Effect of water soluble chitosan on the growth and phenotype of fibroblasts in vitro
- VernacularTitle:水溶性壳聚糖对体外培养成纤维细胞生长及表型的影响
- Author:
Xiangxin CHE
;
Jun CAO
;
Weidong LI
;
Yidong WANG
;
Ping WU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;11(31):6282-6285
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Studies have demonstrated that chitosan can indirectly promote the proliferation of fibroblast and the synthesis of collagen, Using chitosan and specific fibroblasts together to construct tissue-engineering materials for tendon repair may be an adequate way to promote healing and prevent adhesion during the healing process.OBJECTIVE: To observe the effect of water soluble chitosan (WSC) on the growth and genotype of 3T3TK fibroblasts.DESIGN: Controlled experiment based on observation.SETTING: Jiujiang University Medical College.MATERIALS: 3T3TK fibroblasts were provided by Cell Bank of Chinese Academy of Sciences. WSC (specification: 60 mesh, 30CPS, Jinan Haidebei Marine Bioengineering Co.,Ltd),DMEM (low sugar) (Gibco Company, USA), fetal bovine serum (FBS, Sijiqing Biological Engineering Institute, Hangzhou), penicillin, streptomycin (Biosharp Company, USA),trypsin (BioEev-Tech Scientific & Technical Co.,Ltd, Beijing).METHODS: This experiment was carried out in the Laboratory for Clinical Application of Biology, Center for Medial Scientific Research, Jiujiang University between November 2004 and April 2006. ①Cells in the log phase were digested with 2.5 g/L trypsin to produce single cell suspension and inoculated into a 96-well culture plate at a density of 6 000 cells /200 μL medium. Five groups were prepared, 5 holes per group. 200 μL cell suspension was added into each well.After 24 hours of cultivation, supernatant was discarded, 200 μL DMEM with 1, 0.1, 0.01 and 0.000 1 g/L chitosan was added respectively in the first four groups. The remaining group was taken as the negative control group, in which 200 μL DMEM medium without chitosan was added. The cell suspension was routinely cultured for 72 hours. The cell viability was measured by CellTilter- GloTM Luminescent Cell Viability Assay according to the manufacturer's protocol. Cells in the log phase were split and inoculated into 24-well culture plates. Four groups were prepared (5 holes per group). 1 mL DMEM medium supplemented with 1, 0.1, 0.01 g/L chitosan was added into the first 3 groups respectively, and the 4th group was set as control group. Hydroxyproline and alkaline phosphatase(ALP) kits were used for detecting the contents of collagen and ALP in the supernatant of fibroblasts.MAIN OUTCOME MEASURES : ①Effect of WSC on viability of fibroblasts cultured in vitro.②Contents of collagen and ALP in the cell culture supernatant of fibroblasts.RESULTS:①Detection results of viability of fibroblasts: There were no significant differences in viability of fibroblasts between each chitosan group and control group (P ＞ 0.05).②Contents of collagen and ALP in the cell culture supernatant of fibroblasts: Hydroxyproline content in the cell culture supernatant of fibroblasts of 1 g/L and 0.1 g/L groups was (7.598±0.770) and (8.366±0.308)mg/L, respectively, which was higher than that of control group [(11.269±0.707)mg/L,P ＜ 0.01]; Collagen content in the 1 g/L and 0.1 g/L groups was(56.703±5.748) and(62.437±2.301)mg/L, respectively, which was lower than that of control group [(84.099±5.280)mg/L,P ＜ 0.01]. With the concentration of chitosan increasing, the collagen content was decreased in a dose-dependent manner. There were no significant differences in ALP activity in the cell culture supernatant between each chitosan group and control group (P ＞0.05).CONCLUSION: WSC does not obviously inhibit the viability of 3T3TK fibroblasts, but can markedly reduce the content of secreted collagen. It is indicated that chitosan can be used as tissue engineering material for tendon repair, and inhibit adhesion formation during tendon repair.
