| BackgroundThe platelet is one of the visible components in the blood of the mammals. It is a small cytoplasm with the biological activity, splitting away from the megakaryocyte with matured bone marrow, with small volume and no cell nucleus. Its main function is solidifying the blood and stopping bleeding, as well as repairing the damaged blood vessel. Clinically, it is mainly applied to the patients with decreasing of the platelet or platelet dysfunction. It must be stored at 22±2℃ with vibration for less than 5 days. It is difficult to ensure the continuous supply of the platelet in clinic. With the development of the platelet separation and storage technology, the freeze-dried platelet has solved the difficulty in storing the fresh platelet in short term. After adding the cryoprotectant dimethyl sulfoxide (DMSO) into the platelet and then freezing, the storage period can be postponed to a year. However, the freeze-dried platelet (FDP)needs to be frozen under the profound hypothermia at-80℃, so it is not suitable for the ware or emergent events. In 1950s, people discovered the way of freezing and drying the platelets. The researchers pre-processed them to improve the antifreeze and anti-dry capability through adding various protective agents. With the constant development of the freeze-dried technology, the advantage of FDP is not only limited to the room-temperature storage, small volume, light weight convenient for the long-distance transportation, etc. Besides, it can release multiple growth factors and accelerate the wound healing.The wound refers that the normal skin tissue causes injury resulting from the external injury factor, such as surgery, external force, heat, current, chemical substances, low temperature, internal factors of the organism (e.g. partial blood supply obstacle, etc.). The wound healing is a complex biological process, mainly including the following stages:inflammatory reaction, cell proliferation, desmoplasia, wound surface shrinkage, wound surface remodeling, etc. These stages were mutually overlapped, and involved in the common participation of multiple repair cells, inflammatory mediator, growth factor, and extra-cellular matrix. Besides, under the control of the organism, they presents highly ordering, integrity and net feature. The disorder of any link will directly affect the progress of he wound healing.Over the years, PRP has been widely applied in the research on the wound recovery, involved in multidisciplinary, such as oral and maxillofacial surgery, ophthalmology, plastic, orthopedics, burn injury, etc. It even presents unique effect on the genetic engineering, cell culture tissue engineering, and anti-aging. The therapeutic effect has been verified and fully affirmed by the foreign and domestic scholars. PRP not only can improve the hemostatic effect and shorten the operation time, but also can alleviate the swelling after the surgery, speed up the wound healing, which has significant value on the wound recovery and tissue reconstruction.PRP. Meanwhile, the test also proves that, the recovery rate of the freeze-dried platelet after hydration can reach 60-70%[4], so that it enables FDP to retain the morphology and function feature of many fresh platelets, which not only extends the storage time of the platelet, but also simplifies the storage method of the platelet. These growth factors can accelerate the proliferation and differentiation of the cells, strengthen the synthesis capability of the collagen, and have important effect on accelerating the matrix synthesis, sedimentation and tissue formation. Moreover, the good synergistic effect between each growth factor can obviously accelerate the wound healing.Clinically, as for the common acute wound, except for the operative incision, skin abrasion, the scald is also a common disease and frequently-occurring disease. Therefore, this project aims at studying the recovery effect of FDP in the scald wound healing through establishing deep second degree scald model in SD rats.ObjectiveApply the freeze-dried platelet (FDP) to deep second degree scald model in SD rats, observe the influence of the FDP on the wound healing, so as to verify the effect of such platelet on the external user for the scald, which provides experiment basis for its clinical application.Method(1) The preparation of the platelet rich plasma (PRP)adopt the whole blood from the volunteer donators, and separate the platelet after it is qualified according to the national standard, carry out 1,500xg centrifugation for 10min, extract upper PRP, make this PRP go through 3,000xg centrifugation for 20min, and then obtain platelet plasma, remove the upper platelet poor plasma, adjust the platelet count of the lower platelet concentrate till the count is adjusted to 1,000x109/L. Obtain PRP through the secondary centrifugation.(2) Freeze drying process of PRPPrepare the pretreatment liquid:adopt NaCl, KCl, CaCl2, MgSO4, NaHCO3, citric acid, sodium citrate, sodium acetate, glucose ultrapure water, mycose, reversible platelet activation inhibitor, adjust PH to 6.6-6.8, adopt 0.22μm filter for filtration.Prepare the freeze-dried buffer solution:add 30% homotype plasma of the total volume into the pretreatment liquid, adjust PH to 6.6-6.8, and use 0.22μm filter for filtration and then reserve it for usage.Perform 1,500xg centrifugation on the platelet for 15min, remove the supernatant and retain the sediment, use the pretreatment liquid in the same volume as the removed supernatant to re-suspend the platelet, and then vibrate it for 4h with 37℃ water bath, to make the platelet keep the suspension state. After 4-hour water bath, perform 1,500xg centrifugation on the platelet suspension for 15min, remove the supernatant and retain the sediment, and use the freeze-dried buffer solution in the same volume s the removed supernatant to re-suspend the platelet. Move the platelet suspension after preprocessing into the siliconization glass bottle (4ml for each bottle), and place them at-80℃ ultra cold storage freezer overnight. In the next day, open the freeze dryer and reduce the temperature to-45℃, and then take the samples out of-80℃ ultra cold storage freezer, and move them into the freeze dryer for vacuum drying. The cold trap temperature is-45±5℃. The vacuum degree is less than 133mbar. Take them out after 24h, seal them, and store them at room temperature.