The Experimen Research Of Frozen Platelet Stored And Preparationed Platelet Gel On Wound Healing

Objective1To observe the quality of pooled human platelet and preparation of platelet gel on cryopreservation agent in vitro.2To observe the effectivity and security of Frozen platelet on wound healing of wistar rats.Methods1Experimental studies in vitro1.1Preparation of cryoprotective agentPreparation of Thrombosol solution (Second Messenger regulator, TS),(50×TS) Thrombosol solution was prepared by12.5mmol/L Amiloride,5mmol/L adenosine and2.5mmol/L sodium nitroprusside that was dissolved in1L pure DMSO, and then frozen platelet at a ratio of1:50, DMSO concentration is2%.50X(2%DMSO+1/2TS) solution was prepared by6.25mmol/L Amiloride,2.5mmol/L adenosine and1.25mmol/L sodium nitroprusside that was dissolved in1L pure DMSO.1.2Preparation of platelet concentration and grouping400ml(ACD-B) of whole blood was collected from health eligble blood donors, and separated to obtain the platelet component by automated blood component separation and preparation machine, then the concentration of platelet was adjusted at1000x109/L. The platelet dilutions were devedied into6groups:fresh platelet (control)、2%DMSO(group A)、5%DMSO(group B)、2%DMSO plus thrombosol(group C)、2%DMSO plus a half of thrombosol(group D) and no add to cryoprotective agent (group E). Each bag was presented5ml platelet, and frozen at-80℃for1、2、4、8weeks (control group was kept at4℃for3days). The frozen platelet were thawed at37℃for5min and tested for platelet count (PLT)、mean platelet volum(MPV)、 PH value and CD62p expression. 1.3Platelet gel preparationPlatelet-rich plasma(PRP) and10%calglucon were mixed in proportion of10:1by the volume rates. Then, detected the contents of growth factor such as PDGF、VEGF、EGF and TGF-β in the gel and the data were for statistical analysis.2Animal experimentals2.1Platelet-rich plasma (PRP) preparation400ml(ACD-B) of whole blood was collected from health eligble blood donors,and separated to obtain the platelet component by automated blood component separation and preparation machine, adjusted the concentration of platelet at1000×109/L, and then frozen PRP at-80℃using2%DMSO. After2months, the frozen platelet were thawed and platelet count (PLT) was tested for680±10×109/L. Frozen or fresh PG was mixed by PRP and10%calglucon in proportion of10:1.2.2Experimental animal models and groupsFifteen whole-thickness skin defects models were done by four diameter1.3cm defects in the left and right sides of the back on each wistar rat. The skin defects animals were randomly devided into3groups:frozen platelet gel gauze(group A)、 fresh platelet gel gauze(group B) and rhEGF gauze(group C). The defect positions were dressed with these3different platelet telae. The telae were changed at days1、2、3、5、7、9、11, and the raw surface and the healing rate were measured. Each group animals were sacrificed at days1、3、5、11, and the tissue specimens were cutted and investigated by HE and immunohistochemical observation.Results1Experimental studies in vitroThe retrieve ratios of groups A～E were93.1±1.5%、95.7±1%、95±1%,93.8±1.5%,64.2±2%, and the control group was56±2%. The results showed that the ratios of groups A～D were significant higher than groups E and control (F=8,697, P=0.000). There were no obvious change among groups A～D from week2to week4, but descended to76±2%at week8. Basic parameters of platelet tested:platelet count(PLT)、mean platelet volum(MPV)、PH value and CD62p expression had no differences among four groups of cryoprotective agent for8weeks(P>0.05), and were significantly better than that fresh and no add groups at1week (P<0.05). Growth factor tested:the growth factor of platelet gels had no differences(P>0.05).2Animal experimentalThe wound grew downwards gradually by following time. The wound of groups A and B were ruddy、clean、no exudation、crusting early、granulation tissue rich、easy bleeding when it was touched and wound healing well. The rate of wound healing had no differences (P>0.05) at dayl、3、5and had significant differences at day7(P=0.017)among groups A～D. Histological observation showed that the healing effect of neogenesis skins of groups A and B were better than groups C and D:detailedly displayed on more plentiful and earlier new vessels appeared、augmented inflammatory cell infiltration、in order and tight up cell line.higher expressions of CD31、CD68and earlier expressions of CD68.Conclusions1The quality of platelet gel had no significant effect for cryoprotective agent frozen platelet within8weeks, and it is better to frozen platelet using2%DMSO.2Frozen PG can promote the wound healing and shows the same healing effect with Fresh PG.