间充质干细胞复合多孔支架修复骨与软骨缺损的实验性研究

发布时间：2018-06-18 02:03

本文选题：骨髓间充质干细胞(MSCs) + 脂肪来源间充质干细胞(ADSCs)　；参考：《浙江大学》2015年博士论文

【摘要】：软骨缺损在运动损伤中非常常见,临床中表现为膝关节疼痛,处理不当可能导致关节绞索、关节畸形等,最终导致骨关节炎的发生,严重影响患者的生活质量。关节软骨本身血供较差,成熟的软骨细胞发生损伤后依靠自体的修复能力有限。目前临床上采用关节软骨钻孔、微骨折等多种治疗方法尝试修复缺损的软骨,但长期临床效果难以让人满意。当软骨损伤累及软骨下骨时,治疗更为困难。由于开放性损伤、慢性骨髓炎和骨肿瘤切除引起的骨缺损是目前临床实践中的另一项难题。治疗骨缺损的金标准取自体骨植骨术,但取自体骨存在供区损伤和供区骨量有限的局限。异体骨移植存在免疫排斥骨愈合缓慢等问题。近些年来,组织工程技术为骨和软骨缺损的治疗提供了新的途径。骨髓间充质干细胞因具有良好的多向诱导分化潜能是目前研究最多的种子细胞。近些年脂肪来源间充质干细胞具有来源丰富、扩增能力强,同时可以多向诱导分化逐渐成为组织工程领域研究热点。三维支架在组织工程中起着支持引导干细胞增殖并提供三维构建模板的作用。近些年来合成材料应用逐步增多,PLGA是目前FDA批准的可以用于人体组织工程的支架材料,具有降解速度可调节和良好的机械性能,但是组织相容性差,很多研究学者尝试对PLGA支架进行修饰改善其组织相容性取得了一定的成效。明胶海绵是另一种目前临床中广泛应用的商业化产品,因其多孔状结构、良好的组织相容性以及可降解性同时也是一种良好的组织工程支架。在软骨修复中软骨下骨占据重要地位,本研究拟采用羟基磷灰石(HA)改善PLGA组织相容性,同时羟基磷灰石具有骨诱导和骨传导有利于软骨下骨的再生。骨发生的方式分为膜内成骨和软骨内成骨,长骨自然发生的方式和骨折保守治疗的愈合方式均为软骨内成骨。研究表明软骨内成骨可以加速骨愈合,有利于再生骨的血管化。本研究拟采用明胶海绵作为多孔支架,采用体外诱导ADSC软骨分化进行体内修复大鼠软骨缺损,进一步明确软骨内成骨在大鼠骨缺损愈合中的作用。本研究主要分为三部分：(1)PLGA/NHA支架的构建以及MSC在支架上的增殖分布；(2)PLGA/NHA多孔支架修复大鼠关节软骨缺损的实验研究；(3)明胶海绵多孔支架复合成软骨诱导ADSC经软骨内成骨修复大鼠节段性骨缺损的实验研究。第一部分PLGA/NHA支架的构建以及MSC在支架上的增殖分布目的：构建PLGA/NHA支架,并研究MSC在支架上的增殖和分布情况。方法：采用热诱导相分离技术制备PLGA/NHA多孔支架,并采用扫描电镜、力学测试等研究材料的特征。取第三代MSCs,通过MTT、DNA定量实验了解细胞在支架上的增殖情况；通过扫描电镜和CM-Dil荧光染色了解MSc在支架上的粘附分布情况。结果：本研究中的PLGA/NHA支架呈多孔状,平均孔隙率为88.3%±2.8%。生物力学测试结果显示PLGA/NHA支架的弹性模量要大于PLGA支架。MTT和DNA定量结果显示MSC在PLGA/NHA多孔支架上的增殖数量显著高于PLGA多孔支架。扫描电镜显示MSCs在PLGA/NHA支架孔壁上铺展良好并分泌大量基质,激光共聚焦显微镜显示MSCs在PLGA/NHA支架上比PLGA多孔支架上细胞数目更多分布更密集。结论：添加纳米HA颗粒可以改善MSC在PLGA支架上的生长、粘附和PLGA支架的机械性能,通过NHA修饰的PLGA可以作为组织工程支架用于修复骨软骨缺损。第2章PLGA/NHA多孔支架修复大鼠关节软骨缺损的实验研究目的：观察PLGA/NHA多孔支架复合骨髓间充质干细胞修复大鼠关节骨软骨缺损以及移植MSc最终分化命运。方法：分离MSC并用CM-Dil进行标记,将MSc接种到PLGA/NHA和PLGA支架上,植入大鼠股骨远端直径1.5mm全层骨与软骨缺损处,分别在术后6周和12周处死,进行大体观察、HE染色、番红染色以及免疫组化染色等进行评价骨软骨修复情况。结果：研究结果发现术后12周PLGA/NHA-MSCs组软骨缺损处再生的软骨为光滑透明软骨,富含GAG和Ⅱ型胶原,但是不含I型胶原。我们通过CM-Dil追踪MSCs研究发现,CM-Dil标记的细胞在术后12周仍位于修复区域。结论：PLGA/NHA复合MSC可以有效的修复关节骨软骨缺损,改善软骨修复质量,移植的MSC可以在骨软骨修复早期大量存活,改善局部再生微环境,而不需要再添加生长因子。