| Platelets are produced from a kind of very large bone marrow cells called megakaryocytes. As megakaryocytes develop into giant cells, they undergo a process of fragmentation that results in the release of small bioactive cytoplasm. As we all know, the mian function of platelets is stick to breaks in the blood vessel wall and also stick to each other to stop bleeding. When there is an injury or cut, it is the platelets that react first to injury, platelets clump onto the fiber surrounding the vessel wall, providing the initial seal to prevent bleeding, and the phospholipid surface for activation of prothrombin complex to promote the formation of blood clots. Platelets make blood clots retracted to become solid tampons, through extending pseudopodia into the fiber mesh and contraction of platelet contractile protein to extrud serum in blood clots. Hemostasis was regarded as a fully understood function of platelets。In recent years, owing to its important role in wound healing, platelets aquired more and more favour and resulted in new central issue. Platelets respond at wound site to initiate procoagulant process, and release their alpha granules after activation, form a rich source of important growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), epidermal growth factor(EGF), transforming growth factor beta (FGF-b) and insulin-like growth factor-I (IGF-I), these growth factors provide the potential to modulate the healing of tissues through stimulating the proliferation of target cells, angiogenesis, increase the synthesis of extracellular matrix and matrix turnover. Platelets is more important and complex than we thought, more and more evidence indicate that in addition to hemostasis, platelets is also important for wound healing.Platelet-rich plasma (PRP) is the routinely application form of platelets, however, it’s can’t be stored beyond five days, and it’s very likely to be contaminated by bacteria then result in sepsis for PRP stored under condition of shaking at (22±2)℃, the inconvenient storage and transportation limits its clinical applicability. In order to solve the problem of the short shelf life of platelets, the experts developed a new way to preserve platelet-frozen, frozen platelets can be stored beyond one year. but, dimethyl sulfoxide (DMSO) was employed during the frozen procedure, concentration of DMSO exceeds a certain criterion will produce toxic effects to body, requirement of large cryogenic refrigeration equipment make it inconvinent to transport. By comparison, freeze-drying, as a long-term preservation method allowing platelets to be stored at room temperature with smaller size and lighter weight, has many advantages such as no need for cooling equipment and convenient transportation. To obtain FDP with integrated structure and function, our team has investigated a variety of pre-lyophilized buffer formulation, freezing and vacuum drying conditions and obtained the optimal freeze-dried procedure of platelets. But the FDP experienced pretreatment, freezing, vacuum drying and re-hydration processes, these prosess do harm to the structure and function of FDP, and result in different characteristics between FDP and PRP in the expression of glycoprotein on the surface of platelet and in vitro experiments. The influences on the hemostasis and wound healing effect of FDP need to be further verified.In this study, we preserved platelets used the freeze-dried procedure optimized by our group previously, we investigated the growth factor levels in the re-hydrated FDP, the result indicating the concentration of some kinds of platelet growth factors in FDP was higher than PRP, to demonstrate the effectiveness of the FDP, we studied the effect of FDP from cell proliferation, acute animal wound healing and trauma hemostasis. In addition, we also evaluate the safety of FDP. Part I section 1 Test concentrations of growth factors in platelets before and after freezing-dried procedureenzyme-linked immuno sorbent assay kits (Quantikine Immunoassay Kit; RayBiotech) were used to measure and compare the differences of PDGF-BB, TGF-β1, VEGF, EGF, IGF-I and FGF-b levels between FDP and the initial PRP used for the preparation of FDP. the result indicating the concentration of PDGF-BB in activated supernatant of FDP:(421.81±44.09) pg/ml is slightly lower than PRP: (390.94±22.43) pg/ml, the difference was not significantly (P> 0.05), other five kinds of growth factors were significantly higher than PRP (P<0.05). According to the results, FDP possess superior ability to PRP to release growth factorsPart I section2 study of the effect of FDP on endothelial cell proliferationWe prepared the FDP following the FDP preparation optimized by our team. To determine the effect of FDP on endothelial cell proliferation, a cell proliferation assay was performed