| BackgroundBacterial infection is one of the main factors affecting wound healing process,of which wound exudate, containing the inflammatory cytokines, proteases and free radicals will slow wound healing. Research and development of novel composite biological antimicrobial dressings have important implications for treating surgery wound infection,that is the inevitable trend of the development of wound dressings. Since the British scholar Winter propose that the wound may heal faster in the moist environment than in dry environment, so people have a new understanding of the wound healing process. The effect on surrounding skin, such as maceration, especially over the sole area, will reduce the host defensive barriers against microbial invasion provided by the thick skin in the region. On the other hand, in a dry condition, cellular activities will be inhibited, an eschar will form, and further tissue necrosis may occur at the wound bed.Basic requirements for wound dressing is:has a certain permeability, less heat and electrolyte loss, reduce protein loss, accelerate wound healing, reduce pain, non-toxic, no antigenicity, and can be used widely, and the price is low.With the development of medical and biological engineering and biology, the ideal wound dressing also should have the following features:①that can absorb exudate of excess, to maintain a certain humidity on contact with the wound surface;②has good easily into a gel, adhesiveness and biocompatibility;③remove the dressing will not cause injury wound again, good biological degradability;④has certain antibacterial activity;⑤the prevention of microbial, harmful particles and other harmful substances contaminated wound;⑥the production and degradation process will not cause environmental pollution. There are reports that choosing modern anti-infective dressing for chronic wound in early stage can effectively resist infections and drainage on local wound.Alginate seaweed fiber,for which possess non-toxic, high water-absorbing gel, high oxygen permeability, good biocompatibility, easy removal of the whole, good biodegradation absorbent, hemostatic properties, can promote wound healing, high ion adsorption and such superior characteristics, have been widely used in biomedical research the. Calcium alginate degrate by enzymatic degradation in vivo,of which time inconsistency reports, but calcium alginate has the complete degradation.Early in1951, Blaine, etc discussed using alginate as the possibility of absorbable hemostatic materials in surgery, also found that alginate has inhibitory effect on bacterial growth.Alginic acid biological dressing extracted from natural seaweed, used non-toxicity material during the machining processing,thus, alginate fiber biological dressing are non-toxic to humans.Alginate can maintain a moist wound environment, conducive to wound healing and regeneration of the skin. Alginate hemostatic performance is good, also can greatly reduce the pain of the wound, much more has relatively low cost. Silver ion because of its strong antibacterial ability, low incidence of bacterial drug resistance was used as antibacterial material has a long history. Silver is thought to have a broad spectrum of antibacterial activity, antibacterial activity against Gram-negative bacteria, Gram-positive bacteria, fungi, part of viruses, or even to showed a strong antibacterial action resistant strain.Research shows that the silver particles not only has anti-inflammatory properties but analgesic effect, which can inhibit the replication of DNA,and has very strong permeability that can penetrate into the subcutaneous tissue incision, will kill pathogens of incision and suture of the body quickly and effectively by rapidly sustained release of silver nanoparticles once contact the wound. Silver compounds because of its wide bactericidal activity and anti-inflammatory effects were used as wound treatment.At present a large number of the occurrence and growth of drug-resistant bacteria promotes silver has been used as an antimicrobial agent again. Alginate dressing added with silver ion perform strong rapid bactericidal capacity, can be used to control the propagation of local bacterial of infection wound. So far, many domestic and foreign manufacturers have launched the nano silver medical antibacterial dressing with nano silver attaching to the medical absorbent gauze or medical nonwovens dressing.Guangdong Baihe medical technology co., LTD has developed a novel composite biological antibacterial dressing that is manufactured calcium alginate fibre added with nano silver,which Showing great significance of prevention and treatment of surgical wound infection, promote wound healing, reduce chronic wound inflammatory response, reduce patient discomfort, shorten the hospitalization time and reduce the burden of treatment.At the same time,It is extremely important to test the antimicrobial properties and biological toxicity of the novel composite biological antibacterial dressing to ensure the safe and effective application.ObjectiveTo develope a novel composite biological antibacterial dressing that is calcium alginate fiber antimicrobial dressing additive of nano silver, and fully evaluate its antibacterial properties, moisture absorption and biological biocompatibility in vitro and in vivo.For it provide the best choice as high-quality dressing for further application in prevention and treatment of surgical infection wounds,and in promoting the healing of wound.Methods1、The preparation of novel composite biological antibacterial dressing adding nano silver to calcium alginate fibers.To prepare nanometer silver alginate fiber by Wet spunning,then prepare novel composite biological antibacterial dressing through process of non-woven spinning. 