摘要：
目的：探讨三维动态培养条件下不同灌注流速对复方壳多糖组织工程真皮生长代谢的影响。方法将人真皮成纤维细胞（HDF）以2×104/ml的终浓度接种到复方壳多糖胶原凝胶中，采用自制的灌注培养装置，培养基分别以50、100和200 ml/min的流速灌注培养12 d，以静态培养为对照。用倒置显微镜观察细胞形态，MTT法检测细胞活力，葡萄糖及乳酸测试盒检测细胞代谢特性，HE染色观察细胞形态和分布，羟脯氨酸测试盒检测胶原合成，同时用改进后的高敏度小张力膜状生物材料力学检测装置检测最大抗张强度。统计分析采用重复测量方差分析、单因素方差分析及LSD⁃t检验。结果灌注组细胞比对照组更密集，第8天时100 ml/min灌注组细胞几乎全部融合成片，200 ml/min灌注组细胞约达80%融合，且有沿培养基流动方向生长的趋势。MTT结果显示，4组细胞增殖活性（A值）都随培养时间的延长先持续上升后逐渐下降（P<0.05），其中灌注组的细胞增殖活性均明显高于对照组，100 ml/min灌注组的最大细胞增殖活性是对照组的1.67倍，且显著高于其他组（均P<0.05）。各组培养基中葡萄糖含量随培养时间延长均下降（P<0.05），第12天时100 ml/min灌注组的葡萄糖含量（1.604±0.038 mmol/L）显著低于对照组（2.205±0.020 mmol/L）、50 ml/min灌注组（1.939±0.037 mmol/L）及200 ml/min灌注组（2.047±0.039 mmol/L），均P<0.05。各组乳酸含量随时间变化的趋势与葡萄糖含量的变化相反，均缓慢上升（P<0.05），但其最大值对真皮内HDF影响不明显。培养第12天，100 ml/min灌注组的羟脯氨酸含量及最大抗张强度均显著高于其余3组，且组间差异均有统计学意义（F=61.512、694.216，均P<0.05）。培养第6、10天，灌注培养条件下细胞在支架中的分布比静态培养时均匀，胞核较大、长，且灌注组细胞数量显著高于对照组（均P<0.05），其中100 ml/min灌注组的细胞数量最多，显著高于其他灌注组（均P<0.05）。结论100 ml/min的灌注流速更适合培养复方壳多糖组织工程真皮，此流速下的细胞活力、细胞分布、代谢速度、胶原合成及最大抗张强度都优于其他组。%Objective To evaluate effects of different medium perfusion rates on the growth and metabolism of composite chitosan⁃based tissue⁃engineered dermis in a dynamic three⁃dimensional culture system. Methods Human dermal fibroblasts (HDFs) at a density of 2 × 104/ml were inoculated onto chitosan⁃collagen composite hydrogels, and then cultured in a self⁃made perfusion culture device with the medium perfusion rates being 50, 100 or 200 ml/min for 12 days(perfusion culture groups), and statically cultivated HDFs served as the control group. An inverted microscope was used to observe morphological changes of HDFs, and methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate proliferative activity of HDFs. Test kits were used to determine glucose and lactate levels to evaluate cellular metabolic activity, and hydroxyproline content was measured to estimate collagen synthesis. An improved highly sensitive mechanical testing device was used to measure the tensile strength of the tissue⁃engineered dermis, and hematoxylin and eosin (HE) staining was performed to observe the morphology and distribution of HDFs. Statistical analysis was carried out by repeated⁃measures analysis of variance, one⁃way analysis of variance and the least significant difference(LSD)⁃t test. Results Compared with the control group, HDFs were distributed more densely in the perfusion culture groups. After 8⁃day culture, HDFs, which tended to grow along the direction of medium flow, grew to almost complete confluence in the 100⁃ml/min perfusion culture group, and to 80%confluence in the 200⁃ml/min group. As MTT assay showed, the proliferative activity of HDFs (expressed as absorbance[A]value)increased initially, but then gradually decreased over time in all the above 4 groups(all P<0.05). Moreover, perfusion culture groups all showed significantly higher cellular proliferative activity compared with the control group, and the maximum cellular proliferative activity in the 100⁃ml/min group was 1.67 times that in the control group, and significantly higher than that in the other perfusion culture groups(both P<0.05). The levels of glucose in the 4 groups all decreased over time(all P < 0.05), and the 100⁃ml/min group(1.604 ± 0.038 mmol/L)showed significantly lower levels of glucose compared with the control group(2.205 ± 0.020 mmol/L, P<0.05), 50⁃ml/min group (1.939 ± 0.037 mmol/L, P<0.05)and 200⁃ml/min group(2.047 ± 0.039 mmol/L, P <0.05)after 12⁃day culture. However, in contrast to glucose, the levels of lactate changed in the opposite direction, and increased gradually over time in all the 4 groups(P < 0.05), but even the maximum level of lactate had no obvious effect on HDFs in the dermis. On day 12, the 100⁃ml/min group showed significantly higher hydroxyproline levels and maximum tensile strength compared with the other three groups(F=61.512, 694.216, respectively, both P<0.05). On days 6 and 10, all the perfusion culture groups showed increased quantities (all P < 0.05) of more evenly distributed HDFs with larger and longer nuclei in scaffolds compared with the control group. Moreover, the amounts of cells were significantly larger in the 100⁃ml/min group than in the other perfusion culture groups(both P<0.05). Conclusions The medium perfusion rate of 100 ml/min is optimal for the culture of composite chitosan⁃based tissue⁃engineered dermis. At this medium perfusion rate, cellular proliferative activity, cell distribution, metabolic rates, collagen synthesis and the maximum tensile strength were more favourable compared with those at other perfusion rates.
