| Burn is one of the major diseases that harm human health and life safety. In clinic, burn was usually treated with gauze bandaging therapy. Wound dressing as a temporary cover for a wound. In the process of wound healing, wound dressing can temporarily replace the skin and it can prevent wound from infection and inflammation. Wound dressing provide a favorable environment for wound healing. According to the dressing form, wound dressing can be divided into film dressing, hydrogel dressing, spray dressing and sponge dressing. Sponge dressing has a relatively large porosity, good wound exudates absorption, and good permeability. Sponge dressing will not cause the accumulation of wound exudates. The characteristic of deep II degree burn is large wound exudates, so select the sponge dressing for further study. Collagen, chitosan, hyaluronic acid and alginate were commonly used in wound dressings. The study select collagen and chitosan as raw material, and combine heparin.Clinical studies showed that heparin can accelerate burn healing. The mechanism of heparin promoting wound healing is as follows:Heparin have an anticoagulation effect. Heparin can improve the microcirculation of the wound, thereby effectively preventing thrombosis, and reducing the degree of burn. Studies have confirmed that heparin can enhance the activity of epidermal growth factor (EGF) and fibroblast growth factor (FGF), and protect them from inactivating. Heparin can also affect the reconstruction of collagen. The heparin and the collagenase form a complex, which regulates the rearrangement of the collagen fibers, so that the wound surface is gradually epithelization. Although heparin has been used in the treatment of burn for decades, its application is still less in China. Because heparin has shorter half-life in the body, the need for frequent administration, and the use of a large number of heparin will produce bleeding side effects. So the clinical use of heparin in the treatment of burn need dermal topical application. Collagen has good biological compatibility, biodegradability and weak antigenicity, so the collagen has good application prospect in the field of biomedical materials and drug delivery. However, the mechanical strength of the collagen sponge with a single component is low, and it is easy to be degraded. By adding cross-linking agent or amino polysaccharide increase the mechanical strength of collagen sponge, and adjust the degradation of collagen sponge. Chitosan has been widely used in medical field since its good biological activity, biocompatibility, biodegradability and antibacterial activity. Chitosan has been widely used in the artificial skin and surgical suture. A large number of studies have confirmed that chitosan has inhibitory effect on bacteria and fungi, such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.In this study, in view of deep II degree burn with large wound exudates, design a novel sponge dressing based on collagen, chitosan and heparin. The ratio of collagen to chitosan was 2 to 8. The collagen and chitosan content of sponge was 1.2%,2.4% and 3.0%. Determine the physicochemical characteristics of different percentage of sponge, including apparent density, porosity, water absorption, moisture-retention and dissolved rate, and select the proper sponge. The antimicrobial activity of the selected sponge was evaluated by measuring the size of the inhibition zone. In addition, the biocompatibility of the sponge was evaluated, including cytotoxicity in vitro, acute systemic toxicity and hemolysis. The full thickness skin excision model and the deep II degree infection burn model were used to study the anti-infection, promoting healing and mechanism of the prepared sponge. The results of the research are as follows:1. This paper has studied the extraction of collagen from pigskin by pepsin. Sigma type I collagen as the standard, by UV spectroscopy, infrared spectrum scanning, SDS-PAGE gel electrophoresis and the content of hydroxyproline determination to confirm the structure of the collagen. The results showed that the extracting collagen was type I collagen.2. Collagen/chitosan sponges (1.2%,2.4% and 3%) with different percentage contents were prepared by two step freeze drying method. Determine the physicochemical characteristics of different percentage of sponge, including apparent density, porosity, water absorption, moisture-retention and dissolved rate. Select the proper sponge, whose physicochemical properties similar to the commercial collagen sponge. In addition, the release curve in vitro of heparin of 2.4% C-C-H sponge was determined, which confirmed that the heparin could release slowly, and there