人脂肪干细胞复合脱细胞软骨基质支架在生物反应器中构建组织工程软骨

Human adipose derived stem cell-seeded articular cartilage acellular matrix in the construction of tissue-engineered cartilage in bioreactor

目的:观察人脂肪干细胞复合脱细胞软骨基质支架在生物反应器中初步构建组织工程软骨的可行性。方法:实验于2005-04/2006-05在解放军总医院骨科研究所完成。脂肪组织和关节软骨均来自膝关节置换术中切除的组织,并经患者知情同意。关节软骨冻干后经粉碎机粉碎,过筛,选取25～38μm大小的软骨微粒。在样品中先加入2.5g/L胰蛋白酶,37℃消化24h,再加入1%Triton X-100震荡72h。将软骨微粒和蒸馏水按1∶3的比例混合后滴加在模板中,置入冷冻干燥机冻干后行紫外线交联。紫外线照射8h完成。最后经25kGy 60Co辐照灭菌完成支架制备。取膝关节置换术中切除的髌下脂肪垫,酶消法获得脂肪干细胞,扩增后复合于脱细胞软骨基质制成圆柱状三维支架上(细胞密度5×1010L-1),置于生物反应器中进行诱导培养,同时设静态培养组作为对照,3周后观测大体形态和组织学形态变化,同时进行组织化学(包括番红花O,阿利新蓝染色)和Ⅱ型胶原免疫组织化学分析。结果:生物反应器组诱导培养3周苏木精-伊红染色显示支架结构消失,只有中心区域残存少量支架结构;静态培养组支架结构尚存在,有少量基质分泌。番红花O染色显示生物反应器组细胞外有大量蛋白聚糖沉积,阿利新蓝染色表明有软骨特异性蛋白多糖的聚集;而静态培养组只有部分区域染色且淡于生物反应器组。Ⅰ型胶原免疫组化的结果显示,在生物反应器组细胞能够合成大量软骨细胞特异性胶原成分,而静态培养组呈弱阳性。结论:生物反应器培养明显促进了脂肪干细胞的增殖与软骨分化,是体外构建组织工程软骨的良好方法。

AIM： To observe the possibility of constructing the tissue engineered cartilage with adipose derived stem cells （ADSCs）-seeded human articular cartilage acellular matrix （ACAM） in the bioreactor. METHODS： The experiment was carded out in the Institute of Orthopaedics, General Hospital of Chinese PLA between April 2005 and May 2006. Adipose tissue and human articular cartilage were harvested from knee replacement operations. The patients knew the fact and agreed to participate in the experiment. Human articular cartilage was crushed into small pieces by muller after lyophilization, and sorted through two different meshes to collect only those specimens measuring 25-38 μm. The specimens were subjected to 2.5 g/L trypsin at 37℃ for 24 hours and 1% Triton X-100 for 72 hours. Admixing the specimens with distilled water as the ratio 1：3 in the mould and then cross-link by ultra-violet for 8 hours after freeze-drying. The scaffolds were accomplished after being irradiated by 25 kGy ^60Co. ADSCs was isolated from infrapatellar fat pad excises in knee joint replacement by collagenase and amplified in plates then seeded in the articular cartilage acellular matrix scaffolds at a final cell density of 5×10^10 L^-1, and induced and cultured in bioreactor. At the same time, the parallel static culture was performed as control group. Gross appearance and histological morphologic change were observed and histochemistry stain including Safranin-O and Alcian blue, type Ⅱ immunohistochemistry stain were analyzed after 3 week. RESUULTS： In bioreactor group, scaffold structures disappeared by hematoxylin-eosin staining after 3-week culture, and only a few scaffold structures in the center. In the static culture group, there were scaffold structure and a little matrix. Safranin-O stain showed that a mass of preteoglycans deposited outside cells in the bioreactor group. Alcian blue stain showed special proteoglycan in cartilage clustered. Only partial staining was found in the static culture group and lighter than that in the bioreactor group. The finding of type Ⅰ immunohistochemistry stain showed that cells could synthesize a mass of special collagen of cartilage in the bioreactor group, whereas it was weakly positive in the static culture group. CONCLUSION： Bioreactor can effuciently promote the ADSCs proliferation and chondrogenic differentiation, and it is a preferable method for cartilage differentiation in tissue engineering in vitro.