Development Of Chitosan-Propolis-PRP Compound And Its Effects On Skin Full-Thickness Wound Healing In Rabbit Model

Background:Platelet rich plasma(PRP) is rich in a variety of cytokines, such as VEGF, TGF-β,EGF and so on. It has positive effects on wound healing, usually used by local injection or used as topical dressing. However cytokines can not be sustained released and only have effects on wound healing in a short period when PRP was used as dressing on wound surface. Chitosan can adsorb the medicine into its microporous which is like a sponge and can sustained-release pharmaceutical active ingredients for a long time. So it is very effective in promoting wound healing. Propolis is our group’s long-term research topic. It is anti-inflammatory, anti-inflammatory and antioxidant. It has been widely recognized in promoting wound healing by researchers at home and abroad. There were some researches that developed the propolis-chitosan compound and applied to clinical and basic research.Therefore, We hope to develop our chitosan-propolis compound so that we can take advantage of chitosan’s adsorption characteristics and propolis’ positive effects on wound healing. Then by absorbing PRP, we get chitosan-propolis-PRP compound. Through the establishment of rabbit’s full-thickness dermal wounds model, observe the effects of chitosan-propolis-PRP compound on wound healing and explore the mechanism.Objective:The present study was done to develop the chitosan-propolis-PRP compound. Detect the chitosan-propolis-PRP compound’s influence on the release of cytokine. And observe its influence on New Zealand rabbit’s wound healing, the process of crawling epithelium,collagen deposition and angiogenesis. Then explore the possible mechanism of chitosan-propolis-PRP conpound on wound healing.Methods:1. Extracted propolis with 50% ethanol, mix the propolis with chitosan, dried it on drying apparatus, then we got chitosan-propolis compound. Observed its microstructure by scanning it with electron microscope.2. Counted the platelet amount in PRP. For enzy-linked immunosorbent assay(ELISA), the PRP was divided into two parts, one was detected and another was used to develop the chitosan-propolis-PRP conpound.3. The rabbits were anesthetized, 1.5cm-diameter circular full-thickness wounds were made at both sides of the new Zealand rabbits. Then divided into four groups, control group,chitosan-propolis compound group, PRP group, chitosan-propolis-PRP compound group.Then wounds was treated with different medicine. At days 3,6,9,12 after the wounds were made, wounds were photographed by digital camera and wound specimens were collected and saved for pathological evaluation in 10% formalin-fixed specimens.4. By hematoxylin-eosin staining, observed the effects of chitosan-propolis-PRP compound on inflammation and epithelial crawling.5. By Masson staining, detected the effects of chitosan-propolis-PRP compound on collagen fibrogenesis and deposition.6. By immunohistochemical staining, detected the effects of chitosan-propolis-PRP compound on Angiogenesis.Results:1.The chitosan-propolis-PRP compound has a Honeycomb porous three-dimensional network structure and it can effectively absorb the medicine into the micropores.2.The platelet concentration was(261.4 ± 27.0 × 109 / L) in whole blood and(1251.0 ±95.8 × 109 / L) in PRP. The VEGF levels in freshly extracted PRP and chitosan-propolis-PRP compound which was frozen for one week were:(976 ± 175 pg / m L)and(863 + 193 pg / m L). That means there are no obvious difference in VEGF levels in two groups.3.At day 3, the wound healing rates in each group were not significant different. At day 6,9,the wound healing rates were, chitosan-propolis-PRP compound group> PRP group>chitosan-propolis compound group> control group. At day 12, the wound healing rates were, chitosan-propolis-PRP compound group> PRP group> chitosan-propolis compound group> control group,however chitosan-propolis compound group and control group had no significant difference(P <0.05).4.By HE staining,we can find that the wounds in control group were swelling serious and have the largest number of inflammatory cells at day 3. However the wound tissue in chitosan-propolis-PRP compound group were not so swelling and have the minimum number of inflammatory cells. At day 12, there are no significant difference in inflammatory cell number in each group’s wound. And the wound in chitosan-propolis-PRP compound group and PRP group were covered by the new epithelium.(Figure 5) The comparison of epithelium creeping distance between each group at day 3 was, PRP group >chitosan-propolis-PRP compound group > chitosan-propolis compound group> control group. The comparison of epithelium creeping distance between each group at day 6 was,chitosan-propolis-PRP compound group> PRP group > chitosan-propolis compound group > control group.5.At day 3, the collagen fibers in chitosan-propolis-PRP compound group were significantly more than the control group. At day 6, the collagen fibers in chitosan-propolis-PRP compound group and PRP group were significantly more than collagen-chitosan compound group and control group. At day 9 the collagen fibers in the chitosan-propolis-PRP compound group were the most.6.At days 3 and 6, the angiogenesis number in granulation tissue is chitosan-propolis-PRP compound group> PRP group> chitosan-propolis group> control group. At day 12, the difference was not significant.Conclusion:1.The chitosan-propolis-PRP compound had a Honeycomb porous three-dimensional network structure and it could effectively absorb the medicine into the micropores.2.The VEGF levels in PRP was not obviously decrease after the PRP was integrated with chitosan-propolis compound and cryopreservation.3.Chitosan-propolis-PRP compound could promote the wound healing by regulating the inflammatory response, promoting epithelial crawling, promoting collagen deposition and angiogenesis.