摘要
关节软骨的自我修复能力极为有限,由于创伤、肿瘤等原因引起的软骨损伤很难自我修复,如果得不到有效的处理韵话,往往会导致进行性的关节退变和骨关节炎的形成。临床上治疗软骨损伤的许多传统方法,如微骨折、软骨下钻孔、骨膜和软骨膜移植术等均存在一定的不足。近年来飞速发展的组织工程技术为关节软骨的修复带来了新的思路。组织工程是应用细胞生物学和工程学的原理,对病损组织的结构和功能进行再生和重建的一门新兴学科,其中新型支架材料的研制开发一直是组织工程的研究热点。本研究采用热诱导相分离的方法制备出新型纳米无定型磷酸钙/聚乳酸(ACP/PLLA)三维多孔支架材料,并与碱性成纤维细胞生长因子(bFGF)相复合来探讨它们在关节软骨修复中的应用前景。
第一部分ACP/PLLA复合材料的制备和表征
目的:通过对新型ACP/PLLA复合材料表征来评价其作为软骨组织工程支架材料的可行性。方法:采用热诱导相分离的方法制备ACP/PLLA复合材料,对该复合材料的显微形貌、孔径、孔隙率和力学性能进行表征。观察支架材料在浸泡PBS液后的形貌变化。同时将bFGF生长因子复合在ACP/PLLA材料上,检测bFGF在PBS溶液中的缓释情况。结果:用热诱导相分离方法制备的ACP/PLLA复合材料是一种三维多孔支架材料,材料孔径在100—200μm之间,孔隙率为91%,材料弹性模量为3.46Mpa。当材料浸泡在PBS液后,材料孔壁表面的ACP颗粒会发生形貌的变化,在原位转变为片状纳米磷灰石晶体。当bFGF复合在ACP/PLLA材料上后,bFGF在溶液中能实现缓释。结论:基于组织工程对支架材料的微观要求,新型ACP/PLLA复合材料可望成为一种理想的软骨组织工程支架材料,它可作为生长因子的载体。
第二部分骨髓间充质干细胞的分离、培养及与复合材料的生物相容性研究
目的:探索兔子骨髓间充质干细胞(bMSCs)的体外分离、培养方法,评价ACP/PLLA复合材料与bMSCs的生物相容性。方法:将抽取的兔子骨髓用密度梯度离心和贴壁培养法分离bMSCs,在体外进行细胞的原代和传代培养,观察细胞的生长情况。将第三代的bMSCs种植在ACP/PLLA和PLLA材料上,用MTT法检测细胞在不同材料上的黏附和增殖情况,用扫描电镜和激光共聚焦显微镜观察细胞在材料中的生长情况和形态改变。结果:分离获得的bMSCs在3天左右贴壁,并逐渐变为梭形,在14天左右细胞长满瓶底,而传代后的细胞在7天左右就长满瓶底。将bMSCs接种至ACP/PLLA和PLLA材料后,细胞在ACP/PLLA复合材料上的黏附率为77.8%,而在PLLA材料中的黏附率为51.1%,两者之间有统计学差异(P＜0.05)。细胞增殖实验结果显示在种植后第3天和第6天,ACP/PLLA材料组和平板培养组的细胞数量明显多于PLLA材料组(P＜0.05)。之后,平板培养组的细胞增殖速度明显减慢,而材料组的细胞仍保持较高的细胞增殖速度,在培养的第10天,ACP/PLLA材料组的细胞数量明显多于PLLA材料组和平板培养组(P＜0.05)。扫描电镜和激光共聚焦显微镜观察证实在ACP/PLLA材料组中bMSCs密度要明显高于PLLA材料组。另外,扫描电镜显示bMSCs在ACP/PLLA材料上黏附良好,并开始分泌细胞外基质,提示bMSCs在材料中具有良好的活力。结论:用密度梯度离心和贴壁培养法可以分离并培养得到bMSCs。与PLLA材料相比,ACP/PLLA复合材料更能促进细胞的黏附和增殖,ACP/PLLA复合材料是一种更具优势的软骨组织工程支架材料。
第三部分ACP/PLLA复合材料联合bFGF在兔子骨软骨缺损修复中的应用研究
目的:探索ACP/PLLA复合材料联合bFGF生长因子修复关节软骨缺损的可行性。方法:在21只骨骼发育成熟的兔子股骨内髁制作直径4mm,深5mm的骨软骨缺损,在缺损处分别植入ACP/PLLA材料和bFGF的复合物、PLLA材料和bFGF的复合物或不做任何处理作为空白对照。在术后4周和12周取材,通过大体观察和组织学切片观察骨软骨的修复情况,并对修复组织进行RT—PCR分析,检测修复组织的基因表达情况。结果:在术后4周时,ACP/PLLA/bFGF组和PLLA/bFGF组缺损内均充满一些不成熟的或纤维样的修复组织,但没有明显的软骨组织形成。在术后12周时,ACP/PLLA/bFGF组软骨缺损表面覆盖有一层典型的透明软骨样组织,在软骨层下面也有一层连续的软骨下骨形成。在PLLA/bFGF组中,修复组织主要为纤维软骨组织,软骨下骨形成不明显。而空白对照组无论在4周还是12周缺损处凹陷明显,表面只覆盖有一薄层纤维组织,提示软骨的自我修复能力很差。RT—PCR分析结果显示在ACP/PLLA/bFGF组中有大量的Ⅱ型胶原和aggrecan基因表达,而在PLLA/bFGF组中只有少量的Ⅱ型胶原表达,没有检测到aggrecan基因。结论:用新型ACP/PLLA复合材料联合bFGF生长因子能在体内修复骨软骨缺损。
It has been well established that articular cartilage has a limited capacity for self-repair. Cartilage lesions, whenever resulting from trauma or due to the tumor, often fail to heal spontaneously and may lead to progressive destruction of the joint and the onset of osteoarthritis. Various methods have been developed to augment its healing response, for example, microfracture, subchondral drilling, perichondrial/periosteal grafts. But these efforts generally seem to be quite limited. Recently, the rapid development of tissue engineering has brought great chance for the cartilage repair. Based on the discipline of cell biology, biomaterial science and engineering, the aim of tissue engineering is to restore the structure and function of tissues lost in the injury or disease. Till now, the development of novel scaffold is the main task in cartilage tissue eingineering. In this study, a novel nano-amorphous calcium phosphate/Poly L-lactic acid (ACP/PLLA) scaffold was developed by a thermally induced phase separation technique. And this tissue engineered scaffold incorporated with basic fibroblast growth factor (bFGF) was introduced to repair articular cartilage defect.
