| Background and ObjectiveWound repair is accomplished by the interaction between the cells involved in the repair and the extracellular matrix(ECM).Collagen is the main component of ECM,which is involved in transduction of signal,transportation of growth factors and cytokines.Meantime,fibronectin(FN)is also an important ECM,which participates in the initiation of fibroblast cell and promotes proliferation,migration,adhesion and differentiation of target cells.Compared with natural protein,the recombinant protein prepared by artificial method has the advantages of poor immunogenicity,wide range of sources,low cost and high activity.In this study,we used recombinant human-like collagen(RHC)and recombinant human-like fibronectin(rhFN)to treat acute wound in C57BL/6 mice individually or in combination,and explored their effects on wound healing.Methods and Processes1.The surface morphologies of RHC and rhFN were observed by scanning electron microscope(SEM).2.The primary dermal fibroblast cells of C57BL/6 mice were identified by immunofluorescence technique,and the effects of RHC and rhFN on proliferation,migration and adhesion of cells were detected by Live/Dead staining,CCK-8 assay,scratch assay and crystal violet staining.3.The acute full-thickness skin defect wounds of C57BL/6 mice were established and treated with drugs and equivalent brine.The effects of RHC and rhFN on the speed and quality of acute wound healing,and the effects on re-epithelialization,collagen deposition,inflammatory regulation and angiogenesis were analyzed by the wound healing rate,H&E and Masson staining as well as immunohistochemical staining.Results1.The SEM showed that RHC and rhFN had irregular but tightly connected reticular structure.2.In vitro experiment:The results of the Live/Dead staining showed that the cell survival rates of all experimental groups were higher than that of control group(more than 100%)at hour 24.The CCK-8 assay indicated that the cell viability of RHC+rhFN group was higher than that of control group on day 3.The scratch assay results showed that the migration rates of all experimental groups were higher than that of control group at hours 12,24 and 36,especially in groups with rhFN.And the crystal violet staining indicated that the adhesion rates of all experimental groups were higher than that of control group at hour 4,especially RHC+rhFN group.3.1n vivo experiment:On post injury day(PID)3,the wound healing rates in RHC,rhFN and RHC+rhFN groups were(63.4±5.2)%,(66.2 ± 3.6)%and(73.3 ±7.1)%,which were higher than(52.6± 3.1)%in control group.On PID 5,the wound healing rates of RHC,rhFN and RHC+rhFN groups were(76.4 ± 5.0)%,(80.0 ±2.0)%and(78.0±7.0)%,which were higher than(63.7 ± 5.5)%in control group(63.7 ± 5.5)%.On PID 10,the re-epithelialization thickness of RHC+rhFN group was higher than that of control group,and the collagen deposition was orderly.At the same time,on PID 5 and 10,the expressions of pro-inflammatory factors(TNF-α,IL-1βand IL-6)in all experimental groups were lower,compared with control group,while the expression of anti-inflammatory factor(TGF-βl)in RHC+rhFN group was higher than that in control group,as well as the expressions of vascular endothelial growth factor(VEGF)and α-smooth muscle actin(α-SMA)in RHC+rhFN group were significantly higher,compared with control group.ConclusionRHC and rhFN both have good reticular structures and their pore sizes are conducive to the attachment of cells to the surface of the material.Meanwhile,RHC and rhFN both promote proliferation,migration and adhesion of cells effectively.At the same time,RHC and rhFN both improve re-epithelialization and collagen deposition,up-regulate pro-inflammatory factors and down-regulate anti-inflammatory factor to maintain the inflammatory balance as well as enhance angiogenesis by increasing the expression of vascular related factors.Besides,the combination of RHC and rhFN has better effect than that of single application. |
PDF Full Text Request