Biological Detection Of Scar Acellular Matrix And The Evaluation Of The Wound Repair Effect In Animals

BackgroundPatients with large scars usually have insufficient normal skin,so it is difficult to completely repair wounds with limited skin or flaps.Split-thickness autografting has the advantages of repeatedly cutting the donor skin.If the wound is covered only with a split-thickness autografting,a second contracture will occur in the transplanted area due to insufficient dermal support.Moreover,the contracture will often be critical than the origin and effect the fuction of recieving area.Dermal scaffolds are the key factors for accelerating skin repair and reconstruction,which provide a microenvironment for cells growth and attachment,guide cells and conduct signals,inhibit over-synthesis of collagen,reduce scar formation and skin pigmentation,and improve the flexibility and viscoelasticity of the repair skin.However,these materials have following issues need to be resolved,including their high cost,the inflammatory response,source limitations,fast degradation,poor nutrient permeability,poor vascularization,complicated extraction steps,poor mechanical properties,degradation products with cytotoxicity,risk of transmitting disease,heterologous virus infection,etc,limiting their clinical applications.Patients with large scars typically undergo scar excision several times.Upon successful use of sAM,which is first generated by scar resection for wound healing after scar excision,it is possible to overcome the limitations of dermal scaffolds like allogeneic or heterogeneous ADM and make better use of patients’ own tissues.It is also consistent with the principle of waste recycling.Scar Acellular Matrix can effectively expand the source of dermal scaffolds and reduce the cost.It can also be used to extract ECM components for the preparation of biosynthetic materials,which promote the development of skin tissue engineering.Objective:1.In this study,we aimed to prepare sAM and ADM without intact nuclear structure.By comparing the structural,biocompatibility and biomechanical characteristics of mature sAM,immature sAM and normal skin ADM,a material basis for animal wound healing experiments was established.2.To investigate the repair effect of these human sAMs on animals’ wound,and study the expression of related genes during wound healing.Methods and Results:1.Preparation and characterization of human scar acellular dermal matrix Methods:We randomly selected human mature scars,human immature scars,and human normal skin from clinic as experimental specimens.Appropriate size and thickness dermal grafts were obtained using a free-hand graft knife or scalpel.Samples were subjected to Trysin+Triton X-100 for cell lysis and disinfectant and then obtain mature and immature sAM and normal skin ADM.Structural and antigenic analysis was performed via 1.HE stain and scanning electron microscopy for tissue structure analysis;2.DAPI and Picogreen for immunogenicity analysis;3.Human epithelial stem cells and skin fibroblast cells were incubated on these materials,and then we observed cells growth characteristic on these materials through light microscopy and scanning electron microscopy;4.The biomechanical characteristics of these specimens,such as strong-stress elongation,Young’s modulus,relaxation slope,relaxation,creep slope,creep and maximum tensile stress were analyzed using a tensile machine,and then the fitted stress-strain,normalized relaxation and creep curves are drawn based on the obtained values.Results:1)Through HE stain,we found that no intact nucleus existed in the three materials.Normal skin ADM fibers were arranged in order,whereas immature scar AM fibers were disordered,and mature scar AM fibers were porous and slightly disordered than normal skin ADM,in which few fibers were similar to normal skin.2)DAPI showed that no intact nucleus exited in three materials.The residual DNA of three kinds of materials was less than 50ng/mg via Picogreen,which showed that the acellular material prepared by the Trypsin+Triton X-100 has low immunogenicity and meet the standards of biological materials3)After incubating human epithelial stem cells and skin fibroblast cells on materials,epithelial stem cells grew in an aggregated state in both normal skin ADMs and mature scar AMs;however,cells adhered only on the surface of immature scar AMs.Skin fibroblasts didn’t grow well in mature scar AM and immature scar AM,which may be related to the culture environment.4)No significant differences were observed in the Young’s modulus or creep slope among three materials,although significant differences in stress-strain elongation,relaxation slope,relaxation and maximum tensile stress creep were noted between immature sAMs and normal skin ADM(p<0.05).While significant differences in relaxation was also noted between immature sAMs and mature sAMs(p<0.05).Only stress-strain elongation was noted significant differences between mature sAM and normal skin ADM.Other values didn’t show significant differences in biomechanics between the two materials.2.Experimental study on animals and the expression of wound healing related genes.Methods:The experimental animal used in