The Effect Of Ecdysterone Combined With Mesenchymal Stem Cells On Wound Healing

1 BackgroundWound healing is a complex biological process of self-repair which involved in many factors of body, and tissue regeneration plays a significant role. Normal wound healing process includes three phases:inflammation phase, cell proliferation phase, and extracellular matrix reconstruction phase.Skin wound often leads to injury on epidermis, dermis and adnexal, namely, it exists cell dysfunction or tissue defect. The progress of wound healing includes stopping bleeding in earlier period, protecting wound surface, controling inflammation, proliferation of kinds of cells and tissue, forming granulation tissue, creeping of the wound limbus, and so on. Proliferation of endothelial cells, epithelial cells and fibroblasts is the key to the formation of granulation tissue, also the key to wound healing.There are many factors affecting wound healing, such as age, nutritional status, wound size, kinds of systemic-diseases and so on. Some factors can result in disunion of wound for a long time, which is counted as the thorny issue of clinical treatment. For the inhibition of cell proliferation caused by necrotic tissue, microbial infection, metabolism and other factors, chronic and refractory wounds always stay in the inflammatory response period, instead of getting into the next period, cell proliferation. Although new technologies and new means come out one after the other, therapeutic efficacy of chronic and refractory wounds such as chronic skin ulcer was still not good. Researches reveal that, all over the world, the number of patients requiring amputation for their chronic skin ulcers will increase by 30% in 2030. Therefore, it is necessary to make more in-depth study and practice to develop effective drugs which could promote wound healing and accommodate clinical requirements and international standards.Mesenchymal stem cells (Mesenchymal stem cells, MSCs) researching is a central issue in many fields. Some reports suggested that MSCs can differentiate into epidermal tissue, bone tissue, cartilaginous tissue, nervous tissue by the effect of different chemicals and cytokine. Meanwhile, MSCs play many roles on wound healing:induce the reproduction of blood vessels; product CD4+chemotactic factor for lymphocytes; express chemokine receptor-CCR7; participate the process of cell migration; supply fibrocytes for burn patients; adjust fibrocellular transportation, generation and differentiation on part of wound by the mechanism of signal transduction between cells, which promote wound healing process and reduce cicatricle. Nakagawa and his colleagues indicated that MSCs combined with fibroblast growth factor can promote the process of cutis wound healing. There was a clinical report about use of MSCs on deep burn patient which suggested that autogeneic MSCs suspension can promote the regeneration of skin and blood vessel by spraiding MSCs suspension on wound surface. Nowadays, there are still many problems about clinical universal use of MSCs, such as the complex technique, undeveloped quality control process and expensive cost. Therefore, it will be a long term before the clinical universal use of MSCs.Ecdysterone (ecdysterone, EDS) is also known asβ-Ecdysone in animals. It is an insect metamorphosis hormone, which could stimulate the insect dermal cell division to generate new skin, result in insect metamorphosis and regulate insect metabolism. But it is found that ecdysterone exists in plants more than in animal. At present, ecdysterone is mostly extracted from plants. Ecdysterone in vertebrates is also the necessary substance. Studies indicate that it also showed a strong pharmacological activity in higher animals, and its side effects were minor. Nowadays, we know its actions include:1) promote nucleic acid and protein synthesis; 2) adjust glucose metabolism; 3) modulate lipid metabolism; 4) regulate gene expression; 5) improve immune function; 6) posses antioxidan activity; 7) promote angiogenesis and establishment of collateral circulation in ischemic area; 8) promote proliferation of varied cells.Foreign extracorporal studies indicated that ecdysterone can promote the cell differentiation of human epidermic cells. The previous investigations of our study group found that ecdysterone can stimulate generation and fission of allantoic veins, degrade vasopermeability of pneumoangiogram, improve the wound healing process of rabbit skin, promote proliferation of human epidermic stem cells and marrow mesenchymal stem cells. On account of the effects in all these systems, the extensive disposition and the its bright application in future, it is fairly feasible to be used in wound healing.The previous investigations of our study group identified that ecdysterone can improve the wound healing process, and ecdysterone paste was produced to be used in wound healing. Simultaneously, we also indicated that ecdysterone can promote proliferation of MSCs. Therefore, New Zealand white rabbits with Deep II degree scald skin on the dorsal are as models in this experiment. It was used to investigate the effect of ecdysterone combined with MSCs on wound healing of cutis in experimental rabbits. And it can provide a basis for the clinical application of ecdysterone in wound healing.2 ObjectiveTo investigate the effect and possible mechanisms of ecdysterone combined with MSCs on wound healing of cutis in experimental rabbits.3 MethodsTaking 18 healthy New Zealand white rabbits, injecting pentobarbital(35mg/kg) into vein of ear for anesthesia, depilating the back fur with 10% sodium sulfide, using iodophor to disinfect the back skin of rabbits. Scald the skin of each rabbit to form a circular wound with diameter 2.0 cm by Temperature Scald Device with constant temperature of 100℃and pressure of 0.5KG for 12 seconds, causing the trauma models. Pathology confirmation of models proved the degree of scald is deepⅡ. Each rabbit has four scald wound, labeled randomly A, B, C and D, respectively as