Part I The synthesis and characterisation of ACP/PLLA hybrid material
Objective: To characterize the novel ACP/PLLA composite and evaluate the feasibility of this material as a scaffold for cartilage tissue engineering. Methods: The novel ACP/PLLA scaffold was developed by the thermally induced phase separation technique. The morphology, porous size, porosity and the mechanical properties of the scaffold was evaluated. The morphology changes were also observed by scanning electron microscope (SEM) when ACP/PLLA was soaked in PBS solution. Furthermore, when the growth factor bFGF was incorporated into ACP/PLLA, the controlled release of bFGF was examined. Results: The novel ACP/PLLA material was a porous three-dimensional scaffold with the pore size of around 100-200μm, porosity of 91% and elastic modulus of 3.46Mpa. When the scaffold was soaked in PBS solution, ACP particles coated on the PLLA pore walls could experience a fast phase transformation and morphological variation to flake-like crystallites. When the growth factor bFGF was incoporated into the scaffold, bFGF could release in a substained manner. Conclusion: The novel ACP/PLLA hybrid material can act as an ideal scaffold for cartilage tissue engineering and vehicle for growth factor.
Part II The isolation, culture of bMSCs and the biocompatibility of ACP/PLLA material with bMSCs
Objective: To investigate the method of isolation, culture of bone marrow-derived mesenchymal stem cells (bMSCs) from rabbits and evaluate their biocompatibility with ACP/PLLA scaffold. Methods: bMSCs were extracted from the bone marrow in rabbits by density gradient centrifugation method and cultured in vitro regularly. When bMSCs reached confluence, they were subcultured. The cells of the 3nd passage were seeded into ACP/PLLA and PLLA scaffolds to investigate the cell attachment and proliferation by MTT assay. The cell morphology was observed by SEM and Confocal Laser Scanning Microscope (CLSM). Results: The obtained primary bMSCs began to adhere at 3 days. Generally, it would take 14 days for the primary cells to reach confluence, and only 7 days for the passaged cells. When bMSCs were seeded into scaffolds, the cell attachment rate in ACP/PLLA was 77.8%, which was statistically higher than that in PLLA scaffold. The cell proliferation assay showed that the cell number in ACP/PLLA group and monolayer group was much higher than that in PLLA group (P<0.05) at 3 and 6 days. Thereafter, the cell proliferation rate in monolayer group slowed down, while the cells in scaffolds still increased rapidly. At 10 days, the cell number in ACP/PLLA group was higher than that in PLLA group (P<0.05). The SEM and CLSM observation further confirmed that the cell density in ACP/PLLA group was obviously higher than in PLLA group. Additionally, the SEM observation revealed that bMSCs adhered tightly onto the pore surface and began to secret the matrix. Conclusion: bMSCs can be obtained from bone marrow by the density gradient centrifugation method. Compared with PLLA scaffold, ACP/PLLA scaffold can promote the cell attachment and proliferation, thus be a more superior scaffold for cartilage tissue engineering.
Part III The experimental study of osteochondral repair by the combination of ACP/PLLA scaffold and bFGF
Objective: To investigate the efficacy of ACP/PLLA scaffold combined with bFGF to repair osteochondral defects in a rabbit model. Methods: Osteochondral defects (4mm in diameter and 5mm in depth) were created in the medial femoral condyles in 21 skeletally mature rabbits. The defects were either implanted with ACP/PLLA loaded with bFGF, PLLA and bFGF composite or left untreated. The rabbits were sacrificed at 4 and 12 weeks after implantation. Samples were evaluated by macroscopic and histological examination. The gene expression of the newly formed tissue was also detected by RT-PCR analysis. Results: 4 weeks after surgery, the defects were filled with fibrous or immature repair tissue in ACP/PLLA/bFGF and PLLA/bFGF groups. No obvious cartilage tissue was observed in both groups. At 12 weeks, a typical layer of hyaline cartilage was formed in the repair tissue in ACP/PLLA/bFGF group. A continuous layer of subcondral bone was also detected below the cartilage layer. However, the newly formed tissue in PLLA/bFGF group was mainly fibrocartilage with no obvious subcondral bone formed. In control group, the defect was still remained and covered by a thin layer of fibrous tissue whenever in 4 and 12 weeks, indicating the poor self-repair ability of cartilage defects. RT-PCR analysis showed that high levels of type II collagen (Col II) and aggrecan message were detected in repair tissue in ACP/PLLA/bFGF group. In contrast, only a small amount of Col II appeared in PLLA/bFGF group with no aggrecan gene expression detected. Conclusion: The osteochondral defects can be successfully repaired by the combination of ACP/PLLA and bFGF.
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