Development And Effect In Skin Wound By LPRF/PVA Dressing

Background The wound dressing is a medical material to protect wounds, prevent infection and promote healing. As the rapid development of chemical engineering technology, biomedical engineering and tissue engineering, the current commercially available dressings,which use the metabolism of biological materials, natural 3D structure and good mechanical properties of engineering material that is a new generation of wound dressing. However, these dressings have a disadvantage: not contain growth factor, so it cannot passive repair wounds or contains growth factor, which only a few or individual. Platelet rich fibrin is the second generation of platelet concentrate produts that has a large number of basic experiment and clinical application prove all has the effect of promoting tissue repair, but there is no relevant report as a wound dressing. Our team has applied PRF to a variety of tissue regeneration, and have a national utility model patent in PRF. So we are playing to develop a new dressing that contained PRF. PVA is a kind of hydrophilic polymers, has good physical and chemical properties and biological compatibility, widely used in biomedicine, food industry. In particular, the preparation issimple, just melting after frozen. So we hope through the combination of the PRF and PVA to develop a new type of wound dressing.Objective Explore the freeze drying of PRF compound PVA hydrogel, and test the mechanical properties, biological compatibility, biodegradable, containing growth factor activity, and study its role in acute full-thickness skin defect repair.Methods 1. Preparing LPRF/PVA dressing: Extract artery of rabbit ears in sterile, then high-speed centrifugal preparation PRF. Then we lyophilized the fresh PRF and grind it into granules(LPRF). A certain quality of LPRF(0.5g, 1g, 2g,) put into PVA hydrogel under magnetic stirring and in 45??50?, then the mixture into 12 or 24 hole in 0.5ml or 0.25 ml per hole, and refrigerated 24 h in-20?,then melting at room temperature. Scanning electron microscopy observate surface morphology of PRF, LPRF, PVA and LPRF/PVA. 2. LPRF/PVA dressing mechanical performance tests: The different concentrations of LPRF/PVA dressing cut into 20 mm x 5mm x 0.3mm, then fixed on the universal testing machine for mechanical testing. 3. LPRF/PVA dressing biocompatibility and biodegradation: According to the containing 30mg/ml, put LPRF/PVA into sterile centrifuge tube that contain serum solution, and then leave it in incubator leaching 24 h under 37?. After 24 h, supernatant fluid co-culture with L929 cell. Through MTT methods detct the cell biocompatibility after culture a period of time. Also we adhere L929 cell into LPRF/PVA directly, through observe the morphology of cell growth to evaluate the biocompatibility of LPRF/PVA, by optical microscope, SEM and fluorescence. Implanted the LPRF/PVA into the wild type C57 mice subcutaneously to test the biodegradation rate. 4. LPRF/PVA dressing release of growth factors and promote cell proliferation: LPRF/PVA dressing in serum-free medium, according to the total of LPRF is 30 mg, then it leaching solution through ELISA to detect the vitro release of vascularendotheial growth factor(VEGF), and platelet derived factors-AB(PDGF-AB). And also study ithe proliferation on HUVEC cell. 5. LPRF/PVA dressing promote healing of skin defect in mice experiment: The subject of acute full-thickness skin defect is wild type C57, and using fixedplate fixed at rim wound. Applicated the blank, PVA, LPRF and LPRF/PVA on wound, after 3, 5, 7, 9d, cut the wound tissue for histological(HE, Masson and saturated picrate Sirius scarlet dye)and immunhistochemical staining(VEGF and CD31).Results 1. Ultrastructure observation: SEM shows: fresh PRF is 3-D collagen network, and LPRF particles appearance is oval, and its long/short axis is 118±10.383nm/81.65±8.591 nm. The surface structure of LPRF/PVA dressing similar to PVA hydrogel, which is a continuous porous structure. The inside wall will closed, if LPRF concentration is 1%. 2. Mechanical performance test results: The Young,s modulus is 6,451±1.551x10-2MPa of 1%LPRF/PVA group, compared to other groups has significant statistical(P<0.05), and the breaking elongation rate is 273.049±24.510% is no difference compared with other group, both of them showed that 1%LPRF/PVA with the excellent mechanical properties. 3. Results cell compatibility and biodegradable: L929 cell culture with different concentrations leach solution of LPRF/PVA, and is determined relative proliferation more than 88% by MTT assay, that is means the toxicity classification is 0?1 level. L929 cell direct adhesion on LPRF/PVA dressing and shows form normal and growth in good condition. All that indicates LPRF/PVA have a good biocompatibility. Buried in mice after subcutaneous 28 d, 1%LPRF/PVA of the degradation rate is between 17%?22%, in the line with the long-term use. 4. In vitro release of growth factors and cell proliferation: Using ELISA method not only detects LPRF/PVA wound dressing can normal release of growth factors, but also the release of the amont of VEGF as it LPRF power groups, but the release of PDGF-AB is less than the amount of LPRF release. LPRFconcentration between 0.05%?0.75%, LPRF and LPRF/PVA are obvious to promote the role of cell proliferation or more than 1.5% will have obvious inhibitory effect. 5. Mice skin defect healing results: Acute postoperative defect model in general view shows: wound deep into muscle membrane layer and no infection appearance and on fixed plate fall off in all observation period that means fixed plate defect model success. After 3, 5, 7, 9, 11 d of acute wound, HE, Masson and picrate Sirius scarlet dye display that in early healing time, inflammation cells and collagen deposition appearance of 1%LPRF/PVA, and collagen is given priority to with bulky closely packed of type I collagen, and faster than other groups into tissue remodeling, so that the wound basic heal completely after 11 days. While other groups like LPRF group to basic healing after 13 days, blank and PVA groups still exist wound during observation.Immunohistochemistry analysis of CD31 and VEGF shows that CD31 and VEGF are significantly well-expressed in the 1%LPRF/PVA group than other groups, blank and PVA group reveals weak positive expression than LPRF group after 7d and 9d. However, the CD31 and VEGF positive expression of 1%LPRF/PVA decreased at 11 d, and LPRF group strong positive than blank and PVA group.Conclusion Successful preparation of LPRF/PVA which combinate with LPRF power and the PVA hydrogel using physical combination that is a new type of dressing which contain a variety of active biological with excellent mechanical properties and biocompatibility, and can promote the healing of tissue for acute full-thickness after verified by animal experiments.