blank control group(group A, only give conventional disinfection), ecdysterone solution group(group B, smear ecdysterone solution with ecdysterone content of 25mg), MSCs suspension group(group C, smear MSCs suspension with cell population of 1-2×105), and ecdysterone combined with MSCs group(group D, smear ecdysterone solution with ecdysterone content of 25mg and MSCs suspension with cell population of 1-2×105). After applying medicine, all the scald wounds were covered with Vaseline gauze, and then bandaged with dry gauze. The wounds were washed with isotonic Na chloride, disinfected with alcohol everyday, and then deal each wound with original modality. Every rabbit was breed separately in cage.18 rabbits were selected randomly, each 6, to observe the wound healing rate and the visual changes of wound in each group on 5,10,15 days after operation. Observe d the histopathological indexes and the inflammatory cell infiltrate degree to assess the therapeutic effect after HE staining and making section preparatio. The measurement data are expressed as Mean±SD. And SPSS 13.0 statistical software was used to analyze the data(using analysis of One-Way ANOVA, Repeated Measures, and the comparison of groups were used LSD or Tamhane's T2 test).4 ResultsThe wound healing rate of each group was increased over time in the period of observation (0-15d). It indicated that the model possess a certain natural healing ability.On 5 days after modeling, the wound healing rates of group B were obviously higher than the group A (P<0.05).There was no significant difference in wound healing rates between group C, D and group A (P>0.05). On 10 days after modeling, there were no significant differences among all groups in the wound healing rates (P >0.05). On 15 days after modeling, the wound healing rates of group B, C and D were obviously higher than group A (P<0.05). The difference between group D and group A was more obvious than between group B, C and group A (P<0.01).There was no significant difference in wound healing rates among group B, group C, and group D(P> 0.05).The visual observations of wound:On 5 days after modeling, there was no apparente purulent infiltration on the wounds of each group. Wound contraction of group B was obvious and the other groups were not.The wound of group A showed small amounts necrotic tissue, small amounts purulent infiltration only covering raw surface, and the tissue under raw surface was dropsical slightly. The raw surface of group B was moist, no apparente purulent infiltration, and its bouncary was clear with normal tissue surrounding. Epidermis was growing toward the centre of raw surface. Wound contraction of group C was not obvious. The purulent infiltration was apparente. The tissue under raw surface was dropsical slightly in some of individuals. Wound contraction of group C was not obvious. The dropsy of tissue under raw surface was not obvious. The purulent infiltration was not apparente. On 10 days after modeling, the wound of all groups were dry and scabed, no infection, and apparente contraction of wound surface and subcutaneous tissue. The wound of group A, C and D could still see exudation. On 15 days after modeling, the wound of group B, C and D could see further wound contraction, dry wound surface, no infection, thick crust on superficies, and small wound surface that not healed. The crusts of all groups were shedding at different level. Compared with the wound of 5 or 10 days after modeling, the wound contraction of group A was conspicuous and the edge of wound formed crust, but the crust did not shed obviously. There were small amounts purulent infiltration under the crust and still large area did not heal.The observation of tissue sections under light microscope:On 5 days after modeling, the wound of each group mainly showed exudation and congestion, little granulation tissue. The inflammatory cell infiltrate degree of group B, D was lower than group A(P<0.01). There was no significant difference in histopathological scores among all groups(P>0.05). On 10 days after modeling, the wound of all groups displayed exudation and congestion still, some granulation tissue, less inflammatory cells, more collagen bundles with order, a few proliferation of epithelial cells. There were no significant differences among all groups in histopathological scores and inflammatory cell infiltrate degree (P>0.05). On 15 days after modeling, the wounds of group B, C and D displayed much granulation tissue, a few inflammatory cells in the junction of dermis and epiderm, more ordered collagen bundles, a large amount of proliferation of epithelial cells and fibroblasts. Much granulation tissue, little inflammatory cells but infiltrated widely can be seen in the raw surface of group A. The proliferation levels of collagen, epithelial cells and fibroblasts in group A were lower than other groups. There were no significant differences among all groups in inflammatory cell infiltrate scores (P>0.05). The histopathological scores of group B,C and D were obviously higher than group A (P <0.01).5 ConclusionsEcdysterone solution, MSCs suspension and Ecdysterone combined with MSCs can all improve the wound healing process in experimental rabbits. EDS can improve the wound healing process in the early time. The mechanism may relate to the local anti-inflammatory effect of EDS that promote the formation of granulation tissue, proliferation of epithelial cells, endotheliocyte and fibroblasts. Thereby, it enhance the speed and quality of wound healing. In addition, on 15 days after modeling, although there was no significant differences in wound healing process between EDS+MSCs group and EDS,MSCs group, the mean of wound healing rates of EDS+MSCs group was higher than EDS group and MSCs group, and P<0.01. Accordingly, we can assume that MSCs combined tith EDS can show its better effect on wound healing process if the days of the treatment with EDS+MSCs get longer and the sample size get larger. The inference could be authenticated in the further experiment.