CAJ | 학술논문

目的制备软骨脱细胞细胞外基质多孔支架,并探讨其与山羊髓核细胞的生物相容性.方法猪关节软骨经研磨、脱细胞、冷冻干燥技术等处理制成三维多孔支架；从山羊腰椎间盘中分离出髓核细胞,培养后获取P1代细胞；四甲基偶氮唑蓝(MTT)检测支架浸提液毒性；将髓核细胞以5×106/ml的密度接种在支架上体外培养48 h,通过倒置显微镜、HE染色、死活细胞染色(LIVE/DEAD染色)、扫描电镜观察细胞在支架上的黏附及活性.结果软骨脱细胞基质多孔支架在敷水状态下光滑透明,分离的髓核细胞呈典型的软骨细胞样形态；MTT检测各组间增殖,其差异不具有统计学意义(P＞0.05)；倒置显微镜、电镜观察髓核细胞呈球状或短梭形均匀地贴附在支架内部,HE染色观察可见髓核细胞均匀分布在支架内部,LIVE/DEAD染色显示全部为绿色荧光(活细胞),未见红色荧光(死细胞).结论软骨脱细胞基质多孔支架在组成上与髓核组织相似,与山羊髓核细胞具有良好的生物相容性,可以作为髓核组织工程的支架材料.
목적 제비연골탈세포세포외기질다공지가,병탐토기여산양수핵세포적생물상용성.방법 저관절연골경연마、탈세포、냉동간조기술등처리제성삼유다공지가；종산양요추간반중분리출수핵세포,배양후획취P1대세포；사갑기우담서람(MTT)검측지가침제액독성；장수핵세포이5×106/ml적밀도접충재지가상체외배양48 h,통과도치현미경、HE염색、사활세포염색(LIVE/DEAD염색)、소묘전경관찰세포재지가상적점부급활성.결과 연골탈세포기질다공지가재부수상태하광활투명,분리적수핵세포정전형적연골세포양형태；MTT검측각조간증식,기차이불구유통계학의의(P＞0.05)；도치현미경、전경관찰수핵세포정구상혹단사형균균지첩부재지가내부,HE염색관찰가견수핵세포균균분포재지가내부,LIVE/DEAD염색현시전부위록색형광(활세포),미견홍색형광(사세포).결론 연골탈세포기질다공지가재조성상여수핵조직상사,여산양수핵세포구유량호적생물상용성,가이작위수핵조직공정적지가재료.
Objective To study the compatibility of acellular cartilage extracellular matrix-derived porous scaffolds with sheep nucleus pulposus cells.Methods Articular cartilage derived from pigs was physically shattered and decellularized,and then made into porous scaffolds with freeze-drying techniques.Nucleus pulposus cells were isolated from the goat lumbar intervertebral disc,and P1 generation were obtained after culturing.The toxicity of leaching liquor from scaffolds was tested by MTT assay.The cells were seeded onto scaffolds with a density of 5 x 106/ml and cultured for 48h in vitro,activity and adhesion for cells on scaffolds were evaluated by inverted microscope,HE staining,LIVE/DEAD staining and scanning electron microscopy.Results Acellular cartilage extracellular matrix-derived porous scaffolds were smooth and transparent,isolated nucleus pulposus cells showed typical chondrocyte-like morphology.MTT assay demonstrated that proliferation among the groups has no significant difference(P＞0.05).Cells showed spherical or short-spindle morphology and attached to the scaffolds evenly under the inverted microscope and scanning electron microscopy,and HE staining confirmed the even attachment of the cells.All the cells showed green fluorescence (live cells) while no red fluorescence (dead cells) was observed after staining with LIVE/DEAD dye.Conclusion The acellular cartilage extracellular matrix-derived porous scaffolds can be used as the nucleus pulposus tissue for sharing similar extracellular matrix composition with nucleus pulposus tissue and possess good cell compatibility with the sheep nucleus pulposus cells.