脱细胞软骨基质来源的多孔支架复合山羊髓核细胞体内初步构建组织工程髓核的实验研究

Construction of tissue engineering nucleus pulposus in vivo by combining acellular cartilage matrix derived porous scaffolds with goat nucleus pulposus cells

[目的]探讨脱细胞软骨基质多孔支架复合PKH26标记的山羊髓核细胞体内异位构建组织工程髓核的可行性。[方法]制备脱细胞软骨基质来源的多孔支架,扫描电镜(scanning electron microscope,SEM)观察、天狼星红染色、HE染色观察、MTT毒性检测;分离山羊髓核细胞,通过倒置显微镜观察、番红O染色、Ⅱ型胶原免疫组化染色进行鉴定;将PKH26标记的山羊髓核细胞接种支架上,体外培养3 d后进行LIVE/DEAD活性染色,将细胞支架复合物置入裸鼠皮下,培养6周,病理切片,荧光显微镜下观察,进行番红O、Ⅰ、Ⅱ型胶原免疫组化染色。[结果]扫描电镜观察支架孔隙相连通且分布均匀,天狼星红染色支架呈黄绿相间色,HE支架淡染,MTT检测细胞增殖曲线无统计学差异(P>0.05);P1代髓核细胞呈软骨样细胞形态,番红O染色、Ⅱ型胶原免疫组化染色均阳性,PKH26标记后的细胞呈红色荧光;体外LIVE/DEAD染色细胞呈绿色荧光,体内培养6周后,带红色荧光的细胞填满支架孔隙,番红O、Ⅱ型胶原免疫组化染色阳性,Ⅰ型胶原免疫组化染色弱阳性。[结论]以脱细胞软骨基质多孔支架复合山羊髓核细胞在体内能够形成组织工程髓核样组织。

[ Objective] To evaluate the feasibility of construction of tissue engineering nucleus pulposus in vivo by combi- ning acellular cartilage matrix porous scaffolds with PKI-I26 labeled goat nucleus pulposus cells. [ Methods ] Porous scaffolds were made of acellular cartilage matrix and evaluated through SEM, Sirius red and HE staining, and toxicity of the scaffolds was assessed by MTr test. P1 generation goat nucleus pulposus cells were identified by safranin O staining and collagen type II immu- nocytochemistry staining. PKH26 labeled cells were seeded onto scaffolds. After 3 d culture in vitro, cell - scaffold hybrids were assessed by LIVE/DEAD staining , then implanted into nude mice subcutaneously for 6w culture. In vivo hybrids were assessed by fluorescence microscope, safranin 0 staining and collagen type Ⅰ、Ⅱ immunocytochemistry staining. [ Results ] Pores in scaffold were evenly distributed and connected under SEM, Sirius red and HE staining showed evenly distributed pores. MTr as- say demonstrated proliferation among the groups had no significant difference （ P 〉 0.05 ） . P1 generation cells showed chondro- cyte- like morphology and stained positively for safranin 0 and collagen type Ⅱ immunocytochemistry. PKH26 labeled cells showed red fluorescence, cells on scaffolds in vitro showed green fluorescence by LIVE/DEAD staining. After 6w in vivo culture, through fluorescence microscope, safranin 0 staining and collagen type Ⅰ、Ⅱ immunocytochemistry staining showed positive. [ Conclusion] Hybrid of acellular cartilage matrix and goat nucleus pulposus cells can produce nucleus pulposus tissue in vivo.