【作者】 杨玲；

【导师】 胡巧玲；

【作者基本信息】 浙江大学 ， 高分子材料， 2020， 博士

【摘要】 壳聚糖(CS)是自然界中唯一的碱性多糖,在生物医学领域已有许多应用,通过对壳聚糖进行化学改性和制备方法的探索,可以制得形式、功能多样的壳聚糖基生物医用材料。然而,现今对于壳聚糖基生物医用材料的可控构筑仍旧十分具有挑战性,具体表现为加工性能差、力学性能不佳、操作麻烦等问题。因此,针对不同的临床需求,选择性构筑结构与性能优异、使用便捷、具有特定功能的壳聚糖基生物医用材料对于解决实际临床问题和拓展壳聚糖材料的应用范围具有十分重要的指导意义。本文选用壳聚糖作为构筑生物医用材料的主要基材,针对不同应用领域的临床痛点,构筑了一系列不同形式、性能优异的壳聚糖基生物医用材料。具体研究内容如下:(1)针对牙周缺损再生存在的非功能性细胞长入而引起的牙根吸收和根骨粘连,进而阻碍新骨组织形成的问题,采用新型的微凝胶自沉积法仿生构筑了具有非对称结构的CS仿生双层复合材料。该双层复合材料兼具多孔层和致密层,其微观结构和力学性能、细胞相容性可满足引导组织再生膜(GTR)的标准,且可通过调控材料的脱乙酰度实现材料性能的调控。利用大鼠颅骨缺损模型将制得的高脱乙酰度组CS仿生双层复合材料与商用Bio-gide~?膜进行比较,Micro-CT和组织切片结果表明CS仿生双层复合材料具有更为优异的骨修复能力和适宜的降解速率。(2)针对肌腱组织自身愈合性能不足、修复过程中易发生腱鞘膜粘连致使修复效果不佳等问题,首次在CS仿生双层复合材料的多孔层引入肌腱干细胞(TSPCs)应用于肌腱修复。采用高分子量、高脱乙酰度制得的CS仿生双层复合材料具有优异的干/湿态拉伸性能,且复合材料对TSPCs细胞成腱性基因和蛋白质的表达具有正向调节作用。利用大鼠跟腱断裂模型对比了不同材料设计组的跟腱修复效果和跟腱粘连情况进行评价。结果表明,负载TSPCs的CS复合材料组相较于空白组和纯CS复合材料材料组在缺损跟腱部位具有更高的I型胶原蛋白(COL1)、肌腱调节蛋白(TNMD)表达量和更为整齐、取向的纤维排列,修复6周得到的跟腱具有更高的生物力学性能和较低的跟腱鞘粘连评分。(3)针对颈动脉大出血和内脏出血的临床应急止血难题,将壳聚糖的止血特性结合天然贻贝粘附蛋白中邻苯二酚基团的湿态粘接特性,构筑了具有可注射、自愈合、快速凝胶化性能的壳聚糖基水凝胶止血粘接剂CCS@gel。CCS@gel在出血部位可快速原位构筑水凝胶(t<15 s),且可通过调控组分浓度对其凝胶化性能进行调控。采用体外力学与粘接性能评价、体内/外相容性评价(细胞、血液、组织)、大鼠脾脏/肝脏出血模型和颈动脉出血模型共同验证其止血封堵效果。与商用纤维蛋白胶相比,CCS@gel对多种抗凝性内脏伤口和颈动脉出血有更为优异的止血效果。此外,由于CCS@gel在生理条件下独特的自收缩特性使其能长期实现对伤口的封堵并促进伤口闭合,对于构筑壳聚糖基应急止血封堵材料具有重要指导意义。(4)针对肿瘤细胞早期检测、发现和治疗的难题,针对肿瘤组织中高还原性微环境,构筑了具有还原响应性的壳聚糖基AIE纳米荧光探针(TCSC NPs),该探针在生理条件下具有良好的水分散性和纳米级的稳定性。在肿瘤细胞还原微环境下,由于羧基的裂解使得AIE探针的水溶性变差,进一步诱导了AIE纳米颗粒的聚集,增强了细胞内滞留,从而增强了对癌细胞的靶向成像和选择性抑制。研究并提出了纳米荧光探针实现其肿瘤特异性增强示踪和抑杀作用的机理。为研究与开发生物相容性、光稳定性良好的肿瘤细胞靶向示踪和生物检测诊断试剂提供了新的思路。更多还原

