脱矿脱细胞骨基质环支架体外构建组织工程化椎间盘纤维环

Demineralized and decellular bone as scaffold for in vitro construction of intervertebral disc anulus fibrosus

目的:探计以脱矿脱细胞骨基质环为支架、纤维环细胞为种子细胞体外培养构建组织工程化椎间盘纤维环的可行性。方法:取兔椎间盘纤维环细胞培养,应用甲苯胺蓝染色和Ⅰ型、Ⅱ型胶原免疫组织化学染色进行鉴定。用纤维蛋白凝胶接种技术将兔椎间盘纤维环细胞接种到经脱矿脱细胞制备的骨基质环支架材料上,体外培养3个月。每月取培养的细胞支架复合体进行大体形态、HE染色光镜检查和扫描电镜观察,并用生化方法检测羟脯氨酸、氨基葡聚糖(GAG)、脱氧核糖核酸(DNA)含量;逆转录聚合酶链反应(RT-PCR)方法检测Ⅰ、Ⅱ型胶原信使核糖核酸(mRNA)表达;免疫组化和蛋白质印迹方法检测Ⅰ、Ⅱ型胶原蛋白表达。结果:培养的第1代细胞甲苯胺蓝染色呈异染性,Ⅰ型、Ⅱ型胶原免疫组化染色均可见阳性表达,表明培养的第1代细胞具有椎间盘纤维环细胞的表型特点。构建的复合体体外培养1、2、3个月时大体呈白色半透明样环状,有光泽,质韧,具有一定弹性,可扭曲;HE染色光镜下见支架孔洞被红染的组织填充,且空洞内的细胞密度逐渐增加;扫描电镜观察材料表面逐渐被组织填充;Ⅰ型、Ⅱ型胶原免疫组化染色均为阳性;培养2个月时复合体羟脯氨酸、GAG、DNA含量明显高于1个月时(P<0.01),3个月时与2个月时比较无显著性差异(P>0.05),各时间点复合体羟脯氨酸、GAG、DNA含量均低于正常纤维环(P<0.05或<0.01);培养1个月时复合体可检测到Ⅰ、Ⅱ型胶原mRNA和蛋白的表达,2个月时与1个月时比较有显著性差异(P<0.01),3个月时与2个月时比较无显著性差异(P>0.05)。结论:以脱矿脱细胞骨基质环为支架、纤维环细胞为种子细胞构建的复合体在体外培养时,细胞能够保持表型特点、逐渐增殖和行使功能,此复合体可被鉴定为类纤维环组织,用其构建组织工程化椎间盘纤维环可行。

Objective：To explore the feasibility of demineralized and decellular bone as scaffold and anulus fibrosus cells as seed cells for in vitro construction of intervertebral disc anulus fibrosus.nethed：Rabbit intervertebral disc anulus fibrosus cells were isolated and cultured in vitro.After tained with toluidine blue and type Ⅰ and Ⅱ collagens immunohistochemistry,the anulus fibrosus cells were seeded into demineralized and decellular scaffolds by fibrin gelatum noculation method,and continued culturing for 3 months in vitro.At the 1tb,2th and 3 month,the cell-scaffold complexes were examined by macroscopy,scanning electron microscopy and histology stained by hematoxylin and eosin （HE）.Hydroxyproline,glycosaminoglycan （GAG） and deoxyribonucleic acid（DNA） were measured with biochemical methods.Messenger ribonucleic acids（mRNA） of type Ⅰ and Ⅱ collagens were determined by reverse transcription-polymerase chain raction （RT-PCR）.Proteins of type Ⅰ and Ⅱ collagens were determined by Western blot and immunohistochemistry.Result：The primary cells displayed intense toluidine blue metachromasia and expressed type Ⅰ and Ⅱ collagens which indicates the ceils retain the phenotypes of the intervertebral disc anulus fibrosus cells.Macroscopically,the cell-scaffold complexes were ring shaped,semitransparent and maleabale which was similar to normal anulus fibrosus as for palpation and texture at each time points.Under light microscope,cell-scaffold complexes revealed more red stained tissues in the pore and the number increased gradually.The pore spaces of the seafolds were filled gradually by tissues observed by scanning electron microscope.Collagen immunohistochemistry demonstrated heterogeneous type Ⅰ and type Ⅱ collagen.The content of hydroxyproline,GAG and DNA in cell-scaffold complexes cuhrured for 2 months was higher than those for l month （P〈0.01）.But there was no significant difference between 2 and 3 months （P〉0.05 ）.Meanwhile , the content of hydroxyproline,GAG and DNA in cell-scaffold complexes was lower than those in normal anulus fibrosus at each time points （P〈0.05,P〈0.01）.The type Ⅰ and typeⅡ collagen mRNA and protein in cell-scaffold complexes could be detected at 1 month.The expression of the type Ⅰ and type Ⅱ collagen mRNA and protein in the cell-scaffold complexes at 1 and 2 months showed significant significance （P〈0.01）,however no significant significance were noted between 2 and 3 months（P〉0.05）.Conelusion：Demineralized and decellular bone as scaffold and anulus fibrosus cells as seed cells for in vitro construction of intervertebral disc anulus fibrosus can retain the phaenotypes,function as proliferation gradually,which can be identified as intervertebral disc anulus fibrosus-like tissues and can be used tissue engineering intervertebral disc anulus fibrosus.