| Objective:To investigate whether porous polymer membrane can be used as dermal scaffold substitute to promote skin wound healing and its possible mechanism.And to provides a new strategy and theoretical study for clinical application of polymer porous biological blend membrane to improve wound healing quality.Methods:The polymer porous biological blend membranes are made of sodium carboxymethyl cellulose(CMC),chitosan(CS)and hyaluronic acid(SH)by proper mixing ratio and process technology.And this part is mainly developed by cooperative units.Skin fibroblasts were obtained by tissue mass culture of human skin.The cultured and expanded human skin fibroblasts were co cultured with polymer porous biological blend membrane.By observing the survival and proliferation of skin fibroblasts on polymer porous biological blend membranes,to verify the biocompatibility and cytotoxicity of the polymer porous biological blend membrane used in this experiment.Usedthe white rabbitsas the animal model,symmetrically manufacturing 4x4cm full-thickness skin wounds in 24 rabbits on both sides of the back parts,then porcine acellular dermal matrix(control group)and the macromolecule porous biomembrane(experimental group)were transplanted respectively.The wound healing was observed and calculated regularly.The wound tissues were taken at 1,2,3,4 weeks after operation(6 samples were randomly selected at each time point),and were randomly divided into 4 parts,including HE staining,CD31 immunohistochemical staining,PCNA immunohistochemical staining and storage reserve.The histological structure,the degradation and the infiltration of inflammatory cells were observed by HE staining,so as to determine the biocompatibility,degradability,immune rejection and cytotoxicity of the implanted dermis scaffold.CD31 immunohistochemical staining was used to observe the proliferation of blood vessels in wound tissue.PCNA immunohistochemical staining was used to observe the proliferation of cells in the wound.Results:Skin fibroblasts were isolated by skin tissue culture.After 3 days of inoculation,the skin fibroblasts were seen crawling from the edge of the adherent skin tissue,showing shuttle type adherent cells.After 5 days of inoculation,the number of skin fibroblastsincreased,showing the appearance of clone.After 7 days of inoculation,the skin fibroblasts placed regularly and polarity.The skin fibroblasts cultured in vitro were co cultured with polymer porous biofilm.After 3 days of inoculation,the skin fibroblasts grew normally on the biofilm,and the appearance restored the shuttle type.The wounds of two groups of rabbits healed within 4 weeks,and healed well without obvious contracture.1 week after injury,there was no significant difference in wound healing rate between the two groups(P>0.05).At the 2 weeks after injury,the wound healing in the control group was faster than that in the experimental group(P<0.05).There was no significant difference in wound healing rate between the two groups at 3 and 4 weeks after injury(P>0.05).The control group was significantly healed after 2 weeks compared with 1 week after injury(P<0.001).The control group was significantly healed after 3 weeks compared with2 week after injury(P<0.01).The experimental group was significantly healed after 3 weeks compared with2 week after injury(P<0.001).Observing the angiogenesis of the wound,2 weeks after injury,the positive rate of CD31 in the control group was higher than that in the same group at 1 weekafter injury(P<0.01).3 weeks after injury,the positive rate of CD31 in the experimental group was higher than that in the same group at 1 week after injury(P<0.01).2 weeks after injury,the positive rate of CD31 in the control group was higher than that in the experimental group(P<0.001).3 weeks after injury,the positive rate of CD31 in the control group was higher than that in the experimental group(P<0.001).4 weeks after injury,the positive rate of CD31 in the control group was lower than that in the experimental group(P<0.05).Observing the cell proliferation of the wound,there was a small amount of cell proliferation in the two groups at 1 weekafter trauma.2 weeks after injury,the number of new cells in the control group increased significantly(P<0.001),but the increase in the number of new cells in the experimental group was not obvious.3 weeks after injury,the number of new cells in the control group decreased slightly,but the number of new cells in the experimental group increased significantly compared with that in the first week(P<0.01).2 weeks after injury,the number of cells in the control group was significantly higher than that in the experimental group(P<0.001).4 weeks after injury,the number of cells in the control group was significantly lower than that in the experimental group(P<0.01).Observing the HE staining of the wound,in the two groups,granulocytic infiltration was seen in the dermis at 1 weeks after trauma.At 2 weeks,a large number of granulocytes infiltrated,and then the number of granulocytes began to decrease.In the control group,the granulocyte infiltration,peak and regression were earlier than those in the experimental group,and the number of granulocytes decreased significantly at 3 weeks.There was obvious biofilm in the experimental group 1 week after trauma.3 weeksafter trauma,the biofilm was filled by surrounding tissues,and some boundaries were blurred.And biofilm and surrounding tissues were fused at 5 weeks.Conclusions:Skin fibroblasts can proliferate normally onthe polymer porous biologic blend membranes,and HE staining showed that the blend membrane degraded well in the wound of white rabbit.It is proved that the highly porous biological blend membrane has good biocompatibility,no obvious cytotoxicity and no obvious rejection.The wound healing of the two groups of rabbits were all good.From the statistical analysis of wound healing rate,we found that the wound healing process of the experimental group was slightly delayed than that of the control group,but all of them played a role in promoting wound healing.CD31 immunohistochemical staining and PCNA immunohistochemical staining showed that the two groups of scaffolds had the effect of promoting angiogenesis and cell proliferation of the wound.CD31 immunohistochemical staining and PCNA immunohistochemical staining showed that the two groups of scaffolds had the effect of promoting angiogenesis and cell proliferation of the wound.Compared with the control group,the rate of angiogenesis and cell proliferation were slightly slower in the experimental group. |
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