摘要
关节软骨以及软骨下骨的损伤是骨科临床的常见疾病。临床对关节软骨损伤治疗的手段不少,但效果都不太理想。研究表明,骨髓基质干细胞作为一种具有多向分化潜能的干细胞,在适当的条件下可以被诱导分化成为软骨细胞和成骨细胞。本实验主要利用骨髓基质干细胞的这一特性,对其进行成软骨和成骨诱导,建立诱导和鉴定体系;并在这基础上,把骨髓基质干细胞与生物支架材料复合培养,在体外构建工程软骨和软骨下骨修复体。本研究主要成果如下:
(1)从人骨髓中成功分离出长梭形、漩涡状分布的贴壁细胞,并通过流式细胞术证明分离并纯化得到的贴壁细胞确实是人骨髓基质干细胞hMSCs。
(2)分离得到的hMSCs在成软骨诱导液的培养条件下高密度培养,得到直径约为0.5 mm的细胞微团。通过阿辛蓝染色、II型胶原的免疫组织化学染色、糖胺聚糖定量qRT-PCR等方法,从组织化学和分子生物学的层面对诱导结果进行检测和验证,最终建立了hMSCs成软骨的诱导和鉴定体系。
(3)分离得到的hMSCs使用成骨诱导液进行单层培养,对成骨诱导21天的细胞进行碱性磷酸酶ALP染色、茜素红染色和I型胶原的免疫组织化学染色、碱性磷酸酶定量和qRT-PCR等,在组织化学和分子生物学的水平上对诱导结果进行检测和验证,最后成功的建立了hMSCs成骨的诱导和鉴定体系。
(4)把得到的hMSCs接种到生物支架材料上。利用前文建立的成软骨诱导和鉴定体系,对COL和COL-HA两种生物支架材料进行比较。结果显示,COL生物支架材料相对于COL-HA生物支架材料更适合用于软骨缺损的修复。
把得到的hMSCs接种到生物支架材料上。利用前文建立成骨诱导和鉴定体系,对COL和COL-HA两种生物支架材料的成骨特性进行比较。比较结果显示,COL-HA生物支架材料相对于COL生物支架材料更适合用于软骨下骨或骨组织缺损的修复。
Articular cartilage and subchondral bone damage is a common orthopedic disease. There are not many treatments that can be used in articular cartilage injury in clinic, yet the effects are not very satisfactory. Studies have shown that bone marrow stromal cells,a kind of pluripotent stem cells,can be induced to differentiate into chondrocytes and osteoblasts under appropriate conditions. This experiment mainly used this feature of bone marrow stromal stem cells to find ways to induce them into chondrocytes and osteoblasts, and also established the induction and appraisal system; Then, on this basis,the bone marrow stromal cells were cultured with the scaffold biomaterials,and the engineering cartilage and subchondral bone restorations were constructed in vitro. The results of this study are as follows:
(1) The adherent cells, with long spindle shapes and whirllike distributions, which were successfully isolated from human bone marrow, have also been proved to be human bone marrow stromal cells (hMSCs) by flow cytometry.
(2) The isolated hMSCs were cultured with high density in the chondrocytes inducing culture solution,then the micromass with a rough diameter of 0.5 mm were obtained.Through alcian blue staining, type II collagen immunohistochemical staining,glycosaminoglycan quantitative, qRT-PCR and other methods,the results of induction were tested on histochemistry and molecular biology levels, ultimately the inducing and identifying systems of hMSCs differentiating into cartilage were established.
(3) The isolated hMSCs were cultured in osteogenic inducing solution by monolayer culture.Cells cultured for 21 days were used to do the experiments of alkaline phosphatase ALP staining, alizarin red staining and type I collagen immunohistochemical staining, quantitative alkaline phosphatase ,qRT-PCR and other methods, the results of induction were tested and verified on histochemistry and molecular biology levels, ultimately the inducing and identifying systems of hMSCs differentiating into osteoblasts were established.
(4) The hMSCs were then seeded in the scaffolds of biomaterials. Using the previously established inducing and identifying systems of hMSCs,the scaffolds of the COL and COL-HA were compared in the properties of differentiating into cartilage. The results showed that COL scaffolds were more suitable for cartilage defect repair than the COL-HA scaffolds.
(5) The hMSCs were seeded in the scaffolds of biomaterials. Using the previously established inducing and identifying systems of hMSCs,the scaffolds of the COL and COL-HA were compared in the properties of differentiating into osteoblasts. The results showed that COL-HA scaffolds were more suitable for the subchondral bone or bone defects repair than the COL scaffolds.
引文
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