CAJ | 학술논문

目的探讨利用自体骨髓间质干细胞外基质(autologous bone marrow mesenchymal stemcell-derived extracellular matrix,aBMSC-dECM)支架体外制备组织工程软骨的可行性.方法取2周龄新西兰大白兔5只,分离、培养骨髓间质干细胞,原代培养4周,收集其分泌的细胞外基质,制备aBMSC-dECM支架.对支架行扫描电镜和HE染色观察.分离培养自体软骨细胞,植入支架内,48 h后对细胞-支架复合物行Live-Dead染色.分别于种植后1、2、4和6周对细胞-支架复合物(组织工程软骨)进行大体观察、体积测量、HE染色、Safranin-O染色、Ⅱ型胶原免疫组织化学染色、Real-TimePCR检测和抗压强度测试.对照为atelocollagen支架.结果 aBMSC-dECM支架呈三维多孔状海绵样结构,孔隙分布均匀,连通性较好,孔径(304.4±108.2) μm,孔隙率93.3％±4.5％.与atelocollagen支架组比较,aBMSC-dECM支架组组织工程软骨呈乳白色,表面光滑有弹性,随观察时间延长体积逐渐增大,软骨细胞数量、蛋白聚糖和Ⅱ型胶原含量逐渐增多,Ⅱ型胶原及Aggrecan的mRNA持续高表达,抗压强度持续增高.结论 aBMSC-dECM支架有利于维持软骨细胞活性和生物学功能,促进组织工程软骨形成.
목적 탐토이용자체골수간질간세포외기질(autologous bone marrow mesenchymal stemcell-derived extracellular matrix,aBMSC-dECM)지가체외제비조직공정연골적가행성.방법 취2주령신서란대백토5지,분리、배양골수간질간세포,원대배양4주,수집기분비적세포외기질,제비aBMSC-dECM지가.대지가행소묘전경화HE염색관찰.분리배양자체연골세포,식입지가내,48 h후대세포-지가복합물행Live-Dead염색.분별우충식후1、2、4화6주대세포-지가복합물(조직공정연골)진행대체관찰、체적측량、HE염색、Safranin-O염색、Ⅱ형효원면역조직화학염색、Real-TimePCR검측화항압강도측시.대조위atelocollagen지가.결과 aBMSC-dECM지가정삼유다공상해면양결구,공극분포균균,련통성교호,공경(304.4±108.2) μm,공극솔93.3％±4.5％.여atelocollagen지가조비교,aBMSC-dECM지가조조직공정연골정유백색,표면광활유탄성,수관찰시간연장체적축점증대,연골세포수량、단백취당화Ⅱ형효원함량축점증다,Ⅱ형효원급Aggrecan적mRNA지속고표체,항압강도지속증고.결론 aBMSC-dECM지가유리우유지연골세포활성화생물학공능,촉진조직공정연골형성.
Objective To evaluate the feasibility of the formation of tissue engineering cartilage in vitro by using autologous bone marrow mesenchymal stem cell-derived extracellular matrix (aBMSC-dECM)scaffold.Methods The autologous bone marrow mesenchymal stem cells were harvested from 5 New Zealand white rabbits which are two weeks old.The extracellular matrix was collected after 4 weeks culture and fabricated into aBMSC-dECM scaffold.The scaffold was investigated by a scanning electron microscope and HE staining.The autologous articular chondrocytes were isolated,cultured and seeded into the aBMSC-dECM scaffold.Live-Dead staining was analyzed 48 h after seeding.Gross morphological and volume measurement,HE staining,Safranin-O staining and type Ⅱ collagen immunohistochemistry staining,RT-PCRassay,and compression strength test were operated for the cell-scaffold composite at 1,2,4,and 6 weeks after the cultivation respectively.Atelocollagen scaffold was used as the control group.Results The aBMSC-dECM scaffold was three-dimensional and spongy.The engineered cartilage in aBMSC-dECM scaffold group appeared milk white in color with smooth and glossy surface.Compared with the atelocollagen scaffold group,the volume of engineered cartilage in aBMSC-dECM scaffold group significantly grew with time,the chondrocyte,proteoglycan and type Ⅱ collagen were gradually accumulated,the mRNA of type Ⅱ collage and Aggrecan were constantly expressed,and the compressive strength significantly increased with time.Conclusion The aBMSC-dECM scaffold could enhance the viability and biological function of chondrocytes,and promote engineered cartilage regeneration.It could be a novel candidate scaffold for cartilage tissue engineering.