Mechanically Reinforced Biomaterial Scaffold Regulates The Biological Behaviors Of Dermal Fibroblast And Its Roles In Dermal Regeneration

Background:Burns,diabetic ulcers,venous ulcers and other acute and chronic factors can easily affect the largest organ of the human body-skin,which can cause different degrees of skin wound.When the area of deep skin wound exceeds a certain range,it will be difficult to heal spontaneously and will bring great pain and heavy financial burden to patients and their families.Therefore,the key to the treatment of deep skin wound is early wound closure.The most common clinical treatment is autologous skin transplantation.However,the risk of infection,lack of donor site and secondary injury limit the application of autologous skin transplantation.Dermal substitutes are the template of dermal regeneration,which are very important in skin reconstruction and regeneration.Their appearance provides a new method for the treatment of deep skin wound in clinical settings.Wound repair is a complex yet highly coordinated process involving inflammatory cells,vascular endothelial cells,keratinocytes,fibroblasts,extracellular matrix,growth factors and cytokines.The process of wound healing is classically categorized into 3 overlapping stages-are inflammation stage,proliferation stage and remodeling stage.After skin injury,dermal fibroblasts being an important repair cells undergo several changes such as activation,proliferation,migration,differentiation to synthesize a variety of extracellular matrix(including collagen and polysaccharides)and secreted a variety of cytokines.This biological behavior is considered to be an important part of wound repair.Based on previous studies,it has been found that porous collagen-chitosan(CCS)scaffolds which has good biocompatibility and biodegradability can be used as dermal scaffolds when integrated with PLGA knitted mesh(PLGAm)results with increased mechanical strength,which can induce tissue regeneration in vivo,and has great potential for clinical application.Objective:Building on the foundation of previous studies,the effects of PLGAm/CCS scaffolds on the biological behavior of dermal fibroblasts were investigated in vitro by inoculating human dermal fibroblasts on PLGAm/CCS scaffolds and comparing with collagen-chitosan scaffolds(CCS).In vivo study,PLGAm/CCS scaffolds were implanted in animal wounds to investigate the regeneration and prognosis of dermal substitutes in vivo,which provides support for further study for clinical use of dermal substitutes.Methods:(1)The preparation of enhanced dermal regeneration template:PLGA mesh was placed in the mold and poured over with 0.5%collagen-chitosan solution(mass ratio 9:1).Product was incubated overnight at 4%,freezing for 2 hours at-20%,and then lyophilized for 24 hours to obtain PLGAm/CCS porous scaffolds with thickness of 2 mm.(2)Cell inoculation experiment in vitro:Human dermal fibroblasts were cultured then inoculated onto PLGAm/CCS scaffolds.The changes of alpha-hydroxyproline(alpha-Hp)and collagen type I carboxyl terminal peptide(CICP)levels were detected by ELISA in vitro to observe the collagen synthesis ability of fibroblasts.The effects of PLGAm/CCS scaffolds on fibroblasts were observed by laser confocal microscopy(CLSM);MTT method was used to detect toxicity of PLGAm/CCS scaffolds on fibroblasts.(3)PLGAm/CCS scaffolds were implanted into SD rats in vivo.The effects of PLGAm/CCS scaffolds on wounds in SD rats were observed by HE staining and immunohistochemistry.(6)Using RT-q PCR and Western blot(WB)to analysis the effects of PLGAm/CCS scaffold transplantation on the expression of CD31,Col ?,Col? and Elastin in neonatal rat tissues.Results:(1)PLGAm/CCS scaffolds have good mechanical strength and suitable three-dimensional porous structure.(2)After PLGAm/CCS and CCS scaffolds were inoculated with dermal fibroblasts,the contents of alpha-Hp and CICP in the supernatant increased with elapse of culture time,and there was significant difference between the two groups(P<0.05).It indicated that PLGAm/CCS scaffolds could promote collagen synthesis and secretion of dermal fibroblasts.(3)The content of fibroblasts in PLGAm/CCS scaffolds was significantly higher than that in other scaffolds(P<0.05).The results suggested that PLGAm/CCS scaffolds could promote the adhesion and growth of fibroblasts.(4)MTT results showed that PLGAm/CCS scaffolds had good biocompatibility,and the presence of PLGA knitted mesh had little effect on cell viability.(5)In vivo transplantation experiments,HE staining and immunohistochemistry demonstrated that PLGAm/CCS scaffolds could integrate to surrounding tissues,and the number of neovascularization increased significantly(P<0.05).Collages could be seen arranged in a orderly fashion,which was more conducive to tissue regeneration.(6)5 days after implantation,RT-q PCR and WB results showed the expression of CD31,Col I,Col III and Elastin in PLGAm/CCS scaffold group was significantly higher than that in CCS scaffold group(P<0.05).The results showed that PLGAm/CCS scaffolds could induce rapid cell growth,promote angiogenesis and promote the formation of new tissues.Conclusion:Enhanced dermal regeneration template(PLGAm/CCS)has high mechanical strength and good biocompatibility.It has little effect on the proliferation of dermal fibroblasts and can promote synthesis and secretion of collagen and other extracellular matrix.PLGAm/CCS scaffolds transplanted in vivo can promote angiogenesis,collagen fibers formation and deposition.Collagen was arranged in an orderly manner this helps to promote wound repair and regeneration.PLGAm/CCS has potential in clinical application.