| Background Burns and trauma can cause extensive full-thickness skin defects,which lead to a series of life-threatening consequences.Therefore,it is very important to repair skin defect quickly.At present,autologous skin grafting is still the standard method for repairing large area skin defects caused by burns.Because of the lack of autologous skin source in patients with extensive burn,it is often necessary to mince split-thickness skin grafts into micrografts to enlarge the expansion ratio,but this is at the cost of reducing the quality of wound healing.Collagen is an important component of extracellular matrix.Compared with synthetic materials,it has excellent properties of biomaterials and has been widely used in the field of biomedical materials.At present,collagen is mostly extracted from animal connective tissue,which has some potential viral hazards and high cost.The recombinant human collagen obtained by introducing human collagen gene into genetically engineered bacteria has high safety,low cost and can be industrialized on a large scale.In addition,the hydrophilic group of recombinant collagen was increased and the water solubility was improved.The hydrogel with 10% collagen concentration could be prepared.Artificial dermis is mainly used as a scaffold to guide cell migration and proliferation and collagen arrangement in the process of dermal repair,to help complete dermal reconstruction and vascularization,to speed up wound healing,and to improve skin elasticity,softness and mechanical wear resistance after wound healing.However,at present,artificial dermis for full-thickness wound repair needs two operations,which increases the pain of patients.In this study,recombinant human collagen was used as raw material to prepare hydrogel which related properties were tested.Then the hydrogel and Pelnac were used as overlay of micrografts to repair the full-thickness skin defect in rats,and the wound healing rate and the quality of wound healing were evaluated.Objective The aim of this study is to prepare recombinant human collagen hydrogel,and to explore the therapeutic effect of recombinant collagen hydrogel and Pelnac as overlay of micrografts for repairing full-thickness skin defect,so as to provide a new therapeutic method for repairing full-thickness skin defect in clinical treatment.Methods 1.Preparation and characterization of recombinant collagen hydrogel 1.1 Collagen hydrogel was prepared by crosslinking alginate with Ca2+ in DMEM/F12 medium with 10% collagen solution;1.2 The gelation state of collagen solution was observed,and the microstructure of hydrogel was observed by SEM;1.3 The swelling and degradation properties in vitro were tested;1.4 By embedding the hydrogel under the skin of rats,the degradation rate of the hydrogel in vivo was detected.2.Effect of recombinant human collagen hydrogel on wound healing-related cells 2.1 CCK-8 method was used to evaluate the toxicity of hydrogel extract to Ha Ca T cells,human fibroblasts and HUVEC;2.2 Cell scratch test and Transwell test were used to evaluate the effect of hydrogel extract on the migration of these three cells.3.Evaluation of the efficacy of recombinant collagen hydrogel or Pelnac as overlay of micrografts for repairing full-thickness skin defect in rats 3.1 Wound healing rates The full-thickness skin defect model of SD rats was made.After the transplantation of micrografts,collagen hydrogel,Pelnac and Vaseline gauze were used as overlay to repair the wound,and Vaseline gauze group was used as control group.The wound healing rates of three groups were compared.3.2 Quality of wound healing 3.2.1 Formation of dermis On postoperative days 7,14,21 and 35,the tissues were harvested and pathological sections were prepared.The formation of dermis in each group was compared by HE and Masson staining,and compared with normal skin tissue;3.2.2 Formation of basement membrane Immunohistochemistry of collagen type IV was used to compare the formation of basement membrane in each group,and compared with normal skin tissue;3.2.3 Deposition of collagen Collagen deposition was detected by Picrosirius red staining,RT-q PCR and WB on day 35,and compared with normal skin tissue;3.2.4 Expression of TGF-beta 1,TGF-beta 3 and alpha-SMA Immunohistochemistry,RT-q PCR and WB were used to detect the expression of TGF-beta 1,TGF-beta 3 and alpha-SMA in each group on day 35.3.3 Mechanism of promoting wound healing 3.3.1 Proliferation of wound cells Ki67 immunohistochemistry was used to compare the proliferation of wound cells in each group,and compared with normal skin tissue;3.3.2 Neovascularization The capillary neovascularization was compared by CD31 immunohistochemical staining;3.3.3 Expression of VEGF The expression of VEGF was detected by immunohistochemistry,RT-q PCR and WB.3.4 HE staining was used to observe the wound healing process of collagen hydrogel or Pelnac as overlay of micrografts respectively.Rusults 1.Preparation and