【作者】 刘钢；

【导师】 敬伟；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2022， 硕士

【摘要】 对于临床上因各种原因导致的骨缺损问题,自体骨移植虽然作为治疗手段的金标准被广泛应用,但仍然存在二次损伤、手术风险等问题,这使得开发新的治疗手段变得十分必要。随着科学技术的发展与进步,对于骨缺损修复重建有了更多的选择,骨组织工程支架植入骨缺损处进行修复重建就是一种更加新颖、有效的方法。然而限制骨组织工程支架发展及应用的是支架内部血管化系统的成功构建。根据已有的文献报道,我们设计了一种新型的骨组织工程再生支架用于增强支架的诱导血管化和成骨的作用。为了避免因免疫排斥的存在导致治疗失败,开发生物相容性良好的支架材料是一个关键的环节。钙磷陶瓷具有与骨组织相同的元素成分,所以在骨组织工程中显示出巨大的临床应用潜力。在陶瓷原材料的选择上,双相磷酸钙(Biphasic calcium phosphate,BCP)是一种具有良好的骨传导与骨诱导活性的钙磷化合物,十分适合用于打印陶瓷支架。此外,我们对陶瓷支架的制造技术也进行了优化,采用了数字化光处理(Digital light processing,DLP)打印工艺进行支架的构建,这种新颖的3D打印工艺解决了传统支架制造的精确性低、可控性差、耗时耗力等问题,可打印出高精确性、可控性及复杂的支架结构。我们通过DLP打印机打印出多孔BCP支架并且在支架中搭载生长因子来进一步增强其诱导成血管和成骨的能力。我们将自兔体内获得的血液进行两次离心获得血小板裂解液(Platelet lysate,PL),并将PL与甲基丙烯酸化明胶(Gelatin methacrylate,GelMA)混合制备PL/GelMA前驱体溶液,最后通过光固化的方法将PL/GelMA加载到支架内,成功制备出PL/GelMA/BCP支架。我们发现在填充了PL/GelMA后,支架的表面形貌也发生了变化,不同于BCP支架的规则排列的大孔径结构,PL/GelMA/BCP支架则表面可见更多的不规则分布的、较小的微孔结构。并且支架的力学性能较未填充PL/GelMA之前也得到了提高。我们还发现PL/GelMA/BCP支架具有良好的生物相容性,人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)在PL/GelMA/BCP支架上培养后表现出良好的粘附性,并且与支架共培养的HUVECs表现出更强的增殖活性。此外,PL中的多种生长因子(Growth factors,GFs)可以从支架中缓慢持续地释放,进而上调HUVECs内成血管相关基因的表达,同时多种因子协同指导细胞的生物学行为,促进细胞形成血管样结构。在植入大鼠背部的PL/GelMA/BCP支架中也发现了更多的毛细血管网形成,表明搭载了PL后BCP陶瓷支架具有的更强的血管化能力。与此同时,在兔的颧弓节段性缺损处植入支架后也表现出相似的结果,在PL/GelMA/BCP支架内部可以发现有更多的胶原纤维形成。再生骨组织的体积定量结果也表明PL/GelMA/BCP支架组内形成了更多的新骨组织。对新形成的骨组织结构进行分析,我们发现在PL/GelMA/BCP支架内部形成的骨小梁具有更大的厚度并且排列更加紧密,这反映出这种新生骨组织具有更强的机械性能。综上,PL/GelMA/BCP支架是一种具有血管化潜能的骨组织工程再生支架。更多还原

【Abstract】 For clinical bone defects caused by various reasons,although autologous bone transplantation is widely used as the gold standard for treatment,there are still problems such as secondary injury and surgical risk,which makes it necessary to develop new treatment methods.With the development and progress of science and technology,there are more options for bone defect repair and reconstruction.Bone tissue engineering scaffolds implanted in bone defects for repair and reconstruction is a more novel and effective method.However,the limitation of the development and application of bone tissue engineering scaffolds is the successful construction of the vascularization system inside the scaffolds.According to the existing literature reports,we designed a novel bone tissue engineering regeneration scaffold to enhance the vascularization and osteogenesis effect of the scaffold.To avoid treatment failure due to the existence of immune rejection,the development of scaffold materials with good biocompatibility is a key link.Calcium-phosphorus ceramics have the same elemental composition as bone tissue,so they show great potential for clinical application in bone tissue engineering.In the selection of ceramic raw materials,biphasic calcium phosphate(BCP)is a calcium-phosphorus compound with good osteoconductive and osteoinductive activities,which is very suitable for printing ceramic scaffolds.In addition,we have also optimized the fabrication technology of the ceramic scaffolds,using a digital light processing(DLP)printing process to construct the scaffolds.This novel 3D printing process solves the problems of low accuracy,poor controllability,time-consuming and labor-intensive manufacturing of traditional scaffolds,and can print highprecision,controllable,and complex scaffold structures.We printed porous BCP scaffolds by DLP printer and carried growth factors in the scaffolds to further enhance its angiogenesis and osteogenesis ability.And then the blood obtained from rabbits was centrifuged twice to obtain platelet lysate(PL).The PL/GelMA precursor solution was prepared by mixing PL with Gelatin methacrylate(GelMA).Finally,the PL/GelMA was loaded into the scaffold by photocuring,and the PL/GelMA/BCP scaffold was successfully prepared.We found that the surface topography of the scaffolds also changed after filling with PL/GelMA.Different from the regularly arranged macropore structure of the BCP scaffold,the PL/GelMA/BCP scaffold showed more irregularly distributed and smaller micropore structures on the surface.And the mechanical properties of the scaffolds were also improved compared to those of unfilled PL/GelMA scaffolds.We also found that the PL/GelMA/BCP scaffolds had good biocompatibility,and human umbilical vein endothelial cells(HUVECs)exhibited good adhesion when cultured on the PL/GelMA/BCP scaffolds.And when co-cultured with the scaffolds,HUVECs showed stronger proliferative activity.In addition,various growth factors(GFs)in PL could be slowly and continuously released from the scaffold,which in turn up-regulated the expression of angiogenesis-related genes in HUVECs.At the same time,a variety of GFs cooperates to guide the biological behavior of cells and promote the formation of blood vessel-like structures.More capillary network formation was also found in PL/GelMA/BCP scaffolds implanted in the back of rats.Therefore,these results indicated that BCP ceramic scaffolds have a stronger ability to induce vascularization after filling with PL/GelMA.At the same time,similar results were shown after implantation of the scaffold at the segmental defect of the zygomatic arch in rabbits,and more collagen fiber formation could be found inside the PL/GelMA/BCP scaffold.The volume quantification results of the regenerated bone tissue also indicated that more new bone tissue was formed in the PL/GelMA/BCP scaffold group.Analyzing the newly formed bone tissue structure,we found that the bone trabeculae formed inside the PL/GelMA/BCP scaffold had a larger thickness and were more densely arranged,which reflected the stronger mechanical properties of the newly formed bone tissue.In conclusion,the PL/GelMA/BCP scaffold is a bone tissue engineering regeneration scaffold with vascularization potential.更多还原