2、Study on antibacterial properties novel composite biological antimicrobial dressings in vitro.This study adopted film method for the detection of novel composite biological antibacterial dressing antibacterial activity on Staphylococcus aureus and Pseudomonas aeruginosa, and compare the antimicrobial properties of novel composite biological antibacterial dressing with calcium alginate dressing adding silver ion and pure calcium alginate dressing. The standard strains of Staphylococcus aureus, Pseudomonas aeruginosa are suspended in TSB culture medium after have been subcultured separately.To configure bacterium liquid in the concentration of1.5×108CFU/mL by the Maxwell turbidimetry, and diluted two times in turn10-fold increments. Pipette200μl of bacteria to inoculated on the surface of antibacterial specimen surface respectively,each of which covered with a sterile PE film and culcured for24hours (each kind of dressing set five parallel samples). To take out the testing samples after24hours,adding20mL eluent to repeatedly washed covering film of each group specimen separately (Pick up the film to flushingwith tweezer),taking200μ1of which inoculated on Nutrient Agar (NA) medium after1min fully shake in vortex oscillation, incubate for24hours at (37±1)℃, count the number of colonies. The experiment was repeated for two times and the results are averaged.3、Study on the hygroscopicity of novel composite biological antibacterial dressing in vitroPut the three kind of dressing aside for24hours after being cut into5cm×5cm size in order to get the balance of fiber moisture regain. Then weigh the initial quality of each sample dressing as W. Put testing dressing and40times heavier A solution prepared British pharmacopoeia which1L distilled water with8.3g sodium chloride and0.277g anhydrous calcium chloride dissolved in in9cm diameter in sterilized petri dish at37℃for30min respectively. Weigh the wet dressing quality (W1) after griped the corner of the dressing in the air with tweezers. Unit mass of fluid absorption rate of dressing==(W1-W)/W (g/g), Per unit area of liquid absorption rate=4(W1-W) g/100cm2.The experiment was repeated for three times and the results are averaged.To determine the distribution of liquid in the fiber and between the fiber.Placed the dressing after imbibition(W1) with gauze bandage in dewatering machine to dewater for15min respectively,then determine the quality as W2which indicate the total quality of dry fiber itself and absorption liquid into the inner fibre. W3stands for the constant dry quality of testing dressing fibre that dried at105℃for4hours after centrifugal dehydration in dehydrator was measured. W1-W2indicates the liquid absorbed in the dressing among fibers,while W2-W3shows internal liquid absorbed into the fibers of dressing.(W1-W2)/W3and (W2-W3)/W3can calculate mass of liquid absorbed into fiber and internal fiber of per gram dry quality dressing.4、We detect the biocompatibility of novel composite biological antibacterial dressing with cell toxicity test, acute systemic toxicity test and haemolysis test according to a series of national standard GB/T16886-2008and ISO10993.12-2009requirements, and compared with calcium alginate dressing with silver ion and pure calcium alginate dressing.Cell toxicity experiment:The experimental group ((novel composite biological antibacterial dressing extract), the negative control group (2%calf serum RPMI1640) and positive control group ((5%dimethyl sulfoxide),3ml of each were placed in the6holes cell culture dish, joining3ml of4x104/ml L-929cell suspension solution,and observe the cell morphology and growth after cultured for72hours in the incubator. Then200ul4x104/mL cell suspension were inoculated on3pieces of96-well culture plates, while cells adherent to culture plates for24hours,abandon liquid cells and added200ul of the experimental group (novel composite biological antibacterial dressing extracts, calcium alginate dressing with silver ion leaching solution and pure calcium alginate dressing extract), negative control group (2%calf serum RPMI1640) and positive control group (5%dimethyl sulfoxide), each extract repeat4holes/plate.Cell morphology and growth were observed after72-hours incubation. Detected the cytotoxicity of experimental group material by MTT assay of L-929cells with relative growth rate (RGR) after cultured for72h.The three experimental group extracts were diluted by gradient according to the volume fraction of75%,50%,25%,10%and1%with2%calf serum RPMI1640. Then take200μl of those liquid respectively to repeat the experiment, using the same2%calf serum RPMI1640as negative control group and5%of the methyl sulfoxide as the positive control group. L-929cell culture relative growth rate(RGR) of72h was detected by MTT assay,then cell toxicity of the change of material was evaluated.Acute systemic toxicity test:Novel composite biological antibacterial dressing extracts were injected into the abdomen of test group mice according to a dose of50ml/kg standard on sterile procedure. was injected with physiological saline as the negative control group and phenol as the positive control group.After administration for7consecutive days To observe the mice breathing, eating, movement and toxicity,and record changes of body weight or death.Hemolysis test:10ml test sample extracts ((novel composite biological antibacterial dressing extracts),10ml physiological saline (negative control group),10ml of distilled water (positive control group), respectively. Observe whether there is significant hemolysis with naked eye after added0.2ml fresh anticoagulant dilution rabbit blood into solution prepared separately.To evaluate blood compatibility of test materials in vitro by the detection of ELISA absorbance.5、To observe the microstructure of novel composite biological antibacterial dressing by scanning electron microscope.The test samples((novel composite biological antibacterial dressing extracts, calcium alginate dressing with silver ion leaching solution and pure calcium alginate dressing extract) were cut into the size of1cm×1cm square,and double fixed specimens with glutaraldehyde and osmic acid after high pressure sterilization, then cover platinum on the sample surface by metal coating method,which were put under scanning electron microscope to observe the microstructure of morphology and distributions of silver ions and silver nanoparticles.Put the experimental samples in1cm×1cm size into sterilized culture tubes with tryptic