梯度降解三维支架材料与肌腱细胞复合培养的实验研究

目的　探讨梯度降解肌腱支架材料 (GDBM)与肌腱细胞的生物相容性及肌腱细胞在三维支架上培养的生物学行为。　方法　将肌腱细胞与梯度降解肌腱支架材料体外复合培养 ,单纯培养为对照组。在 8h、2 4h、72h、7d、2个月进行倒置相差显微镜及扫描电镜形态学观察。 1、2、3、4、5、6、7、8d检测细胞增殖 (MTT法 )。流式细胞仪检测细胞周期、细胞DNA指数及肌腱细胞凋亡。通过3 H Proline掺入实验探讨支架材料对肌腱细胞胶原合成的影响。　结果　肌腱细胞在支架材料上呈梭形复层生长 ,GDBM组肌腱细胞第 2天进入对数增长期 ,倍增时间为 3 .2 5d ,而单纯培养对照组倍增时间为 3 75d。GDBM组与单纯培养对照组比较3 H Proline掺入值差异无显著性 (P >0 .0 5 ) ,说明细胞功能未受影响。与支架材料复合培养的肌腱细胞DNA指数为 0 96,增殖指数比对照组高 2 1% ,提示肌腱细胞在三维支架上生长速度快 ,增殖能力强。　结论　梯度降解肌腱支架材料具有良好的生物相容性 ,可作为肌腱细胞的有效载体应用于组织工程化肌腱的构建。

Experimental studies on gradient degradation biomaterials combined with cultured tenocytes in vitro

Objective To explore the biocompatibility of gradient degradation biomaterials(GDBM) and biological behaviors of cultured tenocytes combined with this three-dimension scaffold in tissue engineering. Methods Tenocyes were cultured combining with GDBM in vitro,while cells cultured in Petri dish as control group. The morphological characters was observed with phase contrast microscope and electron microscope in 8h,24h,72h,7d,2morths;Cell proliferation(MTT) was detected in 1d,2d,3d,4d,5d,6d,7d and 8d;Mitotic cycle,DNA index and apoptosis were detected with flow cytometer. Collagen synthesis were tracked by incorporated radioactive 3H labeled proline into culture. Results The tenocytes attach to GDBM and grow along fibers in spindle-like manner. Cells in GDBM group reach logrithm increase period at the second day. Its doubling time was 3.25d,while cells in control group was 3.75 d. There was no difference of 3H-Proline value between the two groups ( P >0.05),hints that the tenocyte's function was not disturbed. The DNA index of cells of GDBM group was 0.96 and 2.1% higher than the control group,indicating that the tenocytes grow and proliferate faster when being combine cultured on GDBM. Conclusion GDBM show good biocompatibility combined with tenocytes and they are promising extracellular matrix scaffold for cell transplantation in tendon tissue engineering.