Allogeneic Platelet Gel Effecting On Wound Healing And Its Mechanisms

Platelet is one of the visible components in the blood produced by megakaryocytes in the bone marrow hematopoietic tissue, and its main function is to act in physiological hemostasis, to promote blood clotting and to maintain theintegrity of the capillary. Since the 1960s, there has been conclusive evidence that platelets have phagocytosis function of viruses, bacteria and other particles. As we all know, in the past, platelets were used in clinical therapy for patients whose platelets counts were low or platelet functions were abnormal, the method of administration of platelets were intravenous infusion. However, platelet as a multifunctional cell not only plays a role in thrombosis and hemostasis, but also plays an important role in angiogenesis, tissue repair and inflammation. It was found that platelet could be degranulated to release rich growth factors which existed in its a-granule, including platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β),vascular endothelial growth factor (VEGF), endothelial growth factor (EGF),insulin-like growth factor (IGF), and so on. In 1970s, platelet-rich plasma (PRP) was used in the research of wound healing. In 1977, Harke isolated and preparated PRP from the whole blood the first time, and then successfully used PRP for the treatment of cardiac surgery patients. In 1993, Hood AG added thrombin and calcium in PRP to form a gelatinous substance (gel), used the gel instead of the cellulose gel in research,proposed the concept of platelet gel (PG) the first time. For the past few years,platelets used in wound healing studies have a more in-depth and extensive development, involving multiple subjects such as maxillofacial surgery, orthopedic surgery, plastic surgery and cosmetic surgery, even in cell culture (including stem cells culture), genetic engineering, tissue engineering, anti-aging, sports medicine and it reveals a unique role in wound perfect healing. This all reveals that PG has a broad prospect for clinical application.Though the efficacy of PG were fully demonstrated from the experimental to clinical studies in the world, which indicated PG had an important value for wound healing and tissue reconstruction. However, the platelet gel has not been extensively and generally applicated in clinical practice so far because there are many limitations for the PG applications. Firstly, the preservation time of platelet storage is very short,the conditions for platelet storage require high standard, the refractory wounds need frequent dressing and long time for healing. In addition,For preparation of platelet-rich plasma (PRP), the whole blood has to be collected repeatedly from patients and immediately processed, to which the patients and doctors are hard to follow; Secondly, many factors such as the acquisition condition and preparation of technical, environmental requirements can affect PG preparation, making it difficult to use PG in clinical practice. Thirdly, the blood banks and hospitals are independent institutions in our country, and hospitals have no permission to separate and prepare platelets alone. There are no equipment and technique for separating and preparing platelets in the hospitals. The sources of allogeneic platelets is very limited, and so it is difficult to carry out the treatment by utilizating PG. Fourthly,it is difficult to set up standardization of the treatment dosage,and it is not able to effectively evaluate the treatment effect because there are large differences of platelets among individuals in humans.The problems we must face to are how to overcome these shortcomings, and how to promote the use of PG in clinical practice. While allogeneic PG can well overcome these shortcomings. Because allogeneic PG has many superiorities: The use of allogeneic PG is not confine by the conditions of the patient’s themselves, it can be conveniently applicated at any time, it is conducive to the achievement of the quality and standardization, and it is conducive to the realization of the validity of the systematic assessment of treatment. There was a case reported about allogeneic platelet gel with autologous cancellous bone graft for the treatment of a large bone defect abroad. In 2007, Smrke used graft mixture (the allogeneic platelet concentrate which was ABO- and RhD-matched, leukocyte-depleted, irradiated and activated by human thrombin, with autologous cancellous bone graft ) for filling the defect of 45 ml and fixing with an external fixator. Postoperative care was uneventful. After 6 months the graft was incorporated, the bone defect was fully bridged and full weight-bearing capacity was achieved. No side effects were observed and no platelet or HLA class I antibodies were detected. This case report shows that the clinical use of allogeneic platelet gel is feasible.Therefore, so far, there were no case about the allogeneic PG promote wound healing reported at home. In this study, we optimized the preparation condition of allogeneic PG, studied the curative effect of allogeneic PG to wound healing, investigated the contribution of supernatant of allogeneic PG to monocyte. We divided our study into four parts, as described below：I Optimization of preparation conditions of allogeneic platelet-rich plasma admixtureAccompanied by the rapid development of medical technology and medical equipment, the new treatments such as liver transplantation, cardiovascular major surgery, cancer treatment extensively were carried out, leading rapidly increasing needs of the amount of platelets. The application of platelet was unbalance between supply and request. The increasing platelet supply is difficult to meet