A Comparative Study Of CGF Membranes Made By Three Different Compression Methods On The Effect Of The Releasing Of Growth Factors In Vitro And Degradability In Vivo

For the restoration of missing teeth, dental implants are the most popular and reliable methods because of the perfect mastication, aesthetic and pronunciation. With the development of the technology, the osseointegration as the biological basis of implants and the alveolar bone has already meet the needs of the clinical load of mastication. In clinical,the majority implanting cases exhibited different degrees of soft and hard tissue deficiencies, which had a bad effect on the dental implants. To make up for the deficiencies of peri-implant soft and hard tissue for the aesthetic effect after implant restoration, clinicians and patients pay more and more attention.To achieve optimal function and esthetic, the normal form and architecture of the hard and soft tissues before implant placement are necessary. However, a variety of reasons, including alveolar bone atrophy after extraction and congenital malformation, can cause the defect of the alveolar bone, which will affect the implant treatment and the long-term stability. When the guided bone regeneration technique or Immediate implant insertion is carried out after tooth extraction, the insufficient soft tissue usually has a bad effect on soft tissue healing and aestheticrestoration. Additionally, along with the atrophy of keratinized mucosa on the edentulous alveolar ridge, increased levels of plaque and inflammation around implants lead to more prone to bleeding and exhibit greater radiological bone loss and buccal soft tissue recession.To make up for the deficiency of peri-implant soft and hard tissue,the CGF/PRF membranes are usually used to induce the regeneration of the hard and soft tissue. In theory, CGF is produced by centrifuging blood samples with a special centrifuge device, similar to PRF. Nevertheless,the different centrifugation speed permits the isolation of a much larger,denser and richer in growth factors fibrin matrix. In clinical, for compression of the CGF clot to make a CGF membrane, dry gauze or a compressor has been used and then thick tube-like fibrin clots will be shaped to fit the size of the implantation site. However, different compressive methods can lead to different surface topographies,thicknesses and fibrin densities, which may influence cytokines releasing and degradation rate of the fibrin membranes we don’t know clearly.Objective:The objective of this study was to evaluate the differences in surface topographies, thicknesses, fibrin densities, cytokines releasing in vitro and degradation rate in vivo of the fibrin membranes by means of SEM(scanning electron microscope) observation, TEM(transmission electron microscope) observation, Elisa testing and histological evaluation,which freshly prepared CGF clots were compressed into thin membranes by dry gauze, a specialized compressor and a special syringe.Methods:Venous blood samples were collected from healthy volunteers. The CGF clots were compressed to make CGF membranes with dry gauze, a specialized compressor and a special syringe. The membranes were divided into three groups: gauze group, compressor group and syringe group(n=4). The samples were examined with a scanning electron microscope and transmission electron microscope to evaluate the differences in surface topographies and fibrin densities of the CGF membranes. Five milliliters of fresh Dulbecco’s modified Eagle medium(DMEM) was added to CGF membranes, and they were incubated at37℃in a humidified atmosphere of 5%CO2/95% air. The time points to collect exudates were on days 1, 5, 9, 13, 17, 21, 25, 29 and 33. The enzyme-linked immunosorbent assay testing was performed to measure the levels of TGF-β1、VEGF、PDGF-AB.Thirty New Zealand white rabbits were used for this study. Also, the CGF clots were compressed to make CGF membranes with dry gauze, a specialized compressor and a special syringe after centrifugation which the blood samples were collected from central artery in the ear. Each fibrin membrane that trimmed to an uniform size(square shaped specimens,1cm x 1cm) was sutured bilaterally using non-resorbablesutures to lie on a labeled titanium film. The membranes were randomly allocated in the unconnected subcutaneous pouches on the back of each rabbit(n=6). Animals were sacrificed after 1, 2, 3, 4, and 5 weeks.Residues of the membranes were removed with the surrounding connective tissue and fixed in 10% formalin. Histologic sections were stained with haematoxylin eosin staining and Mallory Phosphotungstic acid haematoxylin staining. Thickness of the residual membrane was measured linearly at 10 fields selected at random.Result:1.1 Scanning electron microscopy(SEM) observation1)The surface topographies of the CGF membrane made by three methods were quite different. The CGF membrane compressed by the gauze showed a flat surface on which fibrin arranged intensively and there was few microscopic pore structure characteristic and three-dimensional crossed network. on the contrary, the fibrin of compressor group membrane arranged loosely.On the surface, uniform fibers constituted a three-dimensional crossed network with Large microporosity. Compared with the others, the syringe group showed the medium fibrin arrangement and microporosity.2)The three regions of the SEM observations of the platelets onthe membrane surface showed that the regions which were the distal portion from the red thrombus and the middle portion contained fewer platelets, the region especially which was adjacent to the red blood cell fraction contained much more undamaged platelets.1.2 transmission electron microscope(TEM) observation1)The fibrin densities of the three-group membranes were also quite different. The CGF membranes of the syringe group showed high fibrin density and almost no microporosity in them. The compressor group showed the lowest fibrin density and the gauze group showed medium fibrin density.2)the region which was adjacent to the red blood cell fraction of the TEM observations in the membrane showed three layers clearly:fibrin layer, platelets layer and leukocytes layer. However, the regions which were the distal portion from the red thrombus and the middle portion showed no the same layers.2.The enzyme-linked immunosorbent assay testing1)All the CGF membranes of the three groups could steady controlled-release TGF-β1, VEGF and PDGF-AB to 33 th day, and the release amount hadn’t decreased obviously at different time points.2)At the same time points, there was no significant difference about the release amount of cytokines among the three differentgroups.3.Histological evaluation1)All the CGF membranes of the three groups could degrade completely in five weeks. Membrane thickness of the compressor group was significantly reduced in 2,3, 4 and 5 weeks. Nevertheless,Compared with the 0-week values for the gauze and syringe group,no significant changes could be observed in the first four observation weeks until in the fifth week the membrane degraded completely.2)In the zero week(the control group), the thickness of membranes in the gauze group was the minimum, but there was no difference between the compressor group and the syringe group. At the other same time points, there was no significant effect on the degradation rate of the fibrin membrane for the different compression methods.3)The histological sections showed that the residual membrane inside it still kept compact structure where there was no interconnected porous structure, vascular components or the cells.But among the CGF fragments that on the edge of the membrane,there were mesenchymal cells, fibroblasts and fibrocytes. The inflammation cells and foreign body reaction cells couldn’t be seen.Conclusion:1)Although the surface topographies, fibrin densities and initialthickness of the CGF membrane compressed by three different methods were quite different, there was no significant effect on the the release of the main cytokines in vitro and the degradation rate in vivo.2)The main cytokines could be controlled release steady to 33 th day, and the release amount hadn’t decreased obviously at different time points. As the intensive arranged fibrin and the micropores(1-5μm) which were too small to intrude into the membranes for the vessels, the degradation mechanism of the membranes in vivo was gradually from outside to inside. In the fifth week, the membrane degraded completely.3)The sample in the tube after blood centrifugation with a special CGF centrifuge device can be divided into five layers(from the upper to the lower): platelet poor plasma(PPP) layer, fibrin rich gel layer, platelets layer, leukocytes layer and red blood cell(RBC) layer.