| Objective: By observing the repair effect of full-thickness skin defect of rabbits with acellular dermal matrix with concentrated growth factor,the experimental basis was provided for the clinical use of acellular dermal matrix combined with autogenous concentrated growth factor.Methods:1.Twelve white rabbits from New Zealand were selected and three full-thickness skin defects of 2cm×1cm in length and width to the subcutaneous fascia layer were made on both sides of the spine of the back of the rabbits.A total of 72 defects were divided into three groups Group A—ADM+CGF,Group B—ADM and Group C—normal group,each for 24 defects.2.Preparation of concentrated growth factor(CGF).3.Specimen collection and processing: three rabbits were randomly selected for 1 week,2 weeks,3 weeks,and 4 weeks after the operation.After the rabbits were killed,a full-thickness skin and skin subcutaneous were cut out 0.5 cm from each wound edge.The tissue was immediately put into 4%paraformaldehyde solution.4.General observation: The wound condition was observed before collecting the skin tissue samples,and made statistics and analysis on the healing rate of each wound.5.Histological observation and measure the tissue thickness by HE staining: The number of inflammatory cells,fibroblasts,neovascularization and the arrangement of collagen fibers in epidermis and dermis were observed under light microscope;The thickness of new tissue in each group was measured at each time point.6.Histological observation with Sirius red staining: Different fiber types could be distinguished under a polarized light microscope.Type Ⅰ collagen fibers were thick and their colors were yellow or red;Type III collagen fibers are slender and their colors were green.Image Pro-Plus 6.0 was used to measure the ratio of Ⅰ/Ⅲ collagen fibers in wound skin.7.Perform immunohistochemical staining of TGF-β1 and VEGF,and use Image Pro-Plus 6.0 was uesd to analyze the average optical density of the positive expression of TGF-β1 and VEGF.8.Statistical processing: The data is processed with SPSS21.0 analysis software,and the data is expressed as mean ± standard deviation.The method was single-factor analysis of variance.When the variance is uniform,the LSD method is used to compare the experimental groups in pairs;When not,the Tamhane method was used for pairwise comparison between groups,and the result was statistically significant(P<0.05).Results:1.Gross observation: One week after surgery,group A and B had good healing,with clean surface and partial loose sutures.Group C was full of granulation tissue on the wound surface,with A small amount of oozing blood and purulent secretions.Two weeks after operation,group A had good and smooth healing,while the wound of group B was slightly depressed.Group C had obvious depression and irregular contour.Three or four weeks after operation,most of group A,B and C had healed.Group A and B had better healing with hair,while group C could touch the hypertrophic tissue with tough texture and no hair.2.Histological observation and measure the tissue thickness by HE staining: A week after the operation,the epithelial layer of group A was initially formed,and undegraded ADM and CGF were seen.A large number of inflammatory cells and fibroblasts could be seen in the dermis;collagen fibers were slender and neatly arranged,and more new blood vessels were formed;The epithelial layer of group B is partially formed,and undegraded ADM could be seen,more inflammatory cells infiltrate,and a small amount of new blood vessels were formed;the epithelial layer of group C is not formed,a large number of inflammatory cells and a small amount of new blood vessels are formed,which is obvious at the junction with normal tissue Sunken The epithelial thickness of groups A,B and C were statistically significant compared with the normal group(P<0.05),while that of group A and B were not statistically significant compared with that of group B(P>0.05),and group A and B were statistically significant compared with group C(P<0.05).For two weeks,the epithelial layer of group A was basically formed with a clear layered structure,with a large number of fibroblasts,and the formed collagen fiber bundles were slender and neatly arranged,and a few skin appendages were formed;The layered structure of the epithelium of group B was gradually clear,The new collagen fibers were small and loose,and no skin appendages were formed temporarily.The epidermis of group C is initially formed,and there were still depressions at the junction with normal skin,and the collagen fibers were arranged irregularly;The epithelial thickness of groups A,B and C were statistically significant compared with the normal group(P<0.05),while that of the epithelial thickness among group A,group B,and group C were statistically significant(P<0.05).For three weeks and four weeks,the wounds in groups A,B,and C were cured.The epithelial layers of groups A and B were basically flat and similar to normal tissues,and the epithelial tissue structure was basically mature.The layered structure of the epithelium in group C was gradually clear,and a large number of inflammatory cells and fibroblasts were still visible.The collagen fibers were thick,densely arranged and disorderly,and no obvious skin appendages were formed;At three weeks,The epithelial thickness of groups A,B and C were statistically significant compared with the normal group(P<0.05),while that of the epithelial thickness among group A,group B,and group C were no statistically significant(P>0.05).At four weeks,there was no statistically significant in epithelial thickness between group A and normal group(P>0.05),There were statistical significance in group B and C compared with the normal group(P<0.05).while that of the epithelial thickness among group A,group B,and group C were no statistically significant(P>0.05).3.Sirius red staining of histological observation: for one week,the newly-born collagen fibers in groups A and B were mainly type Ⅲ,and group C was mainly typeⅠcollagen fibers.The ratio of type Ⅰ/Ⅲ collagen fibers in groups A and B were statistically significant compared with that in group C.There was no statistical significance between group A and group B(P>0.05).There was no statistical significance between group A and group B and normal tissue(P>0.05).For two weeks,the ratio of type Ⅰ/Ⅲ collagen fibers among group A,group B,and group C were statistically significant(P<0.05);there was no statistical significance between group A and normal tissues(P>0.05).For three or four weeks,the ratio of typeⅠ/Ⅲ collagen fibers in groups A and B were statistically significant compared with that of group C(P<0.05),and there was no statistical significance compared with normal tissues.4.TGF-β1 expression analysis results: For 1 and 2 weeks,the comparison of A,B,and C is statistically significant(P<0.05),and compared with normal tissues,they are also statistically significant(P<0.05),and the expression level is group A > group B > group C;For 3 weeks,there was statistical significance in pairwise comparison among groups A,B and C(P<0.05),and there was also statistical significance compared with normal tissue(P<0.05).However,TGF-β1 expression level in group C was the highest at this time.For 4 weeks,pairwise comparison among groups A,B and C showed statistical significance(P<0.05),while there was no statistical significance between group A and normal skin(P>0.05).The data of group B and group C were statistically significant compared with that of normal skin(P<0.05).5.Analysis results of VEGF expression: For 1 week,pairwise comparison among groups A,B and C showed statistical significance(P<0.05),while there was no statistical significance between group A and normal skin(P>0.05).Compared with normal skin,group B and group C had statistical significance(P<0.05).For 2 weeks,pairwise comparison of groups A,B and C had statistical significance(P<0.05),and data of groups A,B and C were also statistically significant compared with normal skin(P<0.05).For 3and 4 weeks,there was no statistical significance among the three groups A,B,and C(P>0.05),and the data of the three groups A,B,and C were also not statistically significant compared with normal skin(P>0.05).Conclusions:1.Adm combined with CGF can repair full-thickness skin defects of the rabbits and accelerate the healing speed of wounds.2.ADM combined with CGF can increase epithelial thickness.3.ADM combined with CGF can increase the collagen fibers in the local tissue of the wound in the early stage of skin defect,and reduce the ratio of type Ⅰ/Ⅲ collagen fibers in the later stage,reduce scar formation,and improve the quality of wound healing.4.ADM combined with CGF significantly increased the release of TGF-β1 at the early stage of skin defect healing(1,2 weeks),and stabilized TGF-β1 for 3,4 weeks to facilitate wound healing.5.ADM combined with CGF can increase the release of VEGF and promote the formation of new blood vessels in the process of skin defect healing. |
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