脂肪源干细胞与聚丙烯网片的生物相容性

Biocompatibility of adipose-derived stem cells and polypropylene mesh

目的 观察脂肪源干细胞（ADSC）与聚丙烯网片体内外的生物相容性.方法 制备兔脂肪源性干细胞悬液,行流式细胞术鉴定.取聚丙烯网片浸提液培养ADSC.用MTT法检测24、48、72 h实验组（聚丙烯网片材料浸提液）、阴性对照组（10％FBS的低糖DMEM）与阳性对照组（6.4％苯酚溶液）的细胞活力,评价支架细胞毒性.ADSC传代扩增后,接种到聚丙烯网片支架上,体外培养1周,用扫描电子显微镜观察细胞在支架上黏附生长及增殖.分别将聚丙烯网片、ADSC复合聚丙烯网片植入新西兰大白兔的腹直肌表面,4周后观察网片腐蚀、粘连情况,HE染色进行组织学观察,采用RT-PCR技术动态检测网片周围组织VEGF mRNA的表达水平.结果 ADSC强阳性表达CD90、CD44及CD73 （98.54％、95.32％、98.49％）,低表达CD45、CD34及CD14（1.21％、3.01％、2.14％）.ADSC在聚丙烯网片浸提液中可保持较高的增殖率,24、48、72 h实验组细胞相对增殖率分别为（97.0±12.5）％、（96.0±10.3）％、（101.0±22.8）％,与阴性对照组比较,差异均无统计学意义（均P＞0.05）,与阳性对照组比较,差异均有统计学意义（均P＜0.05）,聚丙烯网片浸提液无细胞毒性.ADSC种植于两种支架材料后生长速度快,聚丙烯网片、ADSC复合聚丙烯网片均有不同程度的腐蚀、粘连,但聚丙烯网片粘连更致密.与聚丙烯网片比较,ADSC复合聚丙烯网片诱导慢性炎性反应轻,炎性反应评分低[0.6（0.3,0.9）比1.1（0.9,1.4）,P=0.001],新生血管形成[2.6（2.1,3.1）比1.7（1.2,2.1）,P=0.000]和成纤维细胞增殖[2.2（0.9,2.6）比0.9（0.6,1.5）,P=0.001]评分更高.ADSC复合聚丙烯网片周围组织VEGF mRNA含量（1.53±0.11）高于聚丙烯网片（0.96±0.05） （t=1 1.005,P＜0.05）.结论 聚丙烯网片支架与ADSC具有良好的生物相容性,可作为脂肪组织工程较理想的生物支架材料.

Objective To investigate the biocompatibility of adipose-derived stem cells （ADSC） and polypropylene mesh in vitro and in vivo.Methods The rabbit ADSC suspension was prepared,and identified by flow cytometry.The polypropylene mesh extract was used to culture ADSC.MTT assay was used to determine the cell viability in the 24,48,72 h experimental groups （polypropylene mesh extract）,negative control group （10% FBS of low glucose DMEM）,and positive control group （6.4% phenol solution）,and evaluate the scaffold cytotoxicity.After passaged and amplified,ADSC were inoculated on polypropylene mesh scaffold for 1-week in vitro culture.The growth and proliferation of ADSC on the scaffold were determined by scanning electron microscope.The polypropylene mesh and ADSC-polypropylene mesh were transplanted in the surface of rabbit rectus abdominis,and the mesh corrosion and adhesion were determined at 4 weeks.Hematoxylin-eosin （HE） staining was used for histological test.RT-PCR was used to dynamically measure the expression levels of VEGF mRNA in tissues surrounding the mesh.Results The ADSC CD90,CD44 and CD73 （98.54%,95.32%,98.49%） were strongly positive expressed,and CD45,CD34 and CD14 （1.21%,3.01%,2.14%） were low expressed.The ADSC could maintain a high proliferation rate in the polypropylene mesh extract.The relative proliferation rate in the 24,48 and 72 h experimental groups were （97.0±12.5）%,（96.0±10.3）%,and （101.0±22.8）%,respectively.There were no statistically significant differences between the experimental groups and the negative control group （all P〉 0.05）.There were statistically significant differences between the experimental groups and the positive control group （all P〈0.05）.No cytotoxicity was found in polypropylene mesh extract.ADSC grew fast after transplanted in the two kinds of scaffold.Both of the polypropylene mesh and ADSC-polypropylene mesh had different degrees of corrosion and adhesion,whereas the polypropylene mesh adhered more tightly.Compared with the polypropylene mesh,ADSC-polypropylene mesh showed mild-induced chronic inflammatory response,low score of inflammatory response [0.6 （0.3,0.9） vs 1.1 （0.9,1.4）,P=0.001],and higher scores of neovascularization [2.6 （2.1,3.1） vs 1.7 （1.2,2.1）,P=0.000] and fibroblastic proliferation [2.2 （0.9,2.6） vs 0.9 （0.6,1.5）,P=0.001].The VEGF mRNA content in the surrounding tissues of the ADSC-polypropylene mesh （1.53±0.11） was higher than that of the polypropylene mesh （0.96±0.05）（t=1 1.005,P〈 0.05）.Conclusion Polypropylene mesh scaffold and ADSC show good biocompatibility,which can be used as an ideal biological scaffold material for adipose tissue engineering.