Security Of Medical Vacuum Sealing Drainage Combined With Ozonated Water

Vacuum sealing drainage technique, also known as the Negative pressure wound therapy or Vacuum assisted closure Technology,which consists of polyethylene alcohol hydration foam, biological semipermeable membrane, drainage tube and negative pressure source. Refers to a after treatment wounds, which filled with a polyethylene alcohol hydration foam and covered with a biological semipermeable membrane, providing a closed treatment of space, negative pressure finally connected response of suction device, to promote a new pure physical therapy of wound healing by intermittent low negative pressure. VSD was invented by Fleischmann and used in USA earliest. In recent years, it has been used in chronic wound healing, open injury of large area skin avulsion and avulsion injury. its clinical value and clinical curative effect has got more and more definite and attention. There are many advantages for wound therapy managed by VSD technique, which can facilitate blood circulation, increase the blood supply, alleviate edema around the wound, accelerate the wound healing, inhibit the bacteria breeding and control infection, offer effectively mechanical traction and stimulate the growth of granulation tissue, promote drainage wound healing, reduce dressing change times, alleviate the pain of patients, ease the workload of the clinicians, short the hospitalization time and reduce medical costs.However, VSD technique also has many disadvantages. For example, the insufficient ability of anti-infection is one aspect of the technology of VSD. There is a frequent phenomenon in clinical application that some patients managed by VSD technique still have different levels of wound infection. These infected wound is often managed by the irrigation of gentamicin solution currently. However, traditional gentamycin solution local flushing, easily leading to drug resistance, resulting in failure for the flap or skin graft.Ozone is a pale blue and stench gas, which constituted by three oxygen atoms. Ozone is discovered and named by Schorbein in1840, which has strong oxidation to oxidize pathogenic microorganisms and harmful substances in the air or water. Ozone water is a new broad-spectrum disinfectant, has many advantages for example convenient, low stimulation, rapid degradation and no pollution etc. At present, ozone water is playing an important role in water treatment, chemical oxidation, food processing and storage, medical and health fields. According to related studies, it also has a good effect on cleaning infected wounds. But there is a lack of experiment reports about the treatment of the wound infection of VSD material combined with ozonated water. There are many questions about relationship between whether VSD material combined with ozonated water can be used in anti-infection and promoting wound healing and their safety and feasibility.Our previous experiments have confirmed the material stability and feasibility of10μg/ml ozonated water combined with VSD. Two main reasons can account for the selection of10μg/ml ozonated water in the experiment. One reason is that previous studies have reported that10μg/ml ozonated water is able to kill most pathogenic microorganism. The other reason is that high concentration ozonated water may bring oxidative injury to normal tissues. Therefore, the concentration of ozonated water should satisfy the demand of killing pathogenic microorganism as well as not cause injury to normal tissue. If it is feasible to combined VSD technique and ozonated water in material science, further explorations will be performed on safety study in vivo and animal experiment, all of which will pave the way for clinical popularization and application.Our experiment is consisted of three parts. Part One included the production of extract preparation of VSD material combined with ozonated water and Intradermal irritation with New Zealand white rabbits. Part Two included the maximum dose response method of Delayed type hypersensitivity experiment with Albino guinea pigs. Part Three included the In vitro cytotoxicity experiment. With different concentrations of extracts liquid to culture cells.According to Magnusson and Kligman grading standards,6level standards are used to identify the feasibility and biological safety of cell cytotoxicity.Part One:Production of extract preparation of10λg/ml ozonated water combined with VSD material and Intradermal irritation experimentPurpose:The aim of chapter one was to produce extract liquid of10μg/ml ozonated water combined with VSD materials for Intradermal stimulation test with New Zealand white rabbits.Materials and methods:1. Production of ozonated water:We poured sterile water for injection into the water storage tank of ozonated water generator (type:Ozonosan Alpha Plus1107). The oxygen valve was opened and10μg/ml ozonated water was produced using ozone bubbling method. Ozonated water was stored in Amber Laboratory Bottle. 