PLGA/NHA复合MSC可以作为一种修复骨软骨缺损的有效组织工程材料,可以进一步在临床上应用。第3章明胶海绵多孔支架复合成软骨诱导ADSC经软骨内成骨修复大鼠节段性骨缺损的实验研究目的：研究明胶海绵多孔支架复合经成软骨诱导的ADSC经软骨内成骨修复大鼠节段性骨缺损的效果。方法：取大鼠腹股沟脂肪按照贴壁法分离ADSCs,将分离的ADSC进行成骨、成脂和成软骨分化诱导进行鉴定。将明胶海绵支架切成方形,扫描电镜观察明胶海绵结构。将ADSCs接种到明胶海绵支架上,并进行DNA定量检测评价ADSC在支架上的增值情况。创建大鼠胫骨骨缺损(2mm)模型。将接种在明胶海绵支架上的ADSC进行成软骨诱导(ADSC-CD)14天,大鼠胫骨缺损处分别采用空白、明胶海绵、明胶海绵-ADSC、明胶海绵-ADSC-CD组进行填充。分别在术后2周、4周和8周取材大鼠胫骨进行X线、microCT影像学评估,进行病理染色评价成骨以及成软骨情况。结果：研究发现我们分离的ADSC可以进行多向分化,ADSC在明胶海绵多孔支架上增殖良好。动物研究发现大鼠骨缺损术后2周明胶海绵-ADSC-CD组已经出现富含GAG的基质和软骨细胞,这表明已经启动软骨内成骨,而空白组和明胶海绵组未见明显GAG基质和软骨细胞出现。术后8周X线和1mircoCT发现明胶海绵-ADSC-CD组胫骨完全愈合,番红O染色表明软骨内骨化已经完成；而明胶海绵-ADSC组胫骨CT仍可见部分骨折线,番红O染色表明仍有部分GAG在钙化改建；空白组和明胶海绵组则骨折线仍清晰可见,番红O染色表明骨折端仍富含GAG软骨基质,表明骨折仍处于软骨内成骨过程中。结论：明胶海绵复合成软骨诱导的ADSC可以加速软骨内成骨,加速骨折愈合,提高骨修复的质量。因为明胶海绵作为支架获取简单,临床可行性强,在临床中应用中有广阔的前景。
[Abstract]:Cartilage defects are very common in sports injury. The clinical manifestation is knee pain. Improper treatment may lead to joint strands and joint deformities, which eventually lead to osteoarthritis and seriously affect the quality of life of the patients. The blood supply of the articular cartilage itself is poor, and the repair ability of the mature soft bone cells is limited after the injury of the mature soft bone cells. At present, a variety of treatment methods, such as articular cartilage drilling and micro fracture, are used to repair the cartilage of the defect, but the long-term clinical effect is difficult to satisfy. It is more difficult to treat the subchondral bone when the cartilage damage involves the subchondral bone. The bone defect caused by the chronic osteomyelitis and bone tumor is the other in the clinical practice. A difficult problem. Autogenous bone grafting is taken for the gold standard of bone defect treatment, but the limitation of donor area injury and limited donor bone volume is found in the autogenous bone. The allograft bone graft has a slow healing of immune rejection.
