基于模量可调的DAFM/PECUU定向混纺纤维层叠支架对纤维环再生的实验研究

Although annulus fibrosus tissue engineering has attracted more and more attention in the treatment of degenerative intervertebral disc disease, it’s restricted to the biochemial properties, organization structure and mechanical characteristics. Our previous study demonstrated that there existed a kind of annulus fibrosus specific stem cells－Annulus Fibrosus-derived Stem Cells(AFSCs) in rabbits, whose differentiation were regulated by polyurethane (PECUU) nanofiber scaffolds with different elastic modulus. Besides, aligned PECUU nanofiber scaffolds could not only induce cell alignment but also could promote the mechanical strength of the scaffolds. Decellularized Annulus Fibrosus Matrix (DAFM) has good biocompatibility and biodegradability. What’s more, it is proved to promote the secretion of extracellular matrix. In the project, we plan to fabricate DAFM/PECUU aligned and blended electrospun scaffolds with different modulus to direct rabbit AFSC differentiating into the similar cell types in the inner, middle and outer regions of annulus fibrosus. Meanwhile, we plan to construct bionic annulus fibrosus tissue through skew laminated way base on gradient elastic modulus which could be successfully applied in the regeneration of defect in annulus fibrosus. The practice of this project will provide theoretical and practical basis for the development of bionic annulus fibrosus tissue engineering close to normal tissue in terms of the biological characteristics and mechanical properties.

虽然组织工程化纤维环在椎间盘退行性疾病的治疗中极具潜力，却往往因支架的生化性能，微观结构及力学性能缺陷而在研究中受到限制。我们前期研究表明，兔的纤维环中存在特异性来源的纤维环源干细胞，其分化受到不同弹性模量聚氨酯(PECUU)的调控，并且具有定向结构的PECUU纳米纤维支架不仅可以诱导细胞定向排布，同时也提高支架力学强度。脱细胞纤维环基质(DAFM)不仅具有良好的生物相容性和可降解性，而且促进细胞外基质分泌。本项目拟采用同轴共纺技术制备具有梯度弹性模量的DAFM/PECUU定向混纺纤维作为支架材料，诱导兔纤维环源干细胞分化成类似于纤维环内中外三区不同的细胞类型，并将混纺纤维按照由内向外弹性模量递增趋势采用斜交层叠的方式予以卷曲成环，从而构建出仿生纤维环组织，以有效应用于纤维环缺损的修复。本项目的实施可为开发生物学特性和力学性能接近正常组织的仿生纤维环提供理论基础和实践依据。

虽然组织工程化纤维环在椎间盘退行性疾病的治疗中极具潜力，却往往因支架的生化性能，微观结构及力学性能缺陷而在研究中受到限制。在本研究中，我们成功制备了具有高亲水性和孔隙率的脱细胞纤维环基质/聚氨酯（DAFM/PECUU）混纺纤维支架。在DAFM/PECUU混纺纤维支架上培养的兔纤维环源干细胞（Annulus Fibrosus-derived Stem Cells, AFSCs）比在PECUU静电纺丝纤维支架上培养的细胞表达和分泌更高水平的Ⅰ型胶原、Ⅱ型胶原和聚集蛋白聚糖。体内植入实验发现DAFM/PECUU支架周围组织的炎症反应较PECUU支架轻。考虑到纤维环的径向弹性模量的梯度差异，我们制备了不同弹性模量DAFM/PECUU混纺纤维支架，发现 AFSCs的纤维环相关基因表达和细胞外基质组成受到基材弹性模量的调控，并有着向纤维环径向各区域组织中所特有的细胞类型分化的趋势。即和纤维环内区相似的低弹性模量的支架上Ⅱ型胶原和聚集蛋白聚糖表达较高，Ⅰ型胶原表达较低；而在和纤维环外区相似的高弹性模量的支架上Ⅱ型胶原和聚集蛋白聚糖表达较低，Ⅰ型胶原表达较高。体内实验表明不同弹性模量DAFM/PECUU混纺纤维支架在体内可以缓慢降解，所引起的免疫反应较为轻微，同时在体实验表明可以有效的对纤维环的缺损进行修复。此外，我们制备了定向DAFM/PECUU混纺纤维支架，发现AFSCs培养在定向混纺纤维支架上细胞可呈定向生长，分泌的细胞外基质也呈定向分布。