| Objective: To study the properpty of collagen-chitosan nanosilver antibacterial wound dressing and observe the effect of the main components on the proliferation and secretion of growth factor and inflammatory factor in human vascular endothelial cells(HUVECs),explore its infuence on partial thickness scald wound healing.Methods: 1 In vitro study,the HUVECs cultured in Dulbecco's modified Eagle medium with10% fetal bovine serum were randomly divided into six groups: collagen-chitosan mixed solution group,small molecule collagen solution group,small molecule chitosan solution group,macromolecular collagen solution group,macromolecule chitosan solution group and nanosilver solution group,according to the different ingredients that composed the dressing with each group provided a series of concentration gradient.MTT assay was used to detect the effect of different concentrations on the proliferation of HUVECs,and the optimal concentration of promoting cell proliferation of each group was selected and acted on cells for 48 h according to the growth curve.Subsequently,the levels of growth factors(VEGF,EGF,PDGF,TGF-?1,b FGF)and inflammatory factors(TNF-?,IL-1?,IL-6,IL-8)in the supernatant were measured by enzyme-linked immuno sorbent assay(ELISA).2 Scanning electron microscopy and X-ray powder diffractometer were used to observe the surface morphologies of collagen-chitosan nanosilver antibacterial wound dressing and the distribution of silver nanoparticles on the surface.Inductively coupled plasma mass spectrometer(ICPMS)was used to analyze the release of silver nanoparticles in the dressing.Simultaneously,we detected the antibacterial activity and water absorbency of the dressing.3 Sixty-six male Sprague Dawley rats were randomly divided into a group N(normal control group)and five experimental groups: group A(silver sulfadiazine group),group B(normal saline group),group C(chitosan dressing group),group D(collagen–chitosan dressing group)and group E(collagen-chitosan-nano-silver dressing group),in which the four partial thickness scald wounds were performed on the back of each rat.The 10 rats in the experimental groups were sacrificed at each time point(1d?3d?7d?10d?14d and 21 d postburn).The wound healing rates were determined for each group.The pathological changes were observed by hematoxylin-eosin(HE)staining.The levels of the growth factors(VEGF,EGFL-7,TGF-?1,b FGF)and inflammatory factors(TNF-?,IL-1?)in the wound tissue were detected by ELISA.Results: 1 The growth curves showed that the cell proliferation enhanced significantly in 1600mg/L collage-chitosan mixed solution group,1600mg/L small molecule collagen solution group,500mg/L small molecule chitosan solution group,4800mg/L macromolecular collagen solution group and 1000mg/L macromolecule chitosan solution group.While the nanosilver solution group had no obvious inhibitory effect on cell proliferation only at the concentration of 2.5mg/L,which indicating that the concentration of nanosilver solution is the highest concentration that had no adverse effect on the cell.The results of ELISA revealed that the levels of growth factors(VEGF,EGF,PDGF,TGF-?1,b FGF)in the cell supernatant in the collage-chitosan mixed solution group were significantly higher than those in the large and small molecule collagen solution group,the large and small molecule chitosan solution group and the blank control group(P <0.05),and the levels of growth factors in the large and small molecule collagen solution groups together with the large and small molecule chitosan solution groups were also significantly increased in comparison to the blank control group(P <0.05).Moreover,there were no significant difference between the nanosilver solution group and the blank control group(P> 0.05).The levels of inflammatory factors(TNF-?,IL-1?,IL-6 and IL-8)in the collage-chitosan mixed solution group were significantly lower than those in the large and small molecule collagen solution groups,the large and small molecule chitosan solution groups and the blank control group(P <0.05),and those in the large and small molecule chitosan solution groups were also significantly declined in comparison to the blank control group(P <0.05).Whereas there were no significant difference between the large and small collagen solution groups,the nanosilver solution group(2.5mg/L)and the blank control group.2 Uniform silver nanoparticles can be detected on the surface of the collagen-chitosan nanosilver dressing and its release rate was approximately 1% with the local concentration far below 2.5mg/L.The 24 h antibacterial rate of the collagen-chitosan dressing on Pseudomonas aeruginosa,Escherichia coli and Staphylococcus aureus was 88.0%,92.0% and 90.7% respectively,with a certain antibacterial effect,but that of the collagen-chitosan nanosilver dressing on these three strains were 99.9%.There were strong water absorbency of the collagen-chitosan nanosilver dressing.3 Compared with group A and B,the dressings of group C,D and E could significantly promote the healing of partial thickness scald wound(P <0.05),and the dressings of group D and E were significantly better than that of group C,the histological examinations of skin tissues by H&E staining also show that infiltrated inflammatory cellular,increased fibrous tissue hyperplasia,significantly improved angiogenesis,and increased differentiatedly and maturely regenerative epithelium.Compared with group N,the levels of growth factors(EGFL7,b FGF,TGF-?1,VEGF)in the wound tissue of all experimental group decreased after scalding(P <0.05).Although decreased levels of VEGF were observed in the first day after injury,those in group D and E were significantly higher than those in other experimental groups(P <0.05).Compared with group N,the levels of EGFL7 in experimental group increased significantly on the 14 th day after scalding(P <0.05).The levels of b FGF in group E were significantly higher than those in other experimental groups on the 10 th day after scalding(P <0.05).Compared with group N,the levels of TGF-?1 in experimental group increased significantly on the 10 th day after scalding(P <0.05),especially those in group D and E were significantly higher than those in other experimental groups(P <0.05).Compared with group N,the levels of inflammatory factors(TNF-??IL-1?)in the wound tissue of all experimental group increased after scalding(P <0.05),among them the difference between group A and B and group N were statistically significant(P <0.05).Conclusion: The mixed solution used to prepare the collagen-chitosan dressing is more effective in promoting the proliferation of HUVECs than its single component.The collagen-chitosan nanosilver dressing has a certain antibacterial activity,and it can promote the healing of partial thickness scald wound and regulate the generation of growth factor and inflammatory response. |
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