Effects Of Cyclic Mechanical Stimulation On Proliferation And Collagen Expression Of Human Dermal Fibroblasts

The healing of skin wound is an orderly complex and dynamic biological procedure. This procedure is dependent on the interaction between fibroblast and extracellular matrix (ECM). Physiologically, human dermal fibroblast is under the mechanical tension. Owing to the effect of skin tension, this mechanical environment is responsible for the biological processes of fibroblast, e.g., proliferation, differentiation, collagen metabolism, gene expression, etc. The understanding on the biological behavior of fibroblast under tension is a fundamental and pivotal knowledge for promoting the wound healing, improving the quality of healing, preventing abnormal scar hyperplasia and dermal chronic ulcer. In this work, we cultivated the human dermal fibroblast (HFB) under a mechanical environment using FX-4000loading system, and investigated the influence of mechanical stimulation on the proliferation of fibroblast and the metabolism of collagen matrix. The main contents are as follows:1. HFB was extracted and cultivated using collagenase digestion method, and then inoculated by the density of3×104/ml in the6holes-flexile cell culture plate with type I collagen. The FX-4000loading system was used to perform the periodically tensile stimulation on the HFB. The loading frequency was0.1Hz. The tensile rates were10%and20%, respectively. The extractions of cells and the corresponding culture supernatant were operated after stretch24hours and48hours,no cyclic mechanical stimulation as the control group.2. Flow cytometry (FCM) was used to determine cell cycle and to analyze the S-stage of cell cycle. The S-stage is responsible for the proliferation of cell. During a cell cycle, the S-stage costs much more time, the cell proliferates more sharply. The experimental results show that compared with the control group, the percentage of S-stage significantly increases in the group of10%stretch with24hours; and then that percentage will decrease over time. This result reveals that fibroblast can respond to the mechanical stimulation that is a10%stretch with24hours. This response changes cell cycle and promotes the proliferation. However, the reduction of proliferation will happen after stretch48hours.3. After the tension test, cells and the corresponding culture supernatant were extracted to determine the expression of mRNA in type Ⅰ and type Ⅲ collagens using the RT-PCR method. The method of ELISA was used to determine the concentrations of type Ⅰ and type Ⅲ collagens in the liquid supernatant of different groups. The experimental results show that the expression of mRNA of fibroblast type Ⅰ collagens are promoted in the groups of10%stretch with24hours and48hours, and20%stretch with24hours. The expression of mRNA of fibroblast type Ⅲ collagens are promoted in the groups of10%stretch with24hours and48hours. The synthesis of type I collagens are promoted in the groups of10%stretch with48hours, and20%stretch with24hours and48hours. The synthesis of type Ⅲ collagens are promoted in the groups of10%stretch with24hours, and20%stretch with24hours and48hours. The extracellular matrix Ⅰ、Ⅲ type collagen metabolism of fibroblast is influenced by mechanical stimulation. This influence is correlated with the rate and time of the stimulation.