Abstract:

Tang Hui, Yan Hongtao, Chen Nian, Yang Hang, Yang Guihong, Wu Jinjin Department of Dermatology, Daping Hospital, The Third Military Medical University, Chongqing 400042, China Corresponding author: Wu Jinjin, Email: wjjjj@163.com 【Abstract】 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.