soy broth (TSB) that have0.2mL bacteria solution of1.5X106cfu/mL Staphylococcus aureus (ATCC29213) dripped.Detect the bacteria distribution in the dressing after cultured for24hours. Results1、Evaluation of antibacterial properties of novel composite biological antibacterial dressing in vitro.Novel composite biological antibacterial dressing has good antibacterial effect on Staphylococcus aureus and Pseudomonas aeruginosa,for the inhibitory rates of which hwere99.7%and99.7%,and the inhibitory rates of calcium alginate dressing containing silver ion on Staphylococcus aureus and Pseudomonas aeruginosa were97.8%and97.0%,and84.9%and84.4%for calcium alginate dressing,respectively.These experiments indicated that3kinds of calcium alginate dressing has certain antibacterial ability to Staphylococcus aureus and Pseudomonas aeruginosa,but novel composite biological antibacterial dressing has obvious advantage in inhibiting and killing of Staphylococcus aureus and Pseudomonas aeruginosa.There are obvious differences between the comparison group (P<0.05).2、Evaluation of novel composite biological antibacterial dressing in vitro.We found that alginate fibers have good water absorption, which show sthe vast majority of absorption of moisture can be retained in the fibers and it is difficult to remove by centrifugal dewatering,when Centrifugal dewatering and drying the testing samples,which demonstrate calcium alginate dressing added with silver ions or silver nanoparticles did not affect the moisture absorption rate of dressing.3、Evaluation of biocompatibility of novel composite biological antibacterial dressing in vitro.Cytotoxicity test:L-929cells cultured in the extract of novel composite biological antibacterial dressing or pure calcium alginate dressing appear good adherent growth, strong refraction,which showed fusiform, no cell loss or cracking cell lesions, RGR of which cultured for72hours were90.5%and95.7%, two groups of cell toxicity was at grade Ⅰ. Given the same circumstance,RGR of extract of calcium alginate dressing with silver ion was36.1%,indicating cell toxicity was grade Ⅲ.Suggested that calcium alginate dressing and novel composite biological antibacterial dressing adding nano silver have good cell compatibility and no cell toxicity; and calcium alginate dressing with silver ions have certain cell toxicity. There is significant difference compared with negative control group (P<0.05).Much more discovered that it shows low cytotoxicity only when the silver ion calcium alginate dressing extract is diluted to the original solution of1/4, for RGR I s78.1%.Acute systemic toxicity test:There were no abnormal symptom of eating,breathing, activitives and excretion on the experimental groups of mice after intraperitoneal injection within7days,while body weight increased,and convulsions, unsteady gait,mania and other toxic reactions were not observed,even with no death.Hemolysis test:The tested dressing extracts do not caused hemolysic reaction in vitro.The hemolysis ratio of novel composite biological antibacterial dressing extracts was1.41%.4、To observe the microstructure of novel composite biological antibacterial dressing by scanning electron microscope.Nano silver particles in novel composite biological antibacterial dressing is in spherical shape with smaller average particle size, evenly distributed in the alginate fibers between the pores.Microstructure of calcium alginate dressing with silver ion shows silver particles with1μ m in size, and less regular on the shape and distribution compared to silver nanoparticles. There is only a small amount of bacteria scattered in the fiber of novel composite biological antibacterial dressing,but calcium alginate dressing with silver ions appears small amount of bacterial distribution and some bacteria colony forming cord-like distribution but not in the fiber cable links, and a layer of bacteria distribution even more connection of bacterial membrane between fibers in calcium alginate dressing was found under scanning electron microscope.Conclusions1、This study is about the preparation of novel composite biological antibacterial dressing by non-woven fabric technology after wet spinning nano silver alginate fiber made by spinning. And it confirm that novel composite biological antibacterial dressing have stronger antibacterial properties, high moisture absorption and excellent biocompatibility in the in vivo and in vitro experiments.2、This paper introduces the production process of novel composite biological antibacterial dressing. Materials can be referred to as antimicrobial materials when its microbial inhibition rate is>90%in accordance with the QB/T2591-2003.Comparing with calcium alginate dressings containing silver ion and calcium alginate dressings, the stronger antibacterial properties of novel composite biological antibacterial dressings were testified through the plate count experiment and obvervation by scanning electron microscope that shows nano silver ion of the novel composite biological antibacterial dressing are uniformly distributed on the calcium alginate fibers.3、Calcium alginate dressing owns good moisture absorption ability.The experiment proved that addiing silver nanoparticles into calcium alginate fiber to made of novel composite biological antibacterial dressing did not affect the ability of absorption of calcium alginate fibers.The novel composite biological antibacterial dressing has strong hygroscopic action what is the same as calcium alginate fiber (P>0.05).4、According to GB/T16886-2008and ISO10993-2009series of standards for the biocompatibility of biomaterials and detection method, the experimental results are fully confirmed that the novel composite biological antibacterial dressings had no cytotoxicity, no acute systemic toxicity, no hemolytic reaction. Compared with calcium alginate fiber, biocompatibility have no significant difference.Novel composite biological antibacterial dressing with excellent biocompatibility provides experimental evidence,for further clinical application as the antimicrobial dressing. |
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