2. The preparation for extract liquid of10μg/ml ozonated water combined with VSD materials:In super-clean bench after sterilization and disinfection, to prepare extract liquid of VSD materials combined with10μg/ml ozonated water with volume ratio1:1and1:2respectively with12hours soaking reaction.3. Intradermal irritation test:With10New Zealand white rabbits, were randomly divided into2experimental groups. Extract liquid were used to perform such biological tests as intracutaneous stimulation test:0.2ml experimental extract liquid, ozonated water, control extract liquid and physiological saline were respectively injected in rabbit spinal’s different areas. Observation erythema and edema of the injection site were recorded,24h,48h,72h and immediately after injection. According to the intradermal irritation score standard, we will calculate the primary irritation scores and the primary irritation index after each time point.Results:The extract liquid of10μg/ml ozonated water combined with VSD materials was successfully produced. The result of two experimental groups of intradermal irritation test results showed no obvious erythema, edema and ulcer reaction and primary irritation score and primary irritation index were0.Conclusion:10μg/ml ozonated water can be produced by the type Ozonosan Alpha Plus1107ozone generator. The results of different concentrations intradermal irritation test of extracts were negative. Our preliminary experimental results prove that materials combined with10μg/ml ozonated water have biological safety and clinical feasibility. Part Two:The extract liquid of10μg/ml ozonated water combined with VSD material for Delayed type hypersensitivity test (DTH)Purpose:The aim of chapter two was to produce extract liquid of10μg/ml ozonated water combined with VSD materials for Delayed type hypersensitivity test (DTH) with Albino guinea pigs.Materials and methods:1. The experimental method and divided experimental groups were as follows: The delayed hypersensitivity of material was assessed in guinea pigs with maximum doses method.20albino guinea pigs were randomly divided into three groups,10rats for experimental group,5rats for ozonated water group and5rats for normal saline group.2. Production of ozonated water:We poured sterile water for injection into the water storage tank of ozonated water generator (type:Ozonosan Alpha Plus1107). The oxygen valve was opened and10μg/ml ozonated water was produced using ozone bubbling method. Ozonated water was stored in Amber Laboratory Bottle.2. The preparation for extract liquid of10μg/ml ozonated water combined with VSD materials:In super-clean bench after sterilization and disinfection, to prepare extract liquid of VSD materials combined with10μg/ml ozonated water with volume ratio1:1with12hours soaking reaction.3. Delayed type hypersensitivity test (DTH):With20Albino guinea pigs were randomly divided into three experimental groups. Different extract liquid of0.2ml were injected in the guinea pig dorsal side, Region A was Freund complete adjuvant mixed with ozonated water, Region B was VSD mixed with ozonated water, Region C was A mixed with B. Skin locally induced reaction and sticking excitation were conducted after7days and14days respectively. Observation with erythema and edema of the injection site of the skin were rccorded after24hours and48hours. Sensitization results were graded according to the Magnusson and Kligman classification.Results:The maximum concentration extract liquid of10μg/ml ozonated water combined with VSD materials was successfully produced. The result of three experimental groups of Delayed type hypersensitivity test showed no sensitization. Results showed no obvious erythema, edema and ulcer reaction. The positive rate was0.Conclusion:The results of different experimental groups of delayed type hypersensitivity test showed no sensitization. Preliminary experimental results prove that materials combined with lOμg/ml ozonated water have biological safety and clinical feasibility.Part Three:The different concentration extract liquid of10pg/ml ozonated water combined with VSD material for vitro cytotoxicity testPurpose:The aim of chapter three was to produce different concentration extract liquid of10μg/ml ozonated water combined with VSD materials for vitro cytotoxicity test with L-929mouse fibroblast cells.Materials and methods:1. The experimental method and divided experimental groups were as follows: The MTT method was used to assess cell proliferation with L-929mouse fibroblast cells. The experimental group were randomly divided into five groups with different concentrations of extract concentration forl:1,1:5,1:10,1:15and1:20, negative control group with RPMI1640medium and positive control group with DMSO medium and ozonated water group. With each group have6holes.2. Production