可降解温敏性可注射改性甲壳素水凝胶的制备及其细胞载体的研究

Scaffold material is the key material in three-dimensional (3D) bioprinting and regenerative medicine. Hydrogels provide favorable microenvironments that promote cell retention and repair of damaged tissue for growing cells in 3D culture to form 3D aggregates. Based on the previous study of degradable thermosensitive polymers we firstly prepared thermosensitive injectable carboxymethyl chitin and hydroxypropyl chitin hydrogels by the homogenous method in NaOH/urea aqueous solution at low temperature, respectively. The obtained polymers which are soluble in water at low temperature become gel quickly at physiological condition at low polymer concentration of 0.5-4%. These modified chitin hydrogels show comparable advantages in thermosensitivity as Matrigel, a commercially available hydrogel widely used for research. However, Matrigel is an animal-derived product that promotes tumorigenicity and the growth of tumor cells in vivo. Furthermore, Matrigel presents some practical limitations in handling and processing, because it requires careful manipulation and must be maintained at constant cold temperature due to its not fully reversible thermoresponsiveness. Preliminary cell and animal experiments indicate that these modified chitin hydrogels are enzymatically degradable, non-cytotoxic and good in vivo biocompatibility. They can be injected and gel quickly inside the animal body and promote 3D cell growth. In this work, in order to study the effect of structure of the modified chitin on thermosensitivity and to explore the mechanism of temperature sensitivity for developing other thermosensitive polymers, a series of modified chitins with different structures will be synthesized. The cell adhesion and mechanical strength of the obtained material will be optimized through further polymer modification and in situ chemical crosslinking after thermal physical gelation to provide good method and strategy to develop suitable injectable polymeric hydrogels for cell delivery. A suitable modified chitin hydrogel will be obtained for in vitro 3D cell culture and clinic application in minimally invasive in situ tissue repair.

支架材料是三维生物打印和再生医学的关键材料，其中水凝胶接近于细胞外基质，对细胞三维生长和体内损伤组织原位修复最为有效。在研究可降解温敏性高分子基础上，我们首次通过低温尿素碱液溶解甲壳素，分别均相合成了温敏性可注射羧甲基和羟丙基改性甲壳素水凝胶。其能低温溶解，体温下快速成胶，且凝胶浓度低（0.5-4%），温敏可控，与已商业化的基质胶性能类似，但却没有基质胶的动物来源致癌风险及不可反复成胶使用的不足。初步的细胞和动物实验表明该类材料在生理条件下可快速成水凝胶，可酶解无毒，可促进细胞三维生长，体内组织相容性好。本项目拟研究改性甲壳素结构与其温敏特性间的关系，探索其温敏机理，为研发其它温敏性高分子提供理论指导。通过进一步改性结合温敏成胶和原位化学交联，优化材料的细胞粘附性和强度，为开发细胞载体用可注射水凝胶提供方法和思路。研制出可望用于体外三维细胞培养和临床原位组织微创修复的改性甲壳素水凝胶材料。

生物支架材料是三维细胞培养、生物打印和再生医学的关键材料，其中水凝胶接近于细胞外基质，对细胞三维生长和体内损伤组织原位修复最为有效，而开发新的温敏可注射可降解高分子水凝胶材料具有重要意义。利用低温尿素碱液水相体系首次均相合成了具有温敏性的低脱乙酰度羧甲基甲壳素、低脱乙酰度温敏性可注射羟丙基甲壳素及其可光交联的温敏可注射改性甲壳素水凝胶等，其能在低温生理水里溶解，体温下快速成胶，且凝胶浓度低（0.5-4%），温敏可控。研究了其结构与其转变温度之间的规律，发现保留较高乙酰基是这些改性甲壳素具有温敏性的共性，并且随着亲水基团的引入转变温度会升高，而引入憎水基团则会降低转变温度，为研发其它温敏性高分子提供了方法和思路。结合温敏成胶和原位化学交联，利用双网络体系，研制了系列新型可原位反应或光交联的可注射改性甲壳素水凝胶，双交联的方法（物理交联和化学交联）能大大提高温敏性水凝胶的力学强度。这些水凝胶在生理缓冲溶液中可长期保持自身结构的完整性，并且可以被溶菌酶生物降解，细胞毒性实验表明这些水凝胶无毒副作用，不影响细胞生长。动物体内注射实验表明这些水凝胶具有在体内可注射可快速原位成胶的能力和良好的生物相容性，这些可注射改性甲壳素水凝胶作为细胞载体开展了体外三维细胞培养、三维细胞打印和动物体内组织修复的研究，研究结果表明该温敏性可降解羟丙基甲壳素水凝胶在医疗器械植入材料、大分子药物和细胞载体等方面，具有很好的应用前景，为开发细胞载体用可注射水凝胶和临床原位组织微创修复材料提供了新的方法和思路。已发表标注SCI论文15篇，其中1区4篇，2区9篇，影响因子大于5的12篇，大于7的8篇；已获中国发明专利授权2项，申请四项；培养并已获得硕士学位毕业5名和博士学位4名。

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