CAJ | 학술논문

目的探讨可缓释诱导因子支架的制备及一期获取自体脂肪来源干细胞(ADSCs)修复猪膝关节软骨缺损的可行性.方法利用冷冻干燥技术制备冻干缓释支架,应用Elisa方法检测支架缓释诱导因子[转化生长因子- TGF-β2 (TGF-β2)、胰岛素样生长因子-1(IGF-1)]的浓度.采用密度梯度离心法分离获取猪ADSCs,取2个细胞支架复合体,体外培养3周后观察细胞在支架内的分布情况及软骨外基质的Ⅱ型胶原和蛋白聚糖的分泌情况.制备猪膝关节7 mm×7 mm正方形软骨缺损,同时一期获取足量ADSCs并与支架复合后一期植入缺损部位,观察软骨缺损的修复效果(实验组)；以猪膝关节微骨折处理作为对照组,每组3只猪.实验动物于术后2、4个月取材,行大体观察及组织学观察.结果冻干缓释支架在体外可以缓慢释放诱导因子,释放浓度未呈现迅速下降趋势.体外培养3周后ADSCs在支架内分布广泛、均匀,软骨特异性指标Ⅱ型胶原及蛋白聚糖呈阳性表达.实验组术后2个月有部分透明软骨修复,术后4个月缺损区被软骨组织样填充修复；HE染色显示有典型的透明软骨结构,但细胞密度较正常软骨组织高.对照组关节软骨缺损未能修复,表面充填纤维组织.结论一期可获取足量用于移植的ADSCs,ADSCs复合在具有诱导分化功能的支架上后可以向软骨细胞分化,从而修复猪膝关节软骨缺损.
목적 탐토가완석유도인자지가적제비급일기획취자체지방래원간세포(ADSCs)수복저슬관절연골결손적가행성.방법 이용냉동간조기술제비동간완석지가,응용Elisa방법검측지가완석유도인자[전화생장인자- TGF-β2 (TGF-β2)、이도소양생장인자-1(IGF-1)]적농도.채용밀도제도리심법분리획취저ADSCs,취2개세포지가복합체,체외배양3주후관찰세포재지가내적분포정황급연골외기질적Ⅱ형효원화단백취당적분비정황.제비저슬관절7 mm×7 mm정방형연골결손,동시일기획취족량ADSCs병여지가복합후일기식입결손부위,관찰연골결손적수복효과(실험조)；이저슬관절미골절처리작위대조조,매조3지저.실험동물우술후2、4개월취재,행대체관찰급조직학관찰.결과 동간완석지가재체외가이완만석방유도인자,석방농도미정현신속하강추세.체외배양3주후ADSCs재지가내분포엄범、균균,연골특이성지표Ⅱ형효원급단백취당정양성표체.실험조술후2개월유부분투명연골수복,술후4개월결손구피연골조직양전충수복；HE염색현시유전형적투명연골결구,단세포밀도교정상연골조직고.대조조관절연골결손미능수복,표면충전섬유조직.결론 일기가획취족량용우이식적ADSCs,ADSCs복합재구유유도분화공능적지가상후가이향연골세포분화,종이수복저슬관절연골결손.
Objective To investigate the feasibility of repairing at one stage defects of porcine articular cartilage of the knee with autologous adipose-derived stem cells (ADSCs) and collagen Ⅰ scaffolds with slow-release inducing factors.Methods We first made collagen Ⅰ scaffolds with slow-release inducing factors using freeze drying technology.The concentrations of slow-release inducing factors(transformation growth factor-β2,insulin-like growth factor-l) were evaluated by Elisa.The porcine ADSCs,obtained by density gradient centrifugation,were seeded onto the collagen Ⅰ scaffolds with slow-release inducing factors for in vitro culture for 3 weeks to observe the cellular distribution and secretion of type Ⅱ collagen and aggrecan within the scaffold.Porcine models of full thickness defects of the knee articular cartilage were created,7 ×7mm in size.ADSCs and collagen Ⅰ scaffolds were implanted into the cartilage defects in the experimental group (3 pigs) while micro-fractures were made in the subchondral bone and treated with absorbable membranes in the control group (3 pigs).Gross observation and histological analyses were conducted 2 and 4months after operation to assess defect healing in the 2 groups.Results The inducing factors were slowly released in the scaffolds with slowly reduced concentrations.The ADSCs distributed extensively and expressions of type Ⅱ collagen and aggrecan were observed in the scaffolds after 3-week in vitro culture.In the experimental group,edges of the articular cartilage defects were filled with reparative hyaline cartilage after 2 months,and the whole defects were repaired by the hyaline cartilage 4 months later.HE staining showed typical cartilaginous structure in the repaired area,though its cellular density was higher than in the normal cartilage.In the control group,defects were not repaired but filled with fibrous tissue.Conclusions Enough autologous porcine ADSCs can be obtained at one stage for implantation.ADSCs seeded onto the collagen Ⅰ scaffolds with slow-release inducing factors may be differentiated into chondrocytes to repair porcine articular cartilage defects.