| Wound products are often favored by patients due to their significant antibacterial and wound healing effects,among which silver has become one of the commonly used materials for wound products due to its unique broad-spectrum antibacterial properties and less drug resistance.From the effects of silver-containing products applied to various types of wounds in clinical practice,silver-containing products have played a positive role in the process of wound healing,which is mainly reflected in their advantages of faster healing speed,better biocompatibility and significant antibacterial effect.With the emergence of nanotechnology,silver has been endowed with more excellent properties,which makes nanosilver-related products go beyond the traditional silver ion products into people’s lives.But soon found that the potential toxicity of silver nanoparticles,therefore,China’s state food and drug administration(CFDA)made nano-silver biomaterials as the third class(the highest level)medical devices,and the registration,approval and supervision of medical devices were stricter,many products with nano-silver or simple silver in their names were removed from shelves one after another,and silver ion products increased accordingly.Therefore,the impact of extensive exposure to silver ion products on the human body has been concerned,and it is urgent for relevant departments to establish a safe and reasonable product content range and quality evaluation system.There are no specific and definite requirements on content and properties,because most silver products as medical devices(rather than the drug)to manage.It is important to evaluate the safety and healing effect of the dose as described by the manufacturer.In order to provide technical reference for market supervision,this subject evaluates the quality and toxic effect of common silver-containing products in the market.Before carrying out the experimental research,the relevant application and therapeutic effect of silver-containing products in wound treatment were understood through reading the literature.When combing the clinical feedback data of domestic silver products,it was found that silver products were often applied to human body in a relatively direct way,and the common forms were liquid,gel and dressing.According to the usage of clinical departments and the market situation of products,five commonly used and common medical products containing silver were selected and purchased as research objects,which were respectively labeled as product A(liquid),B(liquid),C(gel),D(gel)and E(dressing).In this study,the basic properties and silver content of each product were first investigated,and the silver distribution uniformity and release characteristics of dressing product E were studied.Mouse fibroblasts(L929)and human immortalized epidermal cells(HaCat)were used as in vitro evaluation models in the effect study,and the cytotoxic effects of each product were evaluated by MTT test and LDH test.Furthermore,the most widely used product A was selected for animal experiment,and compared with the same type of foreign silver-containing wound product S,the anti-infection and promoting repair effect of each product was evaluated by observing skin irritation,wound healing,silver content,skin pathology and other indicators of mice.The effect of metallothioprotein on metal detoxification has attracted our attention by alleviating cell damage caused by silver-containing products.Metallothionein(MT)is a low molecular weight cysteine-rich intracellular protein,whose special structure endow MT with detoxification ability of heavy metals.In this experiment,the detoxification mechanism of MT was associated with the discovery of damage to cells caused by high-dose silver ion products,and the alleviating effect of MT on oxidative stress damage of cells caused by silver ion products was preliminarily explored.The main results are as follows:1.Detection of silver content in each product: except product D is nanosilver product,products A,B and C are all silver ion products.The silver contents of products A,B,C and D were 256.42±4.29 μg/mL、270.86±3.33 μg/mL、650.88±3.50μg/g、430.14±3.03 μg/g,which were consistent with the labeled concentration.The silver content of each part of the single product was 209.51 ± 1.77 μg /g,and the content deviation was 13.61%,which was relatively uniform.The silver content among the products of E dressing was(208.20±1.05)μg /g,and the deviation of the silver content between the products was 7.42%,which was relatively balanced.The results show that the silver content of each product conforms to its own product labeling standards,but the content of each product varies greatly,so it is significant to give a dose range for regulating the market products.2.In vitro release studies: after 10 min,the average release rates of products A,B,C and D were(83.17±5.86)%、(89.34±5.26)%、(91.06±6.78)%、(58.36±5.96)%respectively.After 30 min,the release rates of each product were(90.91±5.76)%、(93.79±5.13)%、(92.59±6.78)%、(85.97±6.92)% respectively.After 