was no burst release.3. Evaluation of antibacterial activity of self-made sponge dressing and commercial collagen sponge, including Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, which easily infect the wound. The results showed that the self-made sponge dressing had obvious inhibition zone, which could inhibit the growth of these three kinds of bacteria. However, the commercial collagen sponge without obvious inhibition zone, could not inhibit the growth of these three kinds of bacteria.4. Evaluation of the biocompatibility of the sponge, including cytotoxicity in vitro, acute systemic toxicity and hemolysis. The results showed that the self-made sponge dressing had good biocompatibility.5. Evaluation of the effect of 2.4% C-C sponge,2.4% C-C-H sponge and commercial collagen sponge on rat skin incision model. On the 14 days, the wound surface was recorded, and the wound healing rate was calculated by Image J software. And then the skin tissue was removed for HE staining. The results showed that the wound healing rate of negative control group, positive control group,2.4% C-C sponge group and 2.4% C-C-H sponge group was 85.5%,99.8%,98.3%,90.1%, respectively. Pathological biopsy results showed that the best healing was 2.4% C-C sponge group, followed by the positive control group,2.4% C-C-H sponge group, negative control group. The immunohistochemistry results of VEGE and EGF showed that VEGF was expressed in the wound tissues of all groups at 14 days after operation. 2.4% C-C-H sponge group showed the strong positive (+ + +), commercial collagen sponge group and 2.4% C-C sponge group showed moderately positive (+ +), gauze group showed weak positive (+). EGF was expressed in the wound tissues of all groups. The amount of EGF in the wound tissue of the collagen sponge group,2.4% C-C sponge group and 2.4% C-C-H sponge group were moderately positive, and the gauze group was weak positive. The amount of VEGF and EGF of the 2.4% C-C-H sponge group was slightly more than that of the other three groups, and it could promote the wound healing. But in the initial stage of wound healing, heparin of 2.4% C-C-H sponge inhibit platelet aggregation and release, and heparin has anti-inflammatory activity, which will delay wound healing. So at 14 days after operation, the wound healing rate of the 2.4% C-C-H sponge group was slightly lower than that of the 2.4% C-C sponge group.6. Evaluation of the effect of 2.4% C-C sponge,2.4% C-C-H sponge and commercial collagen sponge on a guinea pig burn model. On the 4 and 7 days, the wound surface was recorded, and the wound healing rate was calculated by Image J software. And then the skin tissue was removed for HE staining. The results showed that the wound healing rate of negative control group, positive control group,2.4% C-C sponge group and 2.4% C-C-H sponge group was 19.0%,23.6%,31.0%,34.0%, respectively, on 4 days. On 7 days the wound healing rate of negative control group, positive control group,2.4% C-C sponge group and 2.4% C-C-H sponge group was 36.9%,39.5%,41.2%,57.0%, respectively. Pathological biopsy results showed that there were a large number of pyocyte and inflammatory cells in the wound of gauze group and commercial collagen sponge group, while there was only a small amount of pyocyte and inflammatory cells in 2.4% C-C sponge group and 2.4% C-C-H sponge group. And the best healing was 2.4% C-C-H sponge group, followed by 2.4% C-C sponge group, the positive control group, negative control group. The immunohistochemistry results of VEGF and EGF showed that VEGF was expressed in the wound tissues of all groups at 4 days after operation.2.4% C-C-H sponge group showed the moderately positive (+ +), but commercial collagen sponge group,2.4% C-C sponge group and gauze group showed weak positive (+). EGF was not expressed in the wound tissues of the gauze group. The amount of EGF in the wound tissue of the collagen sponge group.2.4% C-C-H sponge group showed the strong positive (+ + +), and commercial collagen sponge group and 2.4% C-C sponge group showed the moderately positive (+ +). The results showed that the 2.4% C-C-H sponge had anti infection and promoting healing effect on the deep II degree burn infection model.In summary, the sponge has good physical and chemical properties, significant antibacterial activity and good biocompatibility. Through the full-thickness skin defect model and the infected part-thickness burns model, it was confirmed that the sponge can promote wound healing. However, further research about biological safety and the effectiveness and mechanism of promoting wound healing should be carried out. |
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