(3) Observe the ultra-structure of the platelet via the transmission electron microscope (TEM); measure PLT and MPV before the freeze drying of the platelet and after the hydration through the blood cell counter, and calculate the recovery rate of the platelet and the difference on MPV before and after the freeze drying.(4) Establish deep second degree scald model in SD rats:remove the back hair of 40 rats, with the hair removal area of about 4cmx6cm, clean them with water, and observe them for 24h, so as to confirm that there are no abnormal situations on the hair removal parts, such as no red and swelling, inflammation, damage, etc. In the next day, SD rats were under absolute diet for over 8h. Use 10% chloral hydrate intraperitoneal injection with the dosage of 300mg/kg for narcotizing them. After the anesthesia takes effect, fix the rats on the operation platform, and use 75% alcohol to disinfect the skin of the experiment area. Besides, place the aluminum alloy pot on the induction cooker, and also place 50g weight into the pot and heat it till boiling for 5min, and then form round deep second degree scald model with the radius of 1cm. After 3h, perform the tangential excision according to the clinical conventional burn treatment, till the wound surface is turned to red from white. Divide 40 rats that are successfully molded into four groups. Then, apply medicine to them based on different groups. FDP group:re-dissolve the FDP with the homotype plasma, mix them with the calcium gluconate with the proportion of 10:1, and then activate them. Next, spray them on the sterile gauze, make them into platelet gel gauze, and apply it to the wound surface (1ml for each would surface), and bind up with sterile gauze. Fresh-platelet rich in plasma (PRP):adjust the platelet concentration to 1,000×109/L, and activate it with the calcium gluconate at the proportion of 10:1, apply 1ml to each sterile gauze and make platelet gel gauze, apply it to the wound surface, and bind it up with the sterile gauze. Scald ointment group:use the sterile swab for smearing, and bind up with the sterile gauze. Blank group:only use the sterile gauze to bind it up. The medical prescription change time is dl,3,5,7,9,13, and 19 respectively. meanwhile, use the transparent coordinate paper to cover the wound surface, draw the film along the wound edge, calculate the wound surface area, and select the wound tissue with the diameter of 1cm and apply it for HE dyeing.ResultThe influence result of different treatment groups on the scald wound healing rate of the rats indicates that, during the therapy, the scab area of the scald wound surface decreases and the healing rate obviously increases. In 7d, PRP and FDP groups, the treatment makes the scald wound area of the rats obviously decrease, which has obvious wound healing effect and has obvious difference from the experimental control group (P<0.05). However, there is no obvious difference between PRP group and FDP group. It prompts that FDP has the effect of speeding up the early healing of the scald wound surface, which is similar to PRP.The different treatment groups have different influence in the pathologic histology of the rat scald wound surface. Observe the tissue specimen via 200 times electron microscope for 1d:the whole skin ulcer and necrosis; the normal structure disappeared and presented cavity shape; the cellular edema, degeneration and necrosis; collagenous fiber rupture, hinting the scald degree had already reached deep second degree, and the molding was success (see picture).7d after taking the medicine:the epidermis and dermis tissue of FDP group and PRP group had achieved obvious improvement effect; the necrotic tissue and inflammatory exudates of the skin wound surface was remarkably reduced; many new fibroblasts occurred around the blood capillary, and many granulation tissues were formed, while the scald ointment group and blank group still presented that the epidermis was with acute suppurative inflammation, and cellular degeneration and necrosis took place; only few granulation tissues were formed.19 days after taking the medicine:the epidermis of FDP group and PRP group was recovered, and few hair follicle and sebaceous gland were seen; the cellular morphology was well recovered; the collagenous fiber in the dermis tissues were clearly arranged, without dissolution or chaos phenomenon; the scald ointment group and the blank group still had some coagulative necrosis and few granulation tissues, and no hair follicle and sebaceous gland were seen.ConclusionsThe platelet after the freeze drying not only can maintain the effect of accelerating the scald wound healing similar to PRP, but also have the advantages of storage at room temperature, performance stability, convenient transportation, etc. |
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