组织工程化椎间盘构建及其生物学评定

摘要

目的 探讨人骨髓间充质干细胞(BM-MSCs)经诱导转化为人髓核细胞(NPs)并构建组织工程椎间盘的价值.方法 体外培养胎儿NPs及BM-MSCs并种植在聚乳酸-聚羟基乙酸共聚物(PLGA)支架上,倒置显微镜及扫描电镜进行形态学观察.将载有BM-MSCs和NPs的PLGA支架及BM-MSCs和NPs的细胞悬液植入新西兰大白兔椎间盘中,12周后对椎间盘信号强度按Thompson分级进行评定.分光光度法检测蛋白聚糖并免疫组化检测Ⅱ型胶原的表达.结果 BM-MSCs在与NPs共培养后由梭形、多角形变为成纤维细胞样,且两种细胞在PLGA支架表面贴附,形态正常,生长良好;载有BM-MSCs和NPs的PLGA支架在椎间MRI信号维持、蛋白多糖[PLGA支架组含量为(3.93±0.31)mg/100 mg,对照组为(3.52±0.26)mg/100 mg]及Ⅱ型胶原表达上较对照组差异均有统计学意义(P＜0.05).结论 共培养可促使BM-MSCs向NPs转化,PLGA支架为细胞提供良好的生长环境,其力学性能维持和空间结构保障可以满足BM-MSCs与NPs构建组织工程椎间盘的需要,有效延缓了椎间盘的退变.%Objective To investigate the value of bone marrow-mesenchymal stem cells(BMMSCs)transformed by nucleus pulposus(NPs)for construction of tissue engineering disc.Methods BMMSCs and fetal NPs were cultured in vitro,planted on polylactic acid-polyglycolic acid copolymer(PLGA),and observed with inverted microscope and scanning electronic microscope.PLGA scaffolds with adherent BM-MSCs and NPs,as well as BM-MSCs and NPs suspension were implanted into intervertebral discs of New Zealand white rabbits,respectively.Intervertebral signal intensity was evaluated by Thompson grading 12 weeks later.Proteoglycan and type Ⅱ collagen were determined by spectrophotometric method and immunohistochemistry,respectively.Results Spindle or multi-angular BM-MSCs turned into fibro-like phenotype coculture of BM-MSCs and NPs,which grew well with normal morphology when they attached on PLGA scaffolds.There was statistical difference in intervertebral signal intensity,and the expression of proteoglycan and type Ⅱ collagen between PLGA scaffolds group and control group(P＜0.05),the content of proteoglycan was(3.93 ± 0.31)mg/100 mg in the PLGA scaffolds group whereas(3.52 ± 0.26)mg/100 mg in the control group.Conclusions BM-MSCs can be induced into NPs by cocultivation,and PLGA scaffolds can provide good growing conditions,and maintain high mechanical properties and spacial structure which meet the requirement of tissue engineering disc to prevent degeneration.