RADA16-I/TRSAW水凝胶修饰的支架即刻构建组织工程骨及其成骨效果和机制的研究

The resource of a great deal of and highly osteoinductive bone graft is a huge problem during bone tansplantation. Tissue-engineered bone fabricated by autogeneic cells and scaffold has a known and better effectiveness than scaffold alone. However, some issues inhibit its wide application in clinic just like long proliferative time in seeding cells, high technologies in fabrication process, and high requirements on storage and transport. The enriching construct from bone marrow and scaffold by quich fabricated technology shows a good osteoinduction and a promising outlook. At present, low enriching rate and single enriching strategy limit the development of .enriching construct from bone marrow. For raising enriching rate and promoting osteogenesis, a new self-assembling peptide (SAP), RADA 16-I linked with TRSAW, is developed and is used to modify scaffold to improve enrichment of cells from bone marrow and to promote osteogenesis. The potential mechanism incldes that the pore diameter of scaffold will be shrinked by nanoscale fiber of SAP and raise retension of cells and factors from bone marrow; the charges derived from new SAP will increase adhension of cells in bone marrow; the biocompatibility of scaffold will receive benefit from SAP’s similar sructure to extracellular matrix; the osteogenic differentiation triggered by TRSAW will accelerate the osteogenesis of the construct formed by bone marrow and composite scaffold. During osteogenesis of enriching construct trigger by TRSAW, Src kinase plays an important role and interacted with VEGFR2 to induce a series reactions on the osteogenic related cells. The interface modification of new SAP shall renovate the current enriching strategy which depends on the only retension with shrinking pore size. Furthermore, the charges from SAP will increase more enrichment of cells from bone marrow, that has been not focused on before. The study will supply a new stratrgy for bone marrow construct and explore the potential mechanism.

骨髓富集物因含有促成骨的细胞而具有较好的骨修复效果。课题组通过RADA16-I肽来缩小支架材料的孔径提高骨髓中细胞的富集，取得了较好的成骨效果；但手段单一、成骨诱导能力不强是其瓶颈。甲状旁腺素相关蛋白功能肽-TRSAW，带净正电荷兼具成骨诱导能力，而骨髓中细胞表面带负电荷，二者可通过正负电荷相互吸附。构建一种新型的自组装肽RADA16-I/TRSAW，验证是否可通过电荷吸附作用提高细胞的富集效率。锚定支架上的TRSAW更利于局部稳定发挥成骨作用，虽已证实VEGFR2信号通路参与其中，但是二者如何作用并不清楚。本研究通过富集效率检测和信号通路研究验证假说：TRSAW对成骨相关细胞的作用是以Src激酶介导的VEGFR2信号通路实现的。本研究创新性在于首次提出电荷吸附提高骨髓富集的理论及阐明修饰支架的成骨诱导机制，为进一步提高骨髓的富集效率提供新的策略，为骨移植提供了一种新的可供选择的材料。

骨髓中含有大量成骨相关的细胞，具有优异的促成骨作用。传统的骨髓富集材料主要是通过缩小网孔的方式拦截骨髓中的细胞来实现促成骨作用，这种方式措施单一、效果有限。本研究在对支架材料进行修饰时采用可进行自我组装的多肽，其以RADA16-I多肽作为母链，将具有成骨诱导作用的甲状旁腺素相关蛋白的功能肽TRSAW链接到母链上构成RADA16-I/TRSAW，这种肽链可组装成凝胶在局部稳定发挥成骨诱导作用和粘附作用。利用其来修饰HA/β-TCP混合多孔支架，在原有的单一缩小孔径拦截的基础之上，进一步通过TRSAW带有的净正电荷提高对骨髓中带负电荷细胞的富集。研究显示，通过在多肽凝胶末端添加TRSAW可将细胞的粘附率提高20-30%。Zeta电位检测显示，添加TRSAW的修饰支架材料增加了正电荷；通过更换TRSAW为TASAW，去掉了带有正电荷的精氨酸，同时测其Zeta电位也明显降低，伴随着骨髓中细胞的富集能力也明显降低，提示表面修饰的正电荷材料具有增加细胞粘附的作用。同时，作为甲状旁腺素的多肽TRSAW还可以通过与粘附的细胞接触，一方面增加细胞的成骨分化能力；另一方面可持续有效地促进细胞释放成血管因子-1和VEGF。这两种因子的释放可在体外显著增加血管的形成能力。将设计的新型富集材料富集骨髓后移植小鼠骨缺损处，术后的Micro-CT结果显示，相比未添加TRSAW短肽的对照组材料而言，修饰材料的实验组成骨能力更强。Masson及HE染色分析，添加TRSAW的实验组新生骨具有较广的胶原分布，成熟胶原及新生胶原填附于支架材料孔径周围，且孔径中具有较多的细胞存留，表明植骨材料优良的成骨性能，且在胶原附近可见更多的成骨细胞及破骨细胞的分布，展现出较好的成骨修复作用，为进一步拓宽骨髓富集物在成骨修复过程中的应用提供技术支持和实验依据。