| ObjectiveCultured keratinocytes play important roles in burn wound healing and scientific research studies.We aimed to modify the isolation method to avoid over-digestion,maximize the number of isolated epidermal cells and establish a more efficient and innocuous way of cell isolation.To build a method of applying MEEK skin grafting technology to small animals,a self-made simple MEEK machine was designed to cut the skin.To overcome the difficult that the rat wounds was contract easily and was bitten by themselves.A reliable technical scheme was established by modifying the location of wounds and the method of sewing.How to cover wounds caused by extensive deep burns has been an important clinical issue.This study explored a new method using MEEK auto-micrografts in combination with allogenic epidermal cell suspension in the treatment of extensive deep burns.MethodsThis research includes three parts:(1)Efficient isolation and high yield of epidermal cells from foreskin biopsies by dynamic trypsinization,(2)Establish the method of application of MEEK autograft technology and the anti-contracture full-thickness excision skin wound model in rats,(3)MEEK micrografts in combination with allogenic epidermal cell suspension to treat full skin loss in rats.Efficient isolation and high yield of epidermal cells from foreskin biopsies by dynamic trypsinization.Modified group use the trypsin dynamical and reduplicative digestion method to separate cells and control group use the conventional method.Compared the following assessment indexes to evaluate the two methods:(1)The yield of the total number and viable rate of epidermal cells.(2)Flow cytometry analysis of isolated epidermal cells:i)Cell cycle analysis,ii)Cell apoptosis analysis,iii)Detection of CD49f+cells.(3)Cell proliferation assessment(4)Keratinocyte migration assay(5)Senescence-associated?-galactosidase staining.Establish the method of application of MEEK autograft technology and the anti-contracture full-thickness excision skin wound model in rats.The simple MEEK machine consists of a lid with 13 gaps and a large base on top.The material can be aluminum alloy,stainless steel and other metals.The 13 gaps of the lid distributed evenly,In operation,rotary cutter could roll in the gap of the lid and cut the skin.The base is a platform,which is a rectangular shape with no top.Twenty-four SD rats were randomly seperated into three groups equally.Group A:the full-thickness skin defect was establish in traditional method and grafted by MEEK technique;Group B:the same full-thickness skin defect was establish in modified method and grafted by MEEK technique;Group C:the same full-thickness skin defect was establish in modified method without skin-grafts,which was a control group.The wound contraction rates were observed at 7,14 and 21 days post-operation,respectively.The steel wire,suture knot and the growth of autografts were observed at 21 days post-operation.MEEK micrografts in combination with allogenic epidermal cell suspension to treat full skin loss in rats:Animals and Experimental Groups.Female Sprague Dawley(SD)rats(N=48)were randomized into six groups:A:control group;B:MEEK auto-grafts(1:6)group;C:MEEK auto-grafts(1:9)group;D:epidermal cells group;E:MEEK auto-grafts(1:6)+epidermal cells group;and F:MEEK auto-grafts(1:9)+epidermal cells group.Allogenic epidermal cells were supplied by newborn male Wistar rats.Gross wound observation,epithelialization rate(ER),and hematoxylin and eosin(H&E)staining were used to assess wound healing,and Y chromosomes were detected to trace the allogenic epidermal cells.ResultsEfficient isolation and high yield of epidermal cells from foreskin biopsies by dynamic trypsinization.Using the modified method,the cell yield was improved to approximately 3times that of the traditional method.The number of viable cells isolated per gram of adult foreskin epidermal layer tissue was(18.88±13.22)×10~6cells in the control group and(67.34±30.66)×10~6cells in the modified group,and the difference was significant(p<0.001).The rate of viable cells was not significantly different between these two methods(p>0.05).There were no significant differences in the proportion of cells in G2 phase and S phase,the proportion of cells in early apoptosis,the proportion of cells in late apoptosis and necrotic cells,the proportion of CD49f-positive cells or the proportion of senescent cells between the two groups(P>0.05 for all).Moreover,the cell proliferation and cell migration abilities were similar in the two groups.Establish the method of application of MEEK autograft technology and the anti-contracture full-thickness excision skin wound model in rats.In this experiment,the rats were treated with MEEK autografts technology to repair the wound efficiently.The self-designed simply MEEK machine could cut the skin into micro pieces(3mm×3mm)successfully,which the cutting effect satisfied the application on the experiment.The wound contraction ratio(WCR)of the three groups on the 7day,14day and 21day post-operation was calculated and compared:There was statistically significant difference in different days post-operation(F=121.668,P=0.000)and in different groups(F=8.685,P=0.002),the WCR in group A was higher than that in group B(P=0.001),there was no statistically significant difference in the ratio between the group B and group C.Meanwhile,the variation trend of the ratio was not same in the three groups(F=7.408,P=0.000).The total exposed steel wire was 35cm in the group A,16cm in the group B and 13cm in group C on 21 day post-operation.There was statistically significant difference on the number remainder of knots between these groups on the 21day post-operation(P=0.005),which the number was few obviously in group A.At last,the MEEK autografts was survived in group B were better than group A significantly in statistical analysis(P=0.001).MEEK micrografts in combination with allogenic epidermal cell suspension to treat full skin loss in rats.The ER of group E(91.0%±3.8%)was significantly(P<0.05)higher than that of group B(83.0%±8.1%).Similarly,the ER of group F(84.4%±8.0%)was significantly(P<0.05)higher than that of group C(71.4%±9.3%).No significant differences(P=0.664)were observed in group B or group F.The H&E staining showed that there were no obvious differences among groups B,C,E and F.The results of Y chromosome detection demonstrated that the allogenic cells could not survive in the wounds at all.ConclusionsThe modified method was significantly more efficient in dissociating keratinocytes from each unit of skin biopsy,which is particularly important for treating severe burns when donor skin is limited.In this study,the wounds were repaired to prove that the MEEK autografts technology could be applied in the experiment of rats.This means developing MEEK autografts technology for small animals.The establishment of a suitable rat model was closely related to the effect of skin grafting.It is more effectively to establish anti-contracture full-thickness excision skin wound model in rats through the change of wound location and suture.MEEK micro-skin technology,in combination with allogenic epidermal cell suspension,is a promising treatment for extensive deep burns,although further preclinical experiments are needed to prove our findings. |
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