of ozonated water:We poured sterile water for injection into the water storage tank of ozonated water generator (type:Ozonosan Alpha Plus1107). The oxygen valve was opened and10μg/ml ozonated water was produced using ozone bubbling method. Ozonated water was stored in Amber Laboratory Bottle.3. The preparation for extract liquid of10μg/ml ozonated water combined with VSD materials:In super-clean bench after sterilization and disinfection, to prepare extract liquid of VSD materials combined with10μg/ml ozonated water with different volume ratio1:1,1:5,1:10,1:15and1:20with12hours soaking reaction.4. The preparation of the cell culture fluidWith different volume ratio of prepared extracts liquid0.5ml and9.5ml RPMI1640cells culture fluid mixed, which was used to culture cells in experiment. The positive control group was mixed with0.5ml DMSO and9.5ml RPMI1640cells culture fluid. The negative control group also was RPMI1640cells culture fluid and ozonated water group was mixed with0.5ml ozonated water and9.5ml RPMI1640cells culture fluid.5. In vitro cytotoxicity test with MTT methodThe L-929mouse fibroblast cells were made into4×107/Lcell suspension liquid with200μL cell suspension liquid in each hole, and cultured in CO2incubator for24h. Observing the cells adhere to the discard solution, and then washing it with PBS liquid for3times. The96hole plateswere randomly divided into five experimental groups with different concentrations of extract concentration for1:5,1:10,1:15,1:20, negative group (RPMI1640medium), positive group (DMSO) and ozonated water group. Each group has6holes, with every holes was filled with200μL cell suspension liquid. The96hole plates were cultured120h, and then discard the original broth in1,2,3,4,5days respectively. At the same time, adding20μl MTT liquid with concentration of5mg/ml, placed in the CO2incubator for4hours after termination. To absorb the supernatant liquid, joined150μl DMSO in every hole immediately, and posting plates in the slight oscillation for10minutes. Using enzyme immunoassay instrument with490nm wavelength and measuring the absorbance value and recording the results. Calculating the relative cell proliferation rate (RGR) and getting cell cytotoxicity grading.6. Statistical methodThe absorbance and cell relative growth rate values are used with SPSS13system for the single factor analysis of variance for each time point of each group data, and between the two groups was compared with Least Significant Difference method.Results:1. The observation of cell morphologyAfter one day, every group cells were adherent growing. The cells have spindle shaped, strong refraction and well process extended. Positive control group showed more circular suspension and a few cells. After three days, cells in every group have a significant increasing in quantity. We can observe many spindle, triangular and irregular shape of cells. Besides, we also can observe more cells were under a split phase. No significant morphological differences can be observed in the experimental group, negative control group and ozonated water group. But in positive control group, which has a relative small number cells. We can see a lot of suspension cells with dead. After five days, there is still no significant morphological differences can be observed between other experimental group negative control group and ozonated water group. In the positive control group, there has no obvious change compared with before. Based on this we can see that preliminary judgment of materials combined with ozonated water have no cytotoxicity. L-929mouse fibroblast cells were cultured in different experimental groups to measure the value-added rate and to evaluate the cytotoxicity. The datas were analyzed and evaluated according to the criterion of the national standard. While the concentration of1:1experimental group from the beginning to the end of fifth days, the cells were with round, refraction, not growth, a large amount of suspended, slowing down or inhibiting cell growth.2. The Cell proliferation activity of each experimental group at different timeOver time, the A490of experimental group showed increasing trend. The A490of different time point of each experimental group and ozonated water group A490compared with positive control group have significant difference(P<0.05). While there is no significant difference of A490compared with experimental groups and the control group(P>0.05).Conclusion:There is no significant cytotoxicity in experimental group and ozonated water group. The results of different experimental groups of vitro cytotoxicity test with MTT method showed no cytotoxicity. Two kinds of substances mixture with1:1can produce something which can inhibit cell growth. Generally speaking, based on these experimental results, we can conclude that materials combined with10μg/ml ozonated water have biological safety and clinical feasibility.