Effect Of Cryopreservation On Platelet-rich Fibrin

Objective:The aim of this study was to investigate the effects of cryopreservation on the morphology and bioactive factors of platelet-rich fibrin (PRF) through the ultra structure and immunohistochemistry results. At the same time explore the feasibil ity of Cryopreservation PRF’s clinical application.Methods:This report is a randomized observing experiment of platelet-rich fibrin (PRF), di vided into two parts. The first part is extract the venous blood of healthy volu nteers, divided into two. Centrifugal extract the PRF under the same conditions. One marked for the experimental group, the other marked for the control gro up. Put the control group PRF into4%paraformaldehyde; Put the experimental group PRF into frozen pipe (5ml) which be equipped with3ml final concentrati on of5%dimethylsulfoxide (DMSO), penetration25min at room temperature,4℃refrigerator cryopreservated for30minutes, and then moved to the ultra-low-80℃temperature refrigerator save24h, and then placed in liquid nitrogen (-196℃) for a week. Remove the frozen pipe, shaking at38℃in a water bath to t haw3min,2or3times after cleaning using the preheat sterile PBS were placed in4%paraformaldehyde fixed. Two specimens were carried out in general, light mi croscopy, transmission electron microscopy. And both the organizational structure t o be compared and analyzed.The second part also collected samples of healthy volunteers, the same method P reparation of two PRF specimens, placed in the appropriate fixative,the productio n of tissue sections for immunohistochemical observation, and the results were an alyzed and compared.Results:In the first part, Platelet-rich fibrin (PRF), visual observation before and after f reezing are translucent membranous structure, smooth surface, but frozen specime ns less elastic; Observed by light microscope we found that, in platelet-rich fibr in(PRF), red-stained fibrins get together for a loose solid net structure, it conta ins lots of blue-stained leucocytes before and after freezing. Observed by trans mit electron microscope we found that, before freezing, most of platelets relate with several fibrins. We can see the organelles and dense granules in the plat elet. It seems some fibrin straight into the platelets, this part of membrane str uctures of platelets that get close together with the fibrins are disappeared, so me fibrin move highly responsive platelet shape and there is higher density la yer of material between the membrane of platelet. After freezing, most of th e platelet shape irregular or membrane damage, the alpha granules and dense granul es reduced significantly, some of the platelet internal structure completely disappe ared. Take the shape with the platelet membrane shape fibrin and platelet membra ne density slightly higher material stILl exists, no significant change in the fibrin str ucture.In the second part, The immuno-histochemical results show that the platelet-deri ved growth factor (PDGF), transforming growth factor beta (TGF-beta), interleukin-1,4(IL-1,4), tumor necrosis factor (TNF) are expressed both in fresh and froz en PRF. IL-1,1L-4positive reaction site is mainly distributed in the PRF fibrin net work structure. PDGF positive reaction site into a punctate distribution in the fibe r network structure.TGF-β1positive sites are mainly located in the nucleus and cy toplasm of white blood cells. TNF-positive reaction site is mainly located in the c ytoplasm of white blood cells. The express of PDGF、TGF-β1、IL-1、TNF in frozen PRF was not significantly increased compared with fresh PRF(P>0.05), while the express of IL-4in frozen PRF increased significantly(P<0.05).Conclusions:1HE staining of tissue sections and transmission electron microscopy confirmed th at the main structure of PRF freezing is still a porous network structure of fibrin f ormation, it still has to induce cell migration and cell proliferation, thus speeding up the healing process function.2Transmission electron microscopy structure prompted frozen PRF platelet freezing and thawing, the alpha particles are significantly reduced, and its release to the release of growth factors diminished capacity.3Immunohistochemical results showed that PRF growth factor still expression aft er cyopreservation and the content was no significant difference. That still has abi ological basis for the promotion of bone regeneration and soft tissue healing.4HE staining results suggest that the PRF still contain a large number of activated white blood cells, irnmunohistochemical results suggest that the remains of anti-inf lammatory effects of the biological basis of freezing.5The study of cryopreservation of platelet-rich fibrin requires further animal experiments and clinical cases observed because of the phenomenon that less elastic, platelets freeze-thaw after frozen.