兔骨髓基质干细胞诱导分化修复膝关节软骨缺损
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摘要
研究目的：运用组织工程学技术进行骨髓基质干细胞(BMSCs)的提取、培养扩增、诱导分化、关节软骨缺损内植入，初步探索BMSCs在软骨修复中的作用机制及可行性。
研究方法：穿刺抽取2月龄新西兰白兔骨髓，采用密度梯度离心+贴壁培养法分离、纯化和扩增BMSCs，检测第三代BMSCs生长曲线和贴壁率。以转化生长因子β1(TGF-β1)、维生素C和地塞米松为诱导因子，对BMSCs进行平面和立体软骨方向诱导，利用Ⅱ型胶原免疫组化和甲苯胺蓝异染性进行检测。同时检测TGF-β1和IGF-1在联合诱导过程中的作用。取平面诱导4天的BMSCs以2×10~7细胞浓度与海藻酸钠混合，经Ca~(2+)作用形成凝胶珠，随机分为2组，分别应用TGF-β1诱导液和TGF-β1和IGF-1诱导液培养6天。相同方法制作不含细胞的空白凝胶和含未诱导的BMSCs的凝胶复合体，完成植入物准备。于20只兔双侧膝关节股骨内侧髁负重面制作3.5mm×8.0mm×3.0mm的软骨缺损模型，分为4组，每组10个膝，分别为空白凝胶组、未诱导组、TGF-β1诱导组和TGF-β1和IGF-1联合作用组，分别植入相应的细胞凝胶复合体。手术后于2、4、8、12、20周进行取材，然后进行相应的组织学分析。
研究结果：应用密度梯度离心法+贴壁培养法可以分离、纯化、扩增兔BMSCs。在TGF-β1、地塞米松和维生素C的联合诱导下，兔BMSCs可以向软骨细胞表型转化。TGF-β1和IGF-1联合作用下可以促进BMSCs增殖，细胞基质分泌增多。BMSCs在海藻酸钠凝胶中生长良好，缓慢增殖，在诱导条件下，凝胶内部BMSCs可以向软骨细胞表型转化。BMSCs诱导后复合海藻酸钠移植至软骨缺损，可以形成类软骨样组织。
研究结论：兔骨髓基质干细胞可以向软骨细胞表型转化并对软骨缺损的修复有促进作用。
Objective Bone mesenchymal stem cells(BMSCs) were isolated,
purificated, introduced, and transplated into cartilage defect with technology of tissue engineering, by which the machnism and feasibility of BMSCs were investigated initially in cartilage resurfacing.
Methods Bone marrow was aspirated from two-month-old New Zealand
white rabbit. Then BMSCs were isolated and purificated from bone marrow by density gradient centrifuge and adherence culture. When cells grew to 80% confluence, they were harvested and designated as passage 1. These cells were further expanded with 1 : 3 splitting. BMSCs at passage 3 were selected to obtain their proliferation curve and adherence rate. Exposed to transforming growth factor-pi (TGF-pl), vitamin C, and dexamethasone in monolayer and in a three-dimensional matrix, chondrocytic differentiation of mesenchymal stem cells was existed. By the immunohistochemical staining of type Ⅱ collagen and the toluidine blue metachromasia, the effect of differentiation was demonstrated. At the same time, the effects of TGF-pl and IGF-1 in the process of separate or unite introducement could be shown. BMSCs, introduced in medium containing 5ng/ml TGF-β 1 for 4 days, were suspended in 1.2% w/v alginate solution in a density of 2.107 cells per milliliter and dropped into a solution of CaCl2 . The alginate/cell susp
ension gelled immediately and formed spherical beads. They were divided into two groups in random, one exposed to TGF-pl in a three-dimensional matrix, the other to IGF-1 and TGF-pl. By the same way, alginate gel whithout or with BMSCs can be made. Based on the difference of composition, alginate beads were divided into 4 groups. After that, the posterior weight -bearing aspect of the medial femoral condyle was exposed and a 3.5mm 8.0mm full -thickness defect was created with a low-speed burr to a depth of 3.0mm. Then the defects were filled with different alginate beads. At 2, 4, 8, 12, and 20 weeks postoperatively, the rabbits were killed and the distal femora were removed. After decalcified, the specimens were embedded in paraffin
2
and Sum sections were cut . Healing of the defect was investigated histologically using Safranin'O -Fast green staining and toluidine blue staining.
Results From this experience, such results can be obtained: First, rabbit
BMSCs may be isolated and proliferation by density gradient centrifuge and adherence culture. Exposed to TGF-pl, vitamin C, and dexamethasone, BMSCs can express markers of chondrogenesis. Second, it is the combination of TGF-pl and IGF-l that facilitates the proliferation of BMSCs and enhances the synthesis of extracellular cartilage matrix. Third, BMSCs in alginate gel grow well and have a low-rate proliferation. If in inductive culture medium, they can differentiate to type of chondrocyte. Fourth, after transplanted into cartilage defect, alginate/cell gel can transform to tissue like hyaline cartilage.
Conclusion After introduced, BMSCs can express markers of chondrogenesis and facilitate the repair of cartilage defect in rabbit.