using human umbilical vein endothelial cells (HUVEC). Re-hydrated FDP and PRP were actived using thrombin-calcium gluconate, and then they were centrifuged to remove solids matter. The supernatant was used to assay for endothelial cell proliferation. A significant increase in endothelial cells cultured in the presence of FDP, PRP and FBS was seen, increasing the concentration of FDP and PRP activated media in serum free 1640culture media resulted in degenerative in HUVECs proliferation(P<0.05); HUVECs cultured in 5% FDP was significantly higher than 5% FBS at 48h, and similar with PRP and FBS at 24h,72h in the condition of same concentration. Studies show that the supernatants from FDP and PRP and FBS has been shown to be similar potent mitogenic for HUVECs,in the condition of same concentration Part I section3 study of the effect of FDP on wound healing in rats acute woundWound healing is a reconstruction and regeneration progress of a variety of cells and extracellular matrix, it’s consist of four continuous and overlapping phases involves hemostasis, inflammation, proliferation and remodeling. These phases is characterized by inflammatory cells and fibroblast proliferation, angiogenesis, extracellular matrix deposition, and demand the regulation and cooperation of multifarious factors through various biological effects. Previously, we have demonstrated that FDP possess superior ability to PRP to release growth factors, and similar in promoting vascular endothelial cell proliferation with PRP. Further, we want to know whether the FDP can promote wound healing in vivo, full-thickness skin wounds in SD rats were utilized and treated with the FDP, PRP and rhEGF, and compared with group BC without treatment, to evaluate the wound healing effect of FDP. the wound healing effect was monitored by the velocity and quality of healing. With regard to the results, at 7th days, the wound healing was significantly accelerated in all wounds treated with FDP, PRP and rhEGF:(45.08±15.42)%, (46.97±11.48) % and (42.12±11.95)% compared with control group:(31.41±12.84)% (P<0.05), the wound healing rate did not differ significantly among group FDP, PRP and rhEGF; at 10th days, the wound healing in all wounds treated with FDP, PRP and rhEGF: (72.87±13.29)%, (82.7±6.34)% and(71.43±9.81)% compared with control group: (47.06±18.27)%(P<0.05), the wound healing rate of group PRP was significantly higher than group FDP and rhEGF(P<0.05). Histologically, In FDP and PRP group, inflammation was shortened, wounds showed higher velocity of epithelialization, more cells proliferation, angiogenesis and formation and mature of collagen fibers. This study provides evidence that FDP can not only accelerate healing wound but also produce the similar effect to PRP.Part II Experiment study of the hemostatic effect of FDP on injureHemostasis was regarded as a fully confirmed function of fresh PRP, while the effect of FDP after freezing-dried process need experimental verification. In hemostasis assessment, A trauma artery bleeding models of New Zealand rabbit was established, to study the hemostatic effect of FDP, by compared with Freeze-dried cryoprecipitate (FDC) and synergistic actions of FDP and FDC. The results show that, in groups FDP, FDC and FDPC, hemostasis was obtained more rapidly and there was less blood loss than that in group BC (P<0.01); There was significant difference in the hemostatic ability between group FDP and FDC(P<0.01); Compared with FDP and FDC used alone during hemostatic, there was less blood loss and bleeding time in synergistic actions of these (P<0.01). These indicating that FDP can provide a better hemostatic effect than FDC on wounds induced artery bleeding, and the synergistic actions of FDP and FDC possesses better effect than they are used alone.Part III safety assessment of FDPAllogeneic platelets have has been widely used in clinical infusion and wound healing, and obtained satisfactory effect with less side effects reported. some exogenous substances were used during FDP preparation, and freeze-drying process make influence on platelet. To find adverse of these exogenous substances and changes of platelets that may induced by freeze-drying process, We evaluate the safety of FDP by means of single-dose skin stimulation multiple-dose toxicity study, to provide theoretical basis for preclinical studies. The results show that FDP didn’t result in obvious skin stimulation to SD rats and New Zealand rabbits, and no significant toxicity to rabbits after 28 days use, indicating the FDP is safe as a kind of novel biological agents.To conclude, FDP retains the characteristics of the PRP, including growth factor levels, accelerating wound healing and hemostasis. FDP is safe as a kind of novel blood agents. |
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