In recent years, tissue engineering has provided a new way for the treatment of bone and cartilage defects. Bone marrow mesenchymal stem cells are the most widely studied seed cells because of their good multi-directional differentiation potential. In recent years, adipose derived mesenchymal stem cells are rich in source, strong in amplification and can be gradually induced to induce differentiation. The three dimensional scaffold plays a role in supporting the proliferation of stem cells and providing three-dimensional construction templates in tissue engineering. In recent years, the application of synthetic materials has increased gradually. PLGA is a scaffolding material that can be used in human tissue engineering by FDA at present, with degradation speed adjustable and good mechanical properties. But the histocompatibility is poor, and many researchers have tried to modify the PLGA scaffold to improve their histocompatibility. Gelfoam is another commercial product widely used in clinical practice at present. Because of its porous structure, good histocompatibility and biodegradability, it is also a good organization engineering. Bracket.
The subchondral bone occupies an important position in the repair of cartilage. This study intends to use hydroxyapatite (HA) to improve the histocompatibility of PLGA, while hydroxyapatite has bone induction and bone conduction in favor of the regeneration of subchondral bone.
The ways of bone formation are divided into internal and internal osteogenesis of the membrane. The natural occurrence of long bone and the healing method of the conservative treatment are both endochondral osteogenesis. The study shows that the endochondral osteogenesis can accelerate the healing of the bone and facilitate the vascularization of the regenerated bone. This study is to use gelatin sponge as a porous scaffold to induce ADSC cartilage in vitro. Differentiation in vivo to repair cartilage defects in rats, further clarify the role of endochondral ossification in the healing of bone defects in rats.
This study is mainly divided into three parts: (1) the construction of PLGA/NHA scaffold and the proliferation distribution of MSC on the scaffold; (2) experimental study on the repair of articular cartilage defects in rats with PLGA/NHA porous scaffold; (3) experimental study on the segmental bone defect induced by cartilage induced by cartilage by the porous scaffold of gelatin sponge.
The first part is the construction of PLGA/NHA scaffold and the proliferation and distribution of MSC on the scaffold.
Objective: to construct PLGA/NHA scaffold and study the proliferation and distribution of MSC on the scaffold.
Methods: PLGA/NHA porous scaffolds were prepared by heat induced phase separation technique, and the characteristics of the materials were studied by scanning electron microscope and mechanical test. The proliferation of the cells on the scaffold was investigated by MTT, DNA quantitative test, and the adhesion distribution of MSc on the scaffold was investigated by scanning electron microscopy and CM-Dil fluorescence staining in third generations of MSCs.
Results: the PLGA/NHA stent in this study showed a multi pore shape with an average porosity of 88.3% + 2.8%.. The biomechanical test results showed that the elastic modulus of the PLGA/NHA scaffold was greater than the PLGA stent.MTT and DNA quantitative results showed that the number of MSC on the PLGA/NHA porous scaffold was significantly higher than that of the PLGA porous scaffold. The scanning electron microscope showed MSCs in the PLGA/NHA scaffold. The upper wall of the hole was spread well and secreted a large number of substrates. The laser confocal microscope showed that the number of MSCs on the PLGA/NHA scaffold was more denser than the number of cells on the PLGA scaffold.
Conclusion: adding nano HA particles can improve the growth of MSC on PLGA scaffold, adhesion and mechanical properties of PLGA scaffold, and NHA modified PLGA can be used as tissue engineering scaffold for repairing osteochondral defects.
