| The seawater immersed wound would be intensified since the characteristics of seawater,such as low temperature,hyperosmosis and vibrio vulnificus.Common medical dressing had few effect on anti-seawater immerssion,and it has rarely been reported that a dressing used for antiseawater immersed wound.So a dressing can not only promote the healing of seawater immersed wound,but also protect the wound from seawater immersion seemed very imperative.Wound dressing materials should possess essential properties like flexibility,durability,gas permeability,and ability to prevent water loss et al.Many collagen related products have been developed in the past few years for the wound healing purposes.Collagen I,being a major extracellular matrix component,plays a pivotal role in wound healing.Collagen degradation fragments are chemotaxins for cells required for the formation of granulation tissue.Blue shark(Prionace glauca)is very common and widely distributed in the Pacific Oceans and East China Sea.However,shark skin usually was regarded useless leading to a resource waste and environment pollution.And there is rarely report on the properties of collagen from blue shark skin so far.Shark skin collagen(SSC)was isolated via acetic acid dissolving method and shark skin collagen sponge(SSCS)was prepared by lyophilization.Physicochemical properties of SSC and SSCS were evaluated by UV,FT-IR,CD,SDS-PAGE and amino acid composition analysis.The results revealed the triple-helical structure and the high purity of the collagen.The porosity of SSCS was 83.57%,and the swelling capacities were 86.96 g/g in water,16.18 g/g in wound exudate like medium.The water vapor transmission rate of SSCS was 0.0187 g/h.cm2,and the tensile strength was 1.788 N/mm,and the elongation at break was 4.52%.The antioxidative activity of SSC was detected by chemical UV test in vitro.The results showed that the scavenging ratios on hydroxyl was(84±2.1)% at the concentration of 20 mg/mL of SSC,and on DPPH was(18±1.2)% at the concentration of 20 mg/mL of SSC.Total reducing power of SSC was increased as a concentration dependent.The total reducing power of SSC was 0.112±0.002 at the concentration of 20 mg/m L of SSC.The anti-seawater immersed SSCS(SSCS+PU)dressing was prepared by combining the SSCS and PU films.The promoting effects on wound and seawater immersed wound healing of the SSCS and the anti-seawater immersed capability and the promoting effects on wound healing of the SSCS+PU was studied in SD rats model,respectively.Results:(1)Rat back wound model was used to evaluate the effect of SSCS on wound healing.Wound healing results showed that the wound healing rates in SSCS group were 52.1%±0.057 and 57.1%±0.080 on the 3rd and 5th day,which were significantly higher than those in GZ negative control group and CS positive control group(they were 15.9%±0.083,28.4%±0.066 and 22.2%±0.095,36.5%±0.033,respectively)(p ? 0.05).Histopathology showed that SSCS could promote capillary vessels growth in initial stage of wound healing.It could promote re-epithelialization and neogenetic muscle gowth in later period.CD31 immunopositivity results showed that the number of new vessels in SSCS group was 38±4.73 on the 3 rd day,which was significantly higher than those of GZ group(26±3.6,p?0.05)and CS group(27±3.06,).TGF-? immunopositivity results showed that TGF-? expression ratio in SSCS group was 20.1%±3.2 on the 3 rd day,which was significantly higher than those in GZ group of 6.7%±0.7 and CS group of 10.7%±0.5(p ? 0.05).EGF and bFGF immunopositivity results showed that EGF and b FGF expression ratio on the 3 rd and 5 th day in SSCS group were significantly higher than those in GZ group(p?0.05).(2)Seawater immersed wound healing results showed that on the 3 rd and 7 th day,the wound healing rates in SSCS group were 47.2%±0.08 and 81.7%±1.70,respectively,which were significantly higher than those in GZ group and CS group(they were 13.94%±5.50,49.31%±9.10 and 29.40%±1.10,59.25%±6.20,respectively)(p ? 