Study Of In Vitro Degradation And Release Of Partial Growth Factors Of CGF Fibrin Clots And Membranes

Objective:The Concentrated Growth Factors(CGF)fibrin is a completely autologous platelet concentrate,containing lots of fibrins and high concentration of growth factors.It can promote the regeneration and repair of both soft and hard tissue,and is widely used in many medical fields,such as plastic surgery,sports medicine and dentistry.CGF can be utilized directly as a clot or mixed with particulate bone powder to fill a cavity,or compressed into a membrane for clinical use.In this study,CGF clots and CGF membranes were prepared respectively.The histomorphological structure of CGF clots and CGF membranes were observed using optical microscope and scanning electron microscopy,and the characteristics of in vitro degradation and release of partial growth factors were investigated to provide a theoretical basis for the selection of clinical use of CGF clots and CGF membranes.Methods:Exp.1 Preparation and morphological observation of CGF clots/CGF membranesVenous blood samples were collected from 6 healthy volunteers(3 males,3 females).4 tubes of blood were obtained from each volunteer(9ml for each tube).All samples were immediately centrifuged in the CGF centrifuge using the original program.The prepared CGF gels were divided randomly into two groups and respectively made into CGF clots and CGF membranes.To observe the structure of them,HE staining,Wright’s staining and scanning electron microscopy were used.Exp.2 Determination of CGF clots/CGF membranes’degradation curve in vitroVenous Blood samples were collected from 6 healthy volunteers(3 males,3 females).2 tubes of blood were obtained from each volunteer(9ml for each tube).All samples were immediately centrifuged in the CGF centrifuge using the original program.The prepared CGF gels were divided randomly into two groups and respectively made into CGF clots and CGF membranes.After dried to constant weight using the gauze,the initial mass of these two specimens were measured.Then all specimens were immersed into artificial saliva within 37℃ thermostat water bath.Residual mass of these two specimens was measured at the same time every day,until specimens were thoroughly degraded and couldn’t be measured anymore.Accordingly the degradation curve was drawn to compare the degradation rates of CGF clots and CGF membranes,and their in-vitro degradation characteristics were analyzed.Exp.3 Determination of partial growth factors’ concentration of CGF clots and CGF membranes in vitroThe preparation of CGF clots and CGF membranes was the same as experiment 2.CGF clots and CGF membranes were placed in separate wells of a 12-well plates with the addition of 2 ml fresh DMEM,then incubated at 37℃ in a cell incubator.The culture medium was collected at lh,1 d,3d,5 d,7d and 1 Od,with an equal volume of fresh DMEM added back to each well.All collected culture medium were stored at-80℃ before analysis.The concentration of TGF-β1、PDGF-AB、VEGF was quantified using ELISA kits.ResultsExp.1 Preparation and morphological observation of CGF clots/CGF membranesCGF gels are yellow and gelatinous,with a moisturized and shiny surface.CGF clots are pale yellow,semi-translucent and semi-gelatinous,with a smooth and shiny surface.CGF1 membranes are milky white films,with a soft and tough texture,exhibiting a degree of elasticity.The print of the gauze threads can be seen on the surface of CGF membranes.CGF clots and CGF membranes showed similar structures under light microscope:the upper layer mainly consisted of fibrin matrix.The leukocyte layer appeared in the middle with nuclear stained in blue.Red blood cells were located at the bottom.Numerous scattered leukocytes and platelet aggregates were trapped in the fibrin matrix.The fibrin structure of CGF membranes was denser than that of CGF clots.SEM showed that CGF clots were mainly composed of loose and porous fibrin matrix.The fibrin-strands appeared interlaced to each other,forming a three-dimensional network structure with abundant leukocytes and platelets embedded in it.The distribution of platelets and leukocytes,as well as the diameter,porosity and compactness of the fibrin network within the clot was not uniform.A great many RBCs,leukocytes and platelets clusters were found at the yellow/red border.Leukocytes appeared with an irregular surface and seemed quite small,thus could have been mainly lymphocytes.Platelets appeared in irregular shape with pseudopod extending from the surface.CGF membranes were more tightly packed than CGF clots,which had a lower porosity.Fibrins were clearly organized in parallel strands that appeared interlaced to each other.It was hard to distinguish the cellular elements trapped within this condensed network.Exp.2 Determination of CGF clots/CGF membranes’ degradation curve in vitroWith time going on,the mass of CGF clots and CGF membranes varied obviously.The mass of CGF clots showed statistical differences between each time point,P<0.05,which indicated CGF clots continued to degrade.From day 8,the.residual mass of CGF clots was very little andthe degradation rate decreased.CGF clots completely degraded on day 10.The mass of CGF membranes showed statistical differences during the first 6 days of the experiment,P<0.05.CGF membranes completely degraded on day 7.At the beginning of the experiment,there was a statistical difference of mass between CGF clots and CGF membranes,P<0.05.So were each time point following the start of experiment.Exp.3 Determination of partial growth factors’concentration of CGF clots and CGF membranes in vitroTill the end of the experiment,both CGF clots and CGF membranes could slowly and steadily release TGF-1,PDGF-AB and VEGF in vitro.PDGF-AB and TGF-β1 had a constant kinetic release,respectively reaching the maximum release at day 3 and day 5.VEGF had a slow kinetic release,reaching the maximum release at day 7.There was no statistically significant difference in the release of growth factors between CGF clots and CGF membranes at other time points,P>0.05.At a few time points,such as lh,5d,7d of TGF-β1,1h of PDGF-AB,and d10 of VEGF,there were statistically significant differences in the concentration of growth factors released by CGF clots and CGF membranes in vitro,P<0.05.At other time points,there was no statistically significant difference in the release of TGF-1,PDGF-AB and VEGF between CGF clots and CGF membranes,P>0.05.Conclusions:1、Both CGF clots and CGF membranes have a three-dimensional fibrin network structure,the fibrin network of CGF membranes is more compact.2、Both CGF clots and CGF membranes contain a large number of activated leukocytes and platelets.The highest platelet/leukocyte density was found at the yellow/red border,where should be preserved during clinical practice.3、CGF clots and CGF membranes were significantly degraded in the artificial saliva.The degradation period of CGF clots was longer than that of CGF membranes.4、CGF clots and CGF membranes could slowly and continuously release TGF-β1、PDGF-AB and VEGF in vitro.There were no statistically significant differences in the concentrations of TGF-1,PDGF-AB and VEGF released by CGF clots and CGF membranes except several time points.5、In clinical practice,CGF clots and CGF membranes should be reasonably selected according to the purpose of surgery,combined with the physical and chemical properties as well as the biological characteristics of CGF clots and CGF membranes.