研究方法：穿刺抽取2月龄新西兰白兔骨髓，采用密度梯度离心+贴壁培养法分离、纯化和扩增BMSCs，检测第三代BMSCs生长曲线和贴壁率。以转化生长因子β1(TGF-β1)、维生素C和地塞米松为诱导因子，对BMSCs进行平面和立体软骨方向诱导，利用Ⅱ型胶原免疫组化和甲苯胺蓝异染性进行检测。同时检测TGF-β1和IGF-1在联合诱导过程中的作用。取平面诱导4天的BMSCs以2×10~7细胞浓度与海藻酸钠混合，经Ca~(2+)作用形成凝胶珠，随机分为2组，分别应用TGF-β1诱导液和TGF-β1和IGF-1诱导液培养6天。相同方法制作不含细胞的空白凝胶和含未诱导的BMSCs的凝胶复合体，完成植入物准备。于20只兔双侧膝关节股骨内侧髁负重面制作3.5mm×8.0mm×3.0mm的软骨缺损模型，分为4组，每组10个膝，分别为空白凝胶组、未诱导组、TGF-β1诱导组和TGF-β1和IGF-1联合作用组，分别植入相应的细胞凝胶复合体。手术后于2、4、8、12、20周进行取材，然后进行相应的组织学分析。
研究结果：应用密度梯度离心法+贴壁培养法可以分离、纯化、扩增兔BMSCs。在TGF-β1、地塞米松和维生素C的联合诱导下，兔BMSCs可以向软骨细胞表型转化。TGF-β1和IGF-1联合作用下可以促进BMSCs增殖，细胞基质分泌增多。BMSCs在海藻酸钠凝胶中生长良好，缓慢增殖，在诱导条件下，凝胶内部BMSCs可以向软骨细胞表型转化。BMSCs诱导后复合海藻酸钠移植至软骨缺损，可以形成类软骨样组织。
研究结论：兔骨髓基质干细胞可以向软骨细胞表型转化并对软骨缺损的修复有促进作用。
Objective Bone mesenchymal stem cells(BMSCs) were isolated,
purificated, introduced, and transplated into cartilage defect with technology of tissue engineering, by which the machnism and feasibility of BMSCs were investigated initially in cartilage resurfacing.
Methods Bone marrow was aspirated from two-month-old New Zealand
white rabbit. Then BMSCs were isolated and purificated from bone marrow by density gradient centrifuge and adherence culture. When cells grew to 80% confluence, they were harvested and designated as passage 1. These cells were further expanded with 1 : 3 splitting. BMSCs at passage 3 were selected to obtain their proliferation curve and adherence rate. Exposed to transforming growth factor-pi (TGF-pl), vitamin C, and dexamethasone in monolayer and in a three-dimensional matrix, chondrocytic differentiation of mesenchymal stem cells was existed. By the immunohistochemical staining of type Ⅱ collagen and the toluidine blue metachromasia, the effect of differentiation was demonstrated. At the same time, the effects of TGF-pl and IGF-1 in the process of separate or unite introducement could be shown. BMSCs, introduced in medium containing 5ng/ml TGF-β 1 for 4 days, were suspended in 1.2% w/v alginate solution in a density of 2.107 cells per milliliter and dropped into a solution of CaCl2 . The alginate/cell susp
ension gelled immediately and formed spherical beads. They were divided into two groups in random, one exposed to TGF-pl in a three-dimensional matrix, the other to IGF-1 and TGF-pl. By the same way, alginate gel whithout or with BMSCs can be made. Based on the difference of composition, alginate beads were divided into 4 groups. After that, the posterior weight -bearing aspect of the medial femoral condyle was exposed and a 3.5mm 8.0mm full -thickness defect was created with a low-speed burr to a depth of 3.0mm. Then the defects were filled with different alginate beads. At 2, 4, 8, 12, and 20 weeks postoperatively, the rabbits were killed and the distal femora were removed. After decalcified, the specimens were embedded in paraffin
2
and Sum sections were cut . Healing of the defect was investigated histologically using Safranin'O -Fast green staining and toluidine blue staining.
Results From this experience, such results can be obtained: First, rabbit
BMSCs may be isolated and proliferation by density gradient centrifuge and adherence culture. Exposed to TGF-pl, vitamin C, and dexamethasone, BMSCs can express markers of chondrogenesis. Second, it is the combination of TGF-pl and IGF-l that facilitates the proliferation of BMSCs and enhances the synthesis of extracellular cartilage matrix. Third, BMSCs in alginate gel grow well and have a low-rate proliferation. If in inductive culture medium, they can differentiate to type of chondrocyte. Fourth, after transplanted into cartilage defect, alginate/cell gel can transform to tissue like hyaline cartilage.
Conclusion After introduced, BMSCs can express markers of chondrogenesis and facilitate the repair of cartilage defect in rabbit.
引文
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2. Silver FH, Glasgold AI. Cartilage wound healing: an overview. Otolaryngol Clin North Am, 1995, 28: 847-864.
3. Lebaron RG, Athanasiou KA. Ex vivo synthesis of articular cartilage. Biometerials, 2000, 21: 2575-2587.
4. Jackson DW, Simon TM. Tissue engineering principles in orthopaedic surgery. Clin Orthop, 1999, 367S: 31-45.
5. Shapiro F, Kiod S, Gincher G.. Cell origin and differentiation in the repair of full thickness defect of articular cartilage. J Bone Jint Surg(Am), 1993, 74(4): 532-544.
6. Randolph MA, Anseth K, Yaremchuk MJ. Tissue engineering of cartilage[J]. Clin Plast Surg, 2003, 30(4): 519-537.
7. Cancedda R, Dozin B, Giannoni P, Quarto R. Tissue engineering and cell therapy of cartilage and bone. Matrix Biology, 2003, 22: 81-89.
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