0.05).Histopathology showed that more capillary vessels grew in SSCS group on the 3rd day and more collagen fibers and neogenetic muscle tissue grew in SSCS group on the 7 th day.CD31 immunopositivity results showed that on the 3 rd day,the number of new vessels in SSCS group was 88.00±3.61,which was significantly higher than those in GZ group of 33.33±3.05 and CS group of 37.67±2.52(p?0.01).TGF-? immunopositivity results showed that on the 3 rd and 5 th day,TGF-? expressions ratios in SSCS group were 146.33±11.67 and 117.30±9.07,respectively,which were significantly higher than those in GZ group of 58.30±1.53,73.67±5.13 and CS group of 64.00±3.00,72.67±9.50(p ? 0.01).bFGF immunopositivity results showed that on the 3 rd day bFGF expressions ratio in SSCS group was significantly higher than that of GZ group(p?0.05).EGF immunopositivity results showed that on the 3 rd day EGF expressions ratio in SSCS group was significantly higher than those of GZ group(p?0.01)and CS group(p?0.05).The molecular mechanism of promoting seawater immersed wound healing was studied by RT-PCR and western-blot.The results showed that on the 3 rd day,TGF-? mRNA relative expression in SSCS group,CS group,and GZ group were 1.28±0.09,1.76±0.24 and 0.87±0.24,respectively.TGF-? mRNA expression in SSCS group and CS group were significantly up-regulated compared to GZ group(CS vs GZ,p?0.05,SSCS vs GZ,p?0.01).On the 5 th day,integrin-?1 mRNA relative expression in SSCS group,CS group,and GZ group were 1.52±0.12,1.26±0.47 and 1.13±0.35,respectively.Integrin-?1 mRNA expression in SSCS group was significantly upregulated compared to GZ group(p?0.05).Western-blot showed that SSCS and CS could up-regulated expressions of integrin-?1 and a-SMA in wound healing and down-regulated expressions of TGF-?1 in later stage of wound healing.(3)Anti-seawater immersed wound healing results showed that on the 3 rd and 5 th day,the wound healing rates in SSCS+PU group were 25.90%±0.14,38.02%±0.10,which were significantly higher than those in GZ+PU group of 8.17%±0.02 and 15.17%±0.06(p?0.01).Histopathology showed that there were more fibroblasts and fewer inflammatory cells in SSCS+PU group than those in GZ+PU group and CS+PU group on the 5 th day.More capillary vessels and collagen fibers grew in SSCS+PU group on the 8 th day.CD31 immunopositivity results showed that on the 5 th d ay,the number of new vessels in SSCS+PU group was 38.00±7.81,which was significantly higher than those in GZ+PU group of 15.00±2.00 and CS+PU group of 20.33±4.04(p?0.05).TGF-? immunopositivity results showed that on the 5 th day,TGF-? expression ratio in SSCS+PU group was 60.00%±6.24,which was significantly higher than that of GZ+PU group(31.00%±3.61,p?0.01).EGF and bFGF immunopositivity results showed that on the 5 th day EGF and bFGF expression ratio on SSCS+PU group were significantly higher than those of GZ+PU group(p?0.05).According to the national standards(GBT 16886-2000),biosafety of SSCS was evaluated.Cytotoxicity was tested by cck-8.Total body acute toxicity was tested in mouse.Skin irritation was tested in newzealand rabbits.Skin sensitivity was established in guinea pig.The results suggested that the SSCS had no cytotoxicity.100% SSCS extraction could promote proliferation of M3T3 and L929 cells.The proliferation rates of M3T3 and L929 cells were 164%±0.308 and 150%±0.22,respectively.The hemolysis ratio was 0.27% of SSCS.The SSCS had no acute toxicity,no irritation and no sensitivity.The results showed that the SSCS could be regard as a safe dressing.These results suggested that SSCS had a good effect on the seawater immersed wound healing.The SSCS+PU could prevent seawater immersion effectively,and promote the seawater immersed wound healing significantly.The present study was aimed to provide a medical dressing with capability of anti-seawater immersed and promote wound healing to treatment of medium and small-sized wound.It is very important to medical remedy of personnel work on the sea. |
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