还原软骨发生机制支架构建与骨软骨再生

To mitigate the challenges associated with cartilage fibrosis and poor combination of cartilage and subchondral bone in osteochondral tissue engineering, the present study was proposed to construct a polyamino acid-based osteochondral scaffold that could restore the chondrogenesis mechanism to enhance the chondeogenic differentiation of adipose derived stem cells(ASCs). Here we design a scaffold based on amide bonded poly(L-glutamic acid) (PLGA) and chitosan (CS) according to the guide line of designing non-fouling materials. The scaffold possessed high water-uptake ability and supported in-situ adipose derived stem cell (ASC) multicellular spheroids formation to duplicate “condensation” that takes place in vivo during limb development and enhance hyaline cartilage formation; For the purpose of mimicing bone matrix, PLGA-grafted HA(HA-PLGA) composite nanoparticles were prepared by surface-initiated ring-opening polymerization, which were then employed to fabricate subchondral bone scaffold with CS. Meanwhile, akermanite was introduced endow subchondral bone scaffold with osteogenic inducing activity to accelerate bone regeneration. Cartilage scaffold and subchondral bone scaffold were combined tightly through osmosis. Furthermore, a osteochondral scaffold with a oriented cartilage scaffold layer was disigned. Requirements for spheroid formation would be studied. The advantage of spheroid in cartilage formation would be confirmed. And osteochondral regeneration using the present scaffold needed to be evaluated.

骨软骨组织工程存在软骨层纤维化、软骨与下骨结合差的问题。针对这两个问题，申请人提出构建还原软骨发生机制，强化脂肪干细胞(ASCs)软骨向分化的聚氨基酸基骨软骨共修复组织工程支架。聚L-谷氨酸(PLGA)与壳聚糖(CS)化学交联构建弱化细胞与支架黏附作用，驱动ASCs原位自发聚集形成微团的PLGA/CS软骨支架，以强化ASCs软骨向分化和软骨再生；采用“grafting-from”法在纳米羟基磷灰石HA表面接枝PLGA，合成HA-PLGA，模拟下骨基质中无机组分与有机组分的结合，再与CS化学交联构建下骨支架，同时引入镁黄长石赋予下骨支架成骨诱导活性，加快下骨再生以更好地支持软骨再生。利用凝胶渗透作用将软骨支架与下骨支架复合构建具备连续过渡界面的一体化支架。并进一步制备软骨层取向的骨软骨支架。探索微团形成条件，证实微团用于软骨再生的优势，评价一体化支架用于体内关节骨软骨再生情况。

目前，骨软骨组织工程走向临床面临两个挑战：再生软骨多为纤维软骨，缺乏正常透明软骨的力学功能；软骨下骨损伤与软骨损伤修复不同步，影响新生软骨组织的完整性和稳定性。针对以上挑战，本研究提出构建能够差异化负载生长因子的骨软骨一体化支架，该支架软骨层能够抑制纤维基质产生，促进透明软骨再生，下骨层能够负载绑定BMP-2，刺激下骨再生。.项目首次提出原位诱导脂肪干细胞自发聚集形成微团的支架设计策略，以还原软骨发生机制中至关重要的“condensation”过程。该多孔支架由聚L-谷氨酸(PLGA)和壳聚糖(CS)通过酰胺键交联，并结合冷冻相分离技术制备，孔径为180-300 μm。支架具有高溶胀性，可快速吸水。脂肪干细胞(ASCs)接种到支架后并未贴壁黏附于支架孔壁，而是自发聚集形成直径为80-110 μm的多细胞微团。微团内细胞存活率高，且在体外TGF-β1和IGF-1诱导作用下的软骨向分化能力强，表达更多的GAGs和COL II，同时COL I沉积受到抑制。进一步的体内软骨再生结果表明，PLGA/CS支架原位诱导ASCs微团可在兔体内再生形态和功能均接近正常软骨的透明状软骨组织。.为模拟骨基质组成并加速下骨再生，项目提出在纳米羟基磷灰石(nHA)表面键合PLGA链，得到nHA-g-PLGA，以其为原料参与PLGA和CS静电复合支架的构建。以聚乳酸微球作为致孔剂，结合冷冻相分离技术可以制备孔径为200-400 μm的下骨支架。与机械共混nHA制备的支架相比，接枝PLGA提高nHA的分散性，且nHA-g-PLGA可通过静电作用与CS结合，提高支架的力学性能。通过静电复合方式构建的下骨支架残留电荷密度高，可用于负载BMP-2。.ASCs以微团形式分布在软骨层，而贴壁黏附分布在下骨支架孔壁，体外以TGF-β1和IGF-1诱导可启动ASCs的软骨向分化，7 d后植入兔关节骨软骨缺损处进行骨软骨组织重建。体内组织学检测、基质成分检测和生物力学检测结果表明体外启动软骨向分化的ASCs微团能够在体内再生透明软骨组织，而未受到下层BMP-2的影响，同时负载BMP-2的下骨支架能够显著促进下骨组织再生，此外，以一体化支架再生的骨软骨组织具备清晰完整的过渡结构。