the clinical needs. However, the utilization of platelets is less than 10% from whole blood,causing huge waste. In developed western countries, due to the advantage of the equipment condition, they routinely used separator to separate platelets from whole blood. In addition, the time for separating and preparing platelets from whole blood was very short. According to the regulation, we have to prepare platelets from freshly collected whole blood within 6h.According to the first part of the study, we further optimize the preparation conditions of allogeneic platelet-rich plasma admixture. In order to extently utilize the platelet resources of whole blood for clinical application we explorated the buffy-coat method of preparation of manual platelets. In this part, we drawed 400ml of whole blood from 28 healthy blood donors, and then separated and prepared manual single platelet-rich plasma through buffy-coat method (two-step method). In accordance with the acquisition and preparation time, there were four groups. Group A: single platelet-rich plasma were immediately separated and prepared from buffy-coat which were prepared from fresh whole blood collected within 6h (control group); Group B:The first step of the separation was carried out in 6h after the collecton of fresh whole blood and the first step of the separation was proceeded when the buffy-coat was placed for 6h; C: The first step of the separation was carried out in 24h after the collecton of fresh whole blood and the first step of the separation was proceeded when the buffy-coat was placed for 24h; Group D: The separation and preparation of single platelet-rich plasma was proceeded after 6-8h when fresh whole blood was collected.Detect the variation of platelet count (PLT), mean platelet volume (MPV),the leukocyte residual rate, PH, hypotonic shock response (HSR), platelet aggregation rate, and CD62p. Explore the methods to preprare the BC-PC by the study the effect of the time of collection and preparation on the equlity of the manual platelet. Our study found that platelet count (779.9 ± 83.8 × 109/ L) and hypotonic shock response(HSR: 62.61 ± 5.24%) of group B that handmade-PC was prepared when the buffy-coat was placed for 6h were higher than the other three groups ,meanwhile the mean platelet volume (MPV,6.44 ± 0.17 fl) and levels of CD62p (47.67 士 7.40%)were failed. PH value, the HSR and platelet aggregation of group B,C and D are better than the control group A,that was statistically significant(P <0.05),whereas there was no significant difference among the four groups on the expression of CD62p by FACS. The traditional concept that platelets must be prepared within 6-8h by handmade-PC. Simultaneously we sought the concentrated centrifugal conditions of PLT that was 2500g for 10 min.Ⅱ Optimization of preparation conditions of platelet gelNowadays researchers have made some progress of autologous PG useing in bum and plastic surgery, cardiac surgery, chronic ulcers, oral and maxillofacial surgery, ophthalmology and other areas, but the further promotion of the PG was restricted because of the limitations of the PG preparation source and the lack of unified preparation and application of standards. Platelet concentration, ratio of platelets and activator, different types of activators, etc. can affect the biological activity of PG.There is no uniform, standardized criteria of PG preparation methods and conditions at home and abroad,resulting in differences of therapeutic efficacy of PG in clinical treatment. Therefore, the purpose of this part of the study is to find the best PG preparation conditions through comparing different preparation conditions on the biological activity of PG supernatant in vitro.Studies at home and abroad found that the growth factors in the PG supernatant can promote cell proliferation of a variety of cells such as fibroblasts, vascular endothelial cells, keratinocytes. This part of the study we have chosen the vascular endothelial cell ECV304 cell line as a cell model, PG supernatant and ECV304 cell were cocultured. Through the analysis and comparison the effect of PG supernatant to cell proliferation, migration, and determination the levels of VEGF of PG supernatant prepared by different methods, we explore and optimize the PG preparation conditions, including platelet concentration, the proportion of platelets and activator,different activation agents. Our results confirmed that: PG can obviously promote the proliferation and migration of ECV304 cells. Exogenous thrombin shortened the formation time of platelet gel (P<0.05), but there were little effect of different concentrations of thrombin on the formation time of platelet gel the proliferation and migration of ECV304 cell (P>0.05). The effectiveness of PG promotes the proliferation of ECV304 cells according with the increasing platelet concentration,and the effectiveness stabilized when platelet concentration were 1500～2500 × 109 /L. The most appropriate ratio of platelets and the activator was 10:1.VEGF levels of PG supernatant increased according with the increasing of platelet concentration, the difference was significant (P<0.05),There were no differences of VEGF levels between PG supernatant prepared by different concentrations of thrombin (P>0.05).We therefore determined the standard of clinical applications: the acquisition of platelet concentration was 1000×109/L or so, the mix ratio of platelets and the activator was 10:1. Adding activator thrombin or not based on actuality condition in clinic. The results from this study provide an important reference for PG preparation,and standardization, a theoretical basis and data support for the clinical application of PG in future.