosome integrity of the cells, three dimensional cellular organization and structure of TESRUs. Through in vitro and in vivo experiments, observations include the re-epithelialization and dermal formation, basement membrane formation, the dermis revascularization capacity of the TESRUs and the mechanical properties of the new formed skin, while ascertain the whereabouts of the different cellular components. Epidermal cell suspension and the split thickness skin grafts are planned to use as positive control, and the microcarriers, as negative control. TESRUs are expected to have the competency to form dermis, melanin and re-epithelialize the wound. This can circumvent the dilemma between the choices of dermal scaffold and the vascularization which have been haunted in skin tissue engineering for many years, but not satisfyingly resolved as yet. TESRUs, if successfully realized, are also expected to be a new access to the future of skin tissue engineering and regenerative medicine research.

传统的组织工程皮肤在临床应用时难以回避真皮支架的血管化问题，而表皮细胞悬液则缺乏真皮结构，基于此，我们提出了组织工程皮肤再生单元的观点，即具有完备全层皮肤再生能力的最小单元。本研究拟采用可降解大孔微载体分层负载成纤维细胞、表皮及黑素细胞，通过生物反应器的流体力学响应，使不同细胞成分均可以得到的扩增并形成微组织结构。通过计算流体力学方法分析生物反应器流场与再生单元的再生功能关联，得出适合的构建条件；通过实验，观察再生单元中细胞的活力、生长曲线、染色体完整性及再生单元的三维组织学结构，通过体外及体内实验，观察再生单元的上皮化及真皮再生、基底膜形成、真皮的血管化、再生皮肤的力学性能及不同细胞成分的转归，并与细胞悬液及自体断层皮片作为比较，证实再生单元可同步完成真皮再生、上皮化及黑素形成，避免了细胞悬液中没有真皮支架的问题，而且不依赖真皮的血管化，并可为组织工程皮肤及再生医学的研究开辟新的途径。

表皮细胞在组织工程皮肤的构建、烧伤及慢性创面的治疗中有极其重要的地位，是完成创面上皮化的关键细胞；创面治疗中最为理想的表皮细胞应该来源于病人的自身，而在短时间内表皮细胞的大规模获取及大量扩增是限制组织工程皮肤及表皮细胞膜片等应用的主要难题。因此，我们提出了组织工程皮肤再生单元的观点，即具有完备全层皮肤再生能力的最小单元。本研究首先应用CFD技术对搅拌式灌流式生物反应器进行了模拟，从培养室内流体的流动速度、压力、湍流动能及湍流耗散率以及平均粒子停留时间等方面对其进行了考察，通过CFD的模拟我们认为，圆柱形双灌流模式是灌流式组织工程皮肤生物反应器的良好设计模式。其次，我们对表皮细胞的获取、培养基的改进及表皮细胞培养方法、表皮细胞的生物反应器培养以及表皮细胞向创面递呈等环节进行优化。我们发现通过三维环形摆动的动力下对表皮细胞进行消化，可在相同的皮肤标本中获得更多的表皮细胞。通过改良的表皮细胞培养基中，实现表皮细胞、黑素细胞及成纤维细胞的共培养。进而，我们将成纤维细胞与表皮细胞及黑素细胞依次负载于可降解多孔微载体上，置入搅拌式生物反应器内进行培养，通过调整搅拌速度等参数，结合CFD模拟结果，优化并构建组织工程皮肤再生单元，利用可降解多孔微载体可实现表皮细胞大规模扩增。最后，我们通过动物实验发现，将组织工程皮肤再生单元递呈于创面后，可在两周的时间完成创面的上皮化，说明使用组织工程皮肤再生单元在促进创面愈合方面具有明显的优势。