Studies On The Effects Of Enamel Matrix Proteins To Fibroblasts,Keratinocytes And Ulcer Wound Model Of Rabbit Ear

Wound healing after cutaeous trauma were frequently encountered .The process of wound healing refers to a series of interaction of cells and cell factors. Enamel matrix proteins (EMPs) has been largely used for periodontal regeneratioan, and has positive effect. Its tissure regeneration stimulus effct is encouraging. However, there were few researches on the effects of EMPs to cutaeous wound healing. In this research, the biological characters of human fibroblasts and keratinocytes cultured in the nutrient solution containing different concentration EMPs were estimated. Then , EMPs was applied to a ulcer wound model of rabbit ear in order to estimate its effect on cutaneous wound healing. There are three parts involved in this research, as follows:1,Effects of EMPs on the biological characters of human fibroblast cells:Human dermal fibroblast were obtained from human acrobystia and cultured in DMEM medium with 10% FBS. The third to sixth passage cells were used. Different concentration of EMPs (为25,50,100,200μg/mL) were prepared.①In the cell attachment experiment, 0.2ml cells suspension at the concentration of 106/ml was added to the pre-coated 96 well plates which was named group EP1,EP2,EP3,EP4 based on the different concentrations of EMPs. Control is human dermal fibroblast which was added to the un-coated plates. At the time 1.5,3,4.5h after the addition of cells, unattached cells were removed and the attached cells were measured by MTT method. There are significant differences between the control group and experimental group EP2,EP3 and EP4 in the cell attachment experiment( P <0. 05 )②The cell proliferation experiment, 0.2ml cells suspension at the concentration of 5×104/ml was added to the 96 well plates which was named group EP1,EP2,EP3,EP4 based on the different concentrations of EMPs. Control is human dermal fibroblast suspension which didn't contain EMPs. At the time 2,4,6,8d after the addition of cells, unattached cells were removed and the cells were measured by MTT method. in the second day, there are no significant differences between the control group and experimental group; in the fourth day,there are significant differences between the control group and experimental group EP2,EP3 and EP4( P < 0. 05); In the sixth day , there are significant differences between the control group and experimental group EP2,EP3 and EP4( P < 0. 05); in the eighth day , there are significant differences between the control group and experimental group EP2 and EP3 ( P < 0. 05);③In the synthesis of pre-mRNA experiment, 2ml cells at the concentration of 106/ml was added to the 6 well plates which was named group EP1,EP2,EP3,EP4 based on the different concentrations of EMPs. Control is human dermal fibroblast suspension which didn't contain EMPs. At the time 5d after the addition of cells, the synthesis of pre-mRNA was measured by RT-PCR method. There are significant differences between the control group and experimental group EP2,EP3,EP4 in the synthesis of typeⅠcollage pre-mRNA experiment.2 Effects of EMPs on the biological characters of human keratinocytes:Human keratinocytes were obtained from human acrobystia. The third passage cells were used. Different concentration of EMPs (为25,50,100,200μg/mL) were prepared.①In the cell attachment experiment, 0.2ml cells suspension at the concentration of 6×104 /ml was added to the pre-coated 96 well plates which was named group EP1,EP2,EP3,EP4 based on the different concentrations of EMPs. Control is human dermal fibroblast which was added to the un-coated plates. At the time 1.5,3,4.5,6h after the addition of cells, unattached cells were removed and the attached cells were measured by MTT method. There are no significant differences between the control group and experimental group in the cell attachment experiment 1.5h after the innoculation. There are significant differences between the control group and experimental group EP2( P <0. 05 ) 3h after the inoculation; There are significant differences between the control group and experimental group EP2,EP3,EP4 ( P <0. 05 ) 4.5h after the inoculation; There are significant differences between the control group and experimental group EP3,EP4 ( P <0. 05 ) 6h after the inoculation;②The cell proliferation experiment, 0.2ml cells suspension at the concentration of 4×104/ml was added to the 96 well plates which was named group EP1,EP2,EP3,EP4 based on the different concentrations of EMPs. Control is human dermal fibroblast suspension which didn't contain EMPs. At the time 2,4,6,8d after the addition of cells, unattached cells were removed and the cells were measured by MTT method. There are no significant differences between the control group and experimental group in the cell proliferation experiment.③In vitro wound-healing assay: 2ml cells suspension at the concentration of 2×10~5/ml was added to the 12 well plates which was named group EP1,EP2,EP3,EP4 based on the different concentrations of EMPs. Control is human dermal fibroblast suspension which didn't contain EMPs. 5ug/ml Mitomycin was added to plates to inhibit the proliferation of cells. 24h later, healing rate was measured by wound-healing assay. There are no significant differences between the control group and experimental group in the in vitro wound-healing assay.3 Effects of EMPs on the healing of a ulcer wound model of rabbit ear: Four standardized full-thickness skin wounds of 1cm in diameter extending to the perichondrium were made on each side of the ear. 1.5mg /cm2 EMPs+vehicle was applied to left ear each day after operation as experiment group. Vehicle only was applied on right ear in the same way as control. On postwounding days 3,6,9,12,15d, wounds were photographed and software Image J was used to measure the unhealed area and the wound healing rate was caculated. It is considered compeletely accreted when 90 % of wounds healed. Record how many days the wound was compeletely accreted after operation. There are no significant differences between the control group and experimental group 3 and 6 days after operation. There are significant differences between the control group and experimental group 9,12,15days after operation( P <0. 05 ). The average complete healed time of experimental group is 11.4±0.95d,while the average complete healed time of contol group is 11.4±0.95d.There are significant differences between the two ( P <0. 05 ).EMPs can promote the healing of ulcer wound model of rabbit ear. EMPs can stimulate the attachment,proliferation and synthesis of pre-mRNA of typeⅠcollagen of human fibroblasts. EMPs can promote the attachment of human keratinocytes. However, it has no effects on the proliferation and migaration of human keratinocytes. Therefore, we induce that the stimulus effect of EMPs on wound healing mainly rely on fibroblast.