【Abstract】 Chitosan(CS)is the only basic polysaccharide in nature,and has been widely used in biomedical fields.Chitosan-based biomedical materials can be tailored with various forms and versatile functions by chemical modification and exploration of preparation techniques.However,the controllable construction of chitosan-based biomedical materials is still very challenging,which is manifested by its poor processability,inadequate mechanical properties,and operation inconvenience.Therefore,selective construction of CS-based biomedical materials with prominent properties for different clinical demands is of great significance for solving practical clinical problems and expanding the application field of chitosan materials.In this paper,chitosan was selected as the main substrate,and a series of CS-based biomedical materials with different forms and excellent performance were constructed according to the clinical issues in different application fields.Specific research contents are listed as follows:(1)Poor periodontal regeneration is mainly caused by root absorption and bone ingrowth adhesion of non-functional cells,which further hinders the formation of new bone tissue.To solve this problem,a pure CS-based bio-mimetic double-layer composite materials with asymmetric structure was constructed via novel microgel self-deposition technique.This CS composite materials were composed of a porous layer and a dense layer.Its microstructure and mechanical properties can satisfy the criteria for guided tissue regeneration(GTR)membrane.Materials properties could be tune by deacetylation degree(D.D)adjustment.The rat skull defect model was applied to estimate the in vivo bone regeneration performance.The micro-CT and histological results manifested that CS double-layer composites with high D.D exhibited outstanding bone regenerative ability and superior degradation compatibility over the commercialized Bio-gide~?membrane.(2)The poor healing capacity and potential peritendinous adhesion of injured tendons has made tendon repair as a primary clinical concern.Rat tendon stem/progenitor cells(TSPCs)were first incorporated with CS double-layer composites for tendon regeneration.CS double-layer composites with high D.D and molecular weight has excellent tensile properties under dry/wet conditions.It showed positive tendinous gene and protein expression adjustment for TSPCs.The higher production of collagen I(COL1)and tenomodulin(TNMD),higher biomechanical properties and lower adhesion score for Achilles tendon repair model demonstrated that the CS double-layer composites were capable of inducing conspicuous tenogenic differentiation and superior phenotypic maturity.The introduction of TSPCs into the CS scaffold resulted in a synergistic effect on tendon regeneration and yielded better-aligned collagen fibers with elongated,spindle-shaped cells.(3)Aiming for the clinical emergency hemostasis problem of massive carotid arterial bleeding and internal bleeding,a CS-based hydrogel hemostatic adhesive(CCS@gel)were constructed with injectable,self-healing and rapid gelation properties.CCS@gel can form gel rapidly at the bleeding site(t<15 s).In vitro mechanics and adhesion performance evaluation,in vitro compatibility evaluation(cell,blood,tissue),rat splenic/liver bleeding model and carotid artery bleeding model were used to verify the hemostatic plugging effect.Compared with commercialized fibrin glue,CCS@gel had better hemostasis effect on a variety of anticoagulant visceral wounds and carotid bleeding.In addition,due to the unique self-contractile characteristics of CCS@gel under physiological conditions,it could achieve long-term wound sealing and promote wound closure.(4)Considering the early detection of cancer cells,smart systems with stimulus triggered features have become increasingly popular nowadays.On account of the highly reductive micro-environment in tumor tissues,a redox-responsive fluorescent AIE bioprobe based on CS was designed and synthesized.This AIE bioprobe possessed excellent water dispersibility and nano-size stability under physiological conditions.Under the reductive microenvironment of tumor cells,the water dispersibility of the AIE probe becomes worse owing to the cleavage of carboxyl groups.The dispersibility alteration further induces the aggregation of AIE nanoparticles and enhances intracellular retention,allowing for targeted imaging reinforcement and selective suppression of cancer cells.更多还原