vivo was Yorkshire pig.This research was divided into three groups:mature sAM group,immature sAM group and negative control group.We randomly selected some mature and immature scar AMs and implanted into the prepared pig back wound.The wound area was about 2cm × 2cm.And then covered with split-thickness autografting.The experimental materials were collected at 1 month,2 months,3 months,and 6 months after implantation,which were cut along with the epidermis and few subcutaneous tissues.The cell infiltration and collagen fiber arranged structure on wound healing in animals was observed by HE and Masson’s staining.The expression of ECM on wound healing was observed by COLA 1,elastin and fibronectin immunohistochemistry.The expression of wound healing related genes was detected by RT-qPCR.The vessel number was counted through α-SMA immunohistochemistry.Thereafter,we can observe the effect of wound healing treated by sAMs in vivo.Results:1)Both implanted scar AMs appeared to show good biocompatibility and were well-tolerated,no severe infection or graft rejection was observed.The implants,only exhibited mild inflammation at 1 months and 2 months afer implantation,indicated by lots of fibroblasts and inflammatory cells infiltration.At 3 and 6 months after implantation,the porous sAM was gradually filled with new collagen and new vessels,and inflammatory cell infiltration gradually decreased.The sAMs were integrated into the host tissue.The neo-ECM fibers formed in mature scar ADMs compared with those formed in immature sAMs varied more regularly.2)Immunohistochemical results showed that COLA1 expression in three groups peaked at 6 month after surgery,and COLA 1 in the negative group increased faster than the other two experimental groups.For elastin deposition,there was no significant difference between the three groups at the first month after surgery,while elastin in the mature sAM group increased significantly at 2 month,and then remained stable.And the content of elastin in the immature sAM group peaked at 6 month after surgery.Elastin in the negative group were lower at each collected time than other two groups.The content of fibronectin increased with the time of implantation in all three groups.In the first three months after the operation,there was no significant difference in FN expression among the three groups,and at 6 month after surgery,FN in the negative group was higher than that in the other two groups.3)In mature scar AM group,peak TGF-β mRNA expression was observed at 1 month,COLA 1 and Vimentin levels peaked at 6 months after implantation,and MMP-1 and MMP-9 levels were higher at 2 and 6 months.MMP-2 and TIMP1 expression increased at 1 month.In immature scar AM group,TGF-β levels peaked at 2 months after implantation,while COLA1 and Vimentin levels were highest at 6 months after implantation.The MMP-1 and MMP-9 levels were higher at 2 months,and MMP-2 levels increased at 2 and 6 months.TIMP1 expression peaked at 6 months after implantation.4)The vessel luminal structures using α-SMA staining revealed the presence of new microvessels in the periphery of the implanted materials after 1 month,and the density of the blood vessels in mature sAMs was highest at 2 months after implantation,while in immature sAMs,it was highest at 3 months after implantation.Conclusions:1.Our findings showed that mature scar acellular matrix,immature scar acellular matrix and normal skin acellular dermal matrix prepared by Typsin+Triton X-100 has well properties and low antigenicity.In these materials,no intact nucleus was found and the residual DNA was low.Mature sAM is more biocompatible than immature sAM,but slightly inferior to normal skin ADM.Moreover,in biomechanical property,mature sAM was similar to normal skin ADM,showing a slight superior compared with immature sAM.The experimental results reflect that sAM can be used to construct artificial skin in vitro or repair wounds in vivo.It has good application development prospects.2.Covering the wound on the back of experimental animals with mature sAM and immature sAM can effectively promote wound healing,vascularization,and accelerate neo-extracellular matrix remodeling.After sAM implantation,the new tissue had better recoil ability and became softer than the negative group,but it had weaker ability to resist inflammation.In addition,the new tissue in the mature sAM group was much closer to normal skin than that in the immature sAM group.TGF-β in the mature sAM group peaked earlier than that in the immatue sAM group,indicating that the wound healing in the mature sAM group was earlier than that in the immature sAM group.The expression of COLA1 and Vimentin in the two sAM groups were both highest at 6 months after implantation,indicating that the extracellular matrix remodeling was completed in this period.The expression trends of MMP and TIMP are slightly different between mature sAM and immature sAM.Both MMP1 and MMP9 peaked at 2 month after implantation in both sAM groups,and MMP2 in mature sAMgroup peaked earlier than that in immature sAM group,which meaned that mature sAM can promote wound healing faster than immature sAM.In summary,mature sAM has superior repair effect than immature sAM.