Ⅲ Preliminary study of clinical efficacy on wound healing by homologous allogeneic platelet gelChronic skin ulcer is more common in diabetes, cardiovascular disease,nutritional deficiencies, to accept hormonal or cytotoxic therapy, especially the elderly patients who are complicated with DM or bedridden. The reason for the long-term unhealed wound is the normal wound healing function is weak in these patients. Common clinical treatment is dressing debridement, suction, and the use of some special dressings (such as polymer materials coverings, hydrogel dressings,artificial diaphragm, etc.), but the curative effects for chronic skin ulcer is not satisfactory. Growth factor products such as gold due to the skin, bFGF etc have a slightly better effect than the above method,but activity of a single growth factor is not as good as the multiple growth factor. Clinically, for the reason of long-delayed healing of the wound, regular dressing changes, great cost, even it will bring more serious consequences (such as amputation, sepsis, wound infection ) which are difficult for doctors to deal even if a slightest mistake, an new, urgent and effctive treatment is needed to treat difficult to remedy chronic skin ulcer, manage the patients’ pain, and improve the quality of their life.At present clinically the platelet concentrates which is applied to intravenous infusion can be divided into fresh platelets (Fresh platelets), and frozen platelets(Frozen platelets), according to the retention period. In this section we applied fresh platelets, frozen platelet prepared by homologous allogeneic PG (Fresh PG and Frozen PG) to chronic skin ulcer and had a further study on the therapeutic effect. 44 patients were randomly divided into the control group, Fresh PG group and Frozen PG group. Control group adopted routine clinical methods, meanwhile Fresh PG group and Frozen the PG group put the PG Fresh and Frozen PG to use in treatment separately. We observed and recorded the situation of wound healing,measured the area or volume of wound healing for statistical analysis. The statistical results of efficacy evaluation of 44 cases of patients with refractory wounds showed the’treatment effect of Fresh PG group and Frozen PG was better significantly, andand the healing rate of the experiment group was significantly higher than the control group at 3, 6, 12 weeks. The difference was statistically significant. There was not statistically significant difference between Fresh PG group and Frozen PG group. The two groups offered advantages such as short course, long dressing interval and high quality healing. Aboved revealed homologous allogeneic PG applied to the treatment for refractory wounds which was a no adverse reactions, simple, safe and effectiv method was superior to conventional treatment obviously.Ⅳ Effect of PRP on the differentiation of monocytesInflammatory cells, particularly monocytes play an important role in the process of wound healing. Monocytes / macrophages is one type of the major inflammatory cells. Monocytes / macrophages are the body’s first line of defense against foreign pathogens, a group of heterogeneous cells. In different micro-environments,monocytes / macrophages can react differently, and show a different activation state,and differentiated into the subsets with different phenotypic and functional characteristics.At present monocytes / macrophages can be diveded into classically activated macrophages-Ml and alternatively activated macrophages-M2. M1 can produce inflammatory factors and amplify the inflammatory response; otherwise, M2 produces only a low dose of inflammatory factors, inhibits the inflammatory response,and has the ability to promote tissue repair. TNF-alpha of LPS and IFN-gamma may induct monocyte into M1 macrophages; IL-4, IL-10, IL-13 can promote monocytes to differentiate into the M2 macrophage.This part of study,we took monocytes / macrophages as a target,focused on human peripheral CD 14 + monocytes separated by MACS, that were cultured by the mixed AB-type PPP and the PRP (including two kinds of the PRP, platelet count 500×109/L, and 1000×109/L) and study the effect of PRP on the differentiation of monocytes into macrophages. Morphology was observed: we found that with the extension of culturing time cells formed colony-like cell groups with more long spindle-like cells and some non-adherent small round cells under microscope,by contrast the cells which were large, round kind of like fried eggs with rich the cytoplasm particles took the great majority in PPP treatment group . The expression of surface marker by FACS showed, the CD 163, CD309 expression rate of RP500*109/L, PRP and 1000*109/L, treatment group was significantly higher than PPP treatment group. RT-PCR results showed that: compared with PPP group, the expression of IL-10 and TGF-β mRNA increased significantly in PRP500*109/L,PRP1000*109/L,group, simultaneously,iNOs and IL-12P40 mRNA expression were reduced at 24h. The cytokine detection of culture supernatant displayed :the secretion of TGF-β of PRP500*109/L, and PRP1000*109/L, group is higher than the PPP treatment group, meanwhile the CCL-2 secretion lower than the PPP treatment group.According to the results, we proposed: PRP may induce Mo differentiated into M2 macrophages and play a important role in wound repair by M2 macrophages, thereby promoting wound healing, the viewpoint will fertilize the fundamental theory that PRP can promote wound repair, and provide a new theoretical basis and new target for the application of PRP in the treatment of wound with the hope of openning a window for the clinical treatment of trauma with PRP. Its regulation and molecular mechanism required further study.