characterization of recombinant human collagen hydrogel Collagen hydrogel with 10% concentration was successfully prepared;pore structure was observed by SEM;the hydrogel reached swelling equilibrium in 60 minutes and its maximum swelling degree was 1800%;it degraded fastest on day 5-6 in vitro and completely on day 7;it degraded 40% on day 5,80% on day 10 and completely on day 15 in vivo.2.Effect of recombinant human collagen hydrogel on wound healing-related cells Hydrogel extract had little cytotoxic effect on these three cells,and could promote the proliferation of Ha Ca T cells.The survival rate of fibroblasts was above 90%,and that of HUVEC was above 70%.Cell scratch test and Transwell test confirmed that hydrogel extract could promote the migration of these three cells.3.Evaluation of the efficacy of recombinant collagen hydrogel or Pelnac as overlay of micrografts for repairing full-thickness skin defect in rats 3.1 Wound healing rate On postoperative days 7,14 and 21,wound healing rate of hydrogel and Pelnac group was higher than that of Vaseline gauze group.The difference was statistically significant.There was no difference between hydrogel group and Pelnac group.3.2 Quality of wound healing 3.2.1 Formation of dermis On postoperative day 21,dermal tissue was formed in hydrogel and Pelnac group.On day 35,dermal tissue was mature in hydrogel and Pelnac group,while the Vaseline gauze group formed scar tissue with dense collagen deposition;3.2.2 Formation of basement membrane On postoperative day 21,the basement membrane was formed in hydrogel and Pelnac group,and gradually continued on day 35,while no basement membrane was formed on day 35 in Vaseline gauze group;3.2.3 Deposition of collagen On postoperative day 35,collagen deposition of wound in hydrogel and Pelnac group was less than that in Vaseline gauze group.The difference was statistically significant.There was no difference between hydrogel group and Pelnac group;3.2.4 Expression of TGF-beta 1,TGF-beta 3 and alpha-SMA Immunohistochemistry,RT-q PCR and WB confirmed that the expression of TGF-beta 1 and ?-SMA in the wounds of hydrogel and Pelnac group were lower than those of Vaseline gauze group,and the expression of TGF-beta 3 was higher than that of the Vaseline gauze group,the difference was statistically significant.3.3 Mechanism of promoting wound healing 3.3.1 Proliferation of wound cells On postoperative days 7,14 and 21,the number of proliferating cells in hydrogel and Pelnac group was higher than that in Vaseline gauze group.The difference was statistically significant.There was no difference between hydrogel group and Pelnac group;3.3.2 Neovascularization On postoperative day 7,the number of new capillaries in Pelnac group was higher than that in the hydrogel group and Vaseline gauze group,and the difference was statistically significant;3.3.3 Expression of VEGF Immunohistochemistry,RT-q PCR and WB confirmed that the expression of VEGF in Pelnac group was higher than that in hydrogel group and Vaseline gauze group on day 7. 3.4 The dynamic observation of HE staining on the wounds on postoperative days 7,14,21 and 35 confirmed that the wound healing rate in the hydrogel and Pelnac group was faster than that in Vaseline gauze group.Conclusion In the light of all the research on this subject,we can draw the following conclusions: 1.Recombinant collagen hydrogel has been successfully prepared,which has good water absorption and retention properties and suitable degradation rate.The above properties can be adjusted by changing the proportion of raw materials and the degree of crosslinking.The preparation process is simple and the cost is low.2.Hydrogel extract was applied to Ha Ca T cells,fibroblasts and HUVEC,which showed that it had no obvious toxicity on the above three cells,but also promoted the proliferation of Ha Ca T cells and the migration of the above three cells.3.Collagen hydrogel and Pelnac were used to repair full-thickness skin defect in rats.It is proved that both of them can accelerate the proliferation of wound cells,promote the capillary neovascularization,promote the expression of vascular endothelial growth factor in early wound,thereby promoting wound healing;promote the formation of dermal tissue and basement membrane,thereby improving the quality of wound healing;reduce the expression level of TGF-beta 1 and alpha-SMA,increase the expression level of TGF-beta 3,thereby preventing excessive contraction of wound and reducing scar hyperplasia.This is the first time to prove the feasibility of using artificial dermal Pelnac as the overlay of micrograft to repair full-thickness skin defect by one-step approach.In summary,the collagen hydrogel prepared in this study and the one-step approach for full-thickness skin defect reconstruction using minced split-thickness skin grafts with Pelnac overlay provide new ideas for the repair of full-thickness skin defects in clinical treatment. |
PDF Full Text Request