Effect Of DADM Seeded With BM-MSCs On Burn Wound Healing

BackgroundSkin is the largest organ in the human body and covers the body surface.It plays an important role in maintaining the stability of the body’s environment and resisting the invasion of harmful substances from the outside.Burns,trauma and other factors can cause serious skin defects.If not timely and properly treated,the functional recovery in the later period will be a great challenge,affecting the appearance and quality of life of patients,and even endanger the lives of patients.Currently,the treatment of extensive deep burns is usually autologous skin grafting after excision surgery.Autologous skin grafts not only cause damage to the donor area,but also have insufficient autologous skin.Skin substitutes such as acellular dermal matrix(ADM)are a new strategy in the field of wound healing,their clinical application will alleviate these problems to some extent.However,ADM is derived from allogenic or heterogeneous skin,and there is a certain risk of antigenicity and potential microbial infection.Early studies showed that denatured acellular dermal matrix(DADM),the autologous burn skin that was discarded after burn wound excision,was prepared after decellularization and was able to maintain a partial collagen network structure.DADM can be used as a dermal substitute to cover the wound and provide a certain amount of dermal tissue for wound healing with good biocompatibility.Bone marrow mesenchymal stem cells(BM-MSCs)are used as a wound treatment strategy because of their multi-differentiation potential and powerful tissue regeneration ability.They are the ideal seed cells for the current direction of wound repair and regeneration.Based on the above theory,it is speculated that DADM can facilitate the colonization and infiltration of seeded cells,and DADM scaffolds seeded with BM-MSCs can also act as a dermal substitute for the wound sites to promote the wound healing process.At present,despite the increasing number of studies on tissue engineering processing of burned skin,the study of DADM and BM-MSCs-seeded DADM scaffolds for wound healing is still relatively rare.ObjectivePreparing bioactive burn denatured acellular dermal matrix(DADM/MSC)based on DADM scaffolds seeded with BM-MSCs.and its biological properties were evaluated in vitro and in vivo to explore the DADM/MSC scaffold.To explore the feasibility of DADM/MSC scaffold used as a skin substitute for wound,and the application of DADM/MSC scaffold will provide a new strategy for the treatment of large extensive deep burn wounds.MethodsWater scalding method(54 ℃,25 s)was used to prepare deep Ⅱ scald wounds in mice.After 24 hours,scald skin was taken aseptically.Skin was decellularized with 0.02%EDTA-0.25%trypsin solution for 24 hours to make DADM.The tissue structure and collagen composition of DADM were detected by scanning electron microscopy,HE,and immunofluorescence techniques,to confirm that no cell and other components remained.The CM-Dil-labeled mouse BM-MSCs were seeded into DADM scaffolds and cultured in vitro.After 24 hours,biological properties of the DADM/MSC scaffolds were examined,such as the morphology of the scaffold,the proliferation viability of the seeded cells,and the biocompatibility of the scaffolds.Subsequently,normal saline,DADM and DADM/MSC scaffolds were transplanted into the dorsal full-thickness skin wounds of C57BL mice.The wound healing quality was assessed.The histological and immunofluorescence staining were performed after the scaffolds were implanted into the mice to evaluate the effect of DADM/MSC scaffold on wound healing,including epithelization of wound tissue,vascularization,and regeneration of skin appendage.ResultsAfter decellularized process,the appearance of DADM was complete,and in white color with certain elasticity and toughness.HE and immunofluorescence staining showed the process of decellularization was complete,collagen fibers maintain a relatively complete network structure.The seeded BM-MSCs survived in the DADM scaffold,proliferated and infiltrated.The distribution of BM-MSCs was uniform and formed a structure resemble normal dermis.Subcutaneous implantation in vivo showed that the scaffold exhibited good biocompatibility,and the vascularization of DADM/MSC scaffolds is significantly more than DADM scaffolds and began to degrade and synthesized new collagen earlier.No obvious infection or rejection was observed.After saline,DADM,and DADM/MSC scaffolds transplanted into back full-thickness skin wounds of mice,the date demonstrated that no infection was found.The closure time of wound in the DADM/MSC group was significantly shortened,and the epithelialization and vascularization of wound were significantly increased.There was no obvious infection was found during wound healing after implantation of normal saline,DADM,and DADM/MSC scaffolds in the dorsal full-thickness skin wounds of mice in vivo.Compared with DADM group and control group,the healing quality of wounds in DADM/MSC group was significantly improved with obvious epithelialization,vascularization and skin appendage regeneration.Conclusion(1)The collagen of DADM scaffold maintains a certain three-dimensional porous network structure,which is conducive to cell adhesion,colonization and infiltration;(2)The DADM/MSC scaffold shows good biocompatibility,accelerated the vascularization of scaffolds,and may act as a dermal substitute for deep burn wounds;(3)DADM/MSC scaffold conserves as a dermis substitute for mouse full-thickness skin defect wounds,which promotes wound healing by accelerating epithelialization,vascularization,and skin appendage regeneration.