1 h,the release rates of each product were(95.43±6.97)%、(95.20±5.61)%、(94.70±5.84)%、(93.32±7.31)% respectively.The release amounts of the four products reached more than 90% of their labeled content after 1 hour,and the release amounts of the three products A,B and C reached more than 90% after 30 minutes.The results showed that none of the selected silver-containing products in this study had sustained release function,and the content of silver ions in contact with wound cells in clinical application was close to its own content.3.In vitro cytotoxicity evaluation: MTT assay on HaCat cells of each product showed that the cell survival rate of products A and B diluted by 100 times and product C diluted by 100 and 200 times was significantly lower than that of the blank control(P<0.01).The results of MTT assay on L929 cells of each sample showed that compared with the blank control,the cell survival rate of C product diluted by 100 and 200 times was significantly reduced(P<0.01),while the cell survival rate was not significantly reduced under other diluent treatments(P>0.05).Compared with the positive control group,the cell survival rate of the C product diluted by 400 and 800 times was statistically different from that of the A,B and D products at each dilution(P<0.01).MTT results showed that silver-containing products could cause changes in cell viability,and under the same exposure conditions,HaCat cells were more sensitive to the toxicity of silver-containing products than L929 cells.Cellular morphological observation showed that,except for the monomer silver product D,the other silver ion products were: HaCat cells exposed to product A and product B diluted by 100 times and HaCat cells exposed to product C diluted by 100 times and200 times were both able to cause obvious morphological changes in cells 24 hours after the two kinds of cells were infected,with increased cell spacing,smaller cells becoming round and more floating cells.The results of LDH experiment indicated that the silver-containing products with a certain concentration would cause different degrees of damage to the cell membrane of HaCat and L929 cells.Studies have shown that silver products can cause cell membrane damage in both types of cells,which is one of the mechanisms of action of silver products.However,the effects of different products on the two cell membranes were different.The destructive effect of products A and B on HaCat cell membrane still exists after being diluted 200 times,but no obvious damage was found in L929 cells in these two samples.The above studies showed that the higher silver-dose product C may be more likely to induce the decrease of the survival rate of both cells,while the lower silver-dose product A and B was less likely to cause cell death,which might be more conducive to wound healing.Different cells have different sensitivity to silver,and it is suggested that at least two kinds of cells should be selected when evaluating the cytotoxicity of silver ion products.4.Wound healing test in mice: The effects of product A on wound healing were investigated by making mouse skin wound model.The mice that did not use the silver-containing product(blank group)showed enlarged wound area(1.04±0.00)one day later,while the mice that used the silver-containing product did not show enlarged wound area(0.97±0.02,0.94±0.01,respectively).The wound healing relative area of each group reached nearly the same level on the third day(the wound healing relative area was 0.56±0.01,0.57±0.02,and 0.56±0.01,respectively),and there was no significant difference between the groups on the fourth day and after.The products with high silver content also had high silver content entering the wound surface,and the silver content between the wound and the product presented consistency.Pathological examination showed that the skin tissue recovery of the two groups treated with silver-containing products was significantly better than that of the blank group after six days of wound treatment,and the number of fibroblasts was higher in both groups.In terms of the thickness of new epidermis,the result showed that product A group > product S group > the blank control group.The above results showed that the use of silver-containing products immediately after the occurrence of the wound could effectively prevent the continuous expansion of the wound area one day later.Therefore,the application of silver-containing products in the early stage(within three days)is more significant for wound healing of the same type of wound than in the later stage(after three days).For 100 μg/mL and 250 μg/mL doses,at the later stage of wound healing,the difference of wound healing rate caused by the product is not obvious,and the wound prognosis of the two dosages was similar.Product A has slight advantages in the thickness of new granulation.We suggest that silver-containing products should better be set up within the range of 100 μg/mL~250μg /mL. |
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