第三代超声辅助吸脂获取的hADSCs构建组织工程软骨的实验研究

Isolation of Human Adipose-Derived Stromal Cells Using Suction-Assisted or Third-Generation Ultrasound-Assisted Lipoaspiration for Constructing Tissue Engineered Cartilage

目的观察应用第三代超声辅助吸脂技术获取的人脂肪干细胞(hADSCs)与残耳软骨细胞共培养在体内构建组织工程软骨的效果。方法分离培养先天性小耳畸形患者来源的残耳软骨细胞,通过第三代超声辅助吸脂和常规负压吸脂术获取脂肪,并提取hADSCs。实验分为3组:对照组1,PGA/PLA支架接种残耳软骨细胞;对照组2,PGA/PLA支架接种残耳软骨细胞+常规负压抽脂获取的hADSCs;实验组,PGA/PLA支架接种残耳软骨细胞+第三代超声辅助抽脂获取的hADSCs。所有细胞-支架复合物经体外培养4周后植入裸鼠体内,8周后取材。对体外培养4周和体内培养8周的各组标本分别进行大体观察、湿重测量、糖胺多糖含量测定、组织学及免疫组化染色和生物力学检测。结果应用第三代超声辅助吸脂和常规负压吸脂都可获得hADSCs。细胞-支架复合物体外培养4周,对照组1、2与实验组都形成软骨样组织,组织学观察显示软骨特异性ECM沉积,但结构疏松,仍有未降解的PGA支架材料。体内培养8周后取材,对照组1组织学观察显示软骨组织不均一,大部分区域形成成熟软骨组织,但有部分区域组织结构疏松,软骨特异性ECM分泌较少;对照组2和实验组则形成成熟的软骨组织,结构均一,可见大量的软骨陷窝和细胞外基质分泌,支架材料完全降解。湿重、糖胺多糖、生物力学检测结果都显示对照组2和实验组显著优于对照组1,对照组2与实验组无显著差异。结论应用第三代超声辅助吸脂可安全有效地获取hADSCs,与残耳软骨细胞共培养可形成成熟的组织工程软骨。

Objective To investigate the formation of tissue engineered cartilage by culture microtia chondrocytes and ADSCs obtained by suction-assisted lipoaspiration(SAL)or third-generation ultrasound-assisted lipoaspiration(UAL).Methods Microtia chondrocytes were isolated from patients with congenital microtia.ADSCs obtained by suction-assisted lipoaspiration or third-generation ultrasound-assisted lipoaspiration were isolated in vitro.There are 3 groups:Ctrl group 1,microtia chondrocytes were seeded on the PGA/PLA scaffolds;Ctrl group 2,microtia chondrocytes and ADSCs obtained by suction-assisted lipoaspiration were seeded on the PGA/PLA scaffolds;Exp group,microtia chondrocytes and ADSCs obtained by third-generation ultrasound-assisted lipoaspiration were seeded on the PGA/PLA scaffolds.After in vitro culture for 4 weeks and subcutaneous implantation for 8 weeks,the constructs were harvested for gross observation,wet weight measurement,glycosaminoglycan(GAG)quantification,histological and immunohistochemical staining and biomechanical evaluation.Results ADSCs can be obtained by SAL or third-generation UAL.In vitro culture for 4 weeks,chondroid tissues were formed in ctrl group 1,ctrl group 2 and Exp group.Histological observation showed chondrogenic ECM deposition,loosened structure and undegraded scaffolds.In vivo culture for 8 weeks,histological observation of the ctrl group 1 showed that the cartilage tissue was not uniform,most of the regions formed mature cartilage tissue,but some regions had loose tissue structure and less cartilage specific ECM secretion.In ctrl group 2 and Exp group,very mature cartilage tissues were formed with uniform structure,and a large number of cartilage lacunae and extracellular matrix were secreted,and the scaffold materials had been completely degraded.The test result of wet weight,glycosaminoglycan and biomechanics all showed that the ctrl group 2 and Exp group were significantly better than the ctrl group 1,but ctrl group 2 and Exp group revealed no significant difference.Conclusion The third generation UAL can be used to obtain ADSCs safely and effectively.There are no differences in formation of tissue engineered cartilage by culture microtia chondrocytes and ADSCs obtained by SAL or third-generation UAL.