The second chapter is PLGA/NHA porous scaffold for repairing articular cartilage defects in rats.
Objective: To observe the effect of PLGA/NHA porous scaffold combined with bone marrow mesenchymal stem cells on repairing articular cartilage defects and the final differentiation of MSc in rats.
Methods: MSC was separated and marked with CM-Dil. MSc was inoculated on PLGA/NHA and PLGA scaffold. The bone and cartilage defects of the distal femoral diameter of the rat were implanted in the total bone and cartilage defect of the distal femoral diameter of the rat. The gross observation was carried out at 6 weeks and 12 weeks after the operation respectively. The repair of bone and cartilage was evaluated by HE staining, red staining and immunohistochemical staining.
Results: the results showed that the cartilage of PLGA/NHA-MSCs group at 12 weeks after operation was smooth and transparent cartilage, which was rich in GAG and type II collagen, but did not contain I type collagen. We found that CM-Dil labeled cells were still located in the repair area at 12 weeks after the operation by the CM-Dil tracking MSCs study.
Conclusion: PLGA/NHA compound MSC can effectively repair the defect of articular cartilage and improve the quality of cartilage repair. The transplanted MSC can survive in the early period of osteochondral repair and improve the local regeneration microenvironment, but no additional growth factor.PLGA/NHA compound MSC can be used as an effective tissue engineering material for repairing osteochondral defect. For further clinical application.
The third chapter: gelatin sponge porous scaffold combined with cartilage inducing ADSC to repair segmental bone defects in rats by endochondral ossification.
Objective: To study the effect of gelatin sponge porous scaffold combined with cartilage induced ADSC in repairing segmental bone defects in rats.
Methods: the ADSCs of rat's groin fat was separated according to the adherence method, and the separated ADSC was made into bone, fat and chondrogenic differentiation. The Gelfoam scaffold was cut into square, and the gelatin sponge structure was observed by scanning electron microscope. ADSCs was inoculated on the Gelfoam scaffold, and the value added of ADSC on the stent was evaluated by DNA quantitative detection. The rat tibial bone defect (2mm) model was created. The ADSC of the Gelfoam scaffold was induced (ADSC-CD) for 14 days. The shinbone defect of rats was filled with blank, gelatin sponge, Gelfoam -ADSC, and gelatin sponge -ADSC-CD group. The shinbone of rats was taken at 2 weeks, 4 and 8 weeks after the operation, respectively. The X-ray of the rat tibia and microCT shadow were taken respectively. Image evaluation and pathological staining were used to evaluate osteogenesis and chondrogenesis.
Results: the study found that our separated ADSC can be multidifferentiated, and ADSC can proliferate well on the porous scaffold of gelatin sponge. Animal studies found that 2 weeks after the operation of bone defect in rats, the matrix and chondrocytes of the Gelfoam -ADSC-CD group have been found rich in GAG, which indicates that the cartilage has been initiated in the cartilage, but the blank group and the Gelfoam group have not been found. The apparent GAG matrix and chondrocyte appeared. 8 weeks after the operation, X-ray and 1mircoCT found that the tibia of the gelatin sponge -ADSC-CD group was completely healed, and the red O staining showed that the internal ossification of the cartilage was completed, while the shinbone CT in the -ADSC group of gelatin sponge still showed a partial fracture line, and the red O staining showed that there was still some GAG in the calcification, and the blank group and gelatin sponge group were still rebuilt. The fracture line is still clearly visible. The red O staining shows that the fracture end is still rich in GAG cartilage matrix, indicating that the fracture is still in the process of endochondral ossification.
Conclusion: gelatin sponge combined with cartilage induced ADSC can accelerate the endochondral osteogenesis, accelerate the healing of fracture and improve the quality of bone repair. Because gelatin sponge is simple in obtaining the scaffold with a strong clinical feasibility, it has a broad prospect in clinical application.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08

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