| Platelet-rich fibrin(PRF)is the second generation of platelet concentration preparation.PRF is rich in all kinds of growth factors,as an auxiliary additive,often used in stomatology,orthopaedic,traumatology departments.Compared with the first generation platelet concentration preparation platelet-rich plasma(PRP),PRF has its own unique biological characteristics.Autologous fat grafting,as a common technology usually adopted by plastic surgeons,has a history of one hundred years.Autologous fat grafting has the advantages of relatively simple operation,easy to obtain sufficient amount of fat,no immunogenicity as autologous tissue,and is often used in the treatment of tissue volume loss.The acquisition,handling and injection of fat are considered to be the main factors affecting the postoperative fat retention rate.Convenient mechanical refinement of fat is one method of the handling process.Affected by the aforementioned potential factors,there are different degrees of necrosis and absorption after autologous fat grafting,which affects the volume retention rate of fat grafting.Recently,it has become one of the research hotspots to improve the effect of fat grafting by adding auxiliary additives.The auxiliary additives reported in the previous literature include chemicals,growth factors,adipose stem cells,stromal vascular fraction and platelet concentration preparations.ObjectiveThe biological characteristics of platelet-rich fibrin were studied,including gross observation,structural observation,component analysis and factor release in vitro.The effects of platelet-rich fibrin combined with different forms of fat grafting were evaluated and compared in animal experiments.MethodsTwenty milliliters of blood was collected from elbow vein of healthy volunteers using disposable collection containers.Then the blood sample was immediately placed in the ordinary desk centrifuge and centrifuged with parameter of 3000rpm/10min.First,the obtained PRF went through general observation.Then,PRF and the membrane were fixed in glutaraldehyde solution respectively.The structure and cell composition of the two were observed by scanning electron microscope(SEM).In addition,the PRF of the corresponding cases were cultured in DMEM solution in vitro.The samples at 10 time points were collected,and the release of PRF factors in vitro was determined by suspension chip method.In vivo experiment,the animal suture model of immunodeficient nude mice established by our team was used.The experiment was divided into chylous fat group and granular fat group.The experimental group and control group of chylous fat group were chylous PRF and chylous NS,and the experimental group and control group of granular fat group were granular PRF and granular NS.Chylous fat and granular fat were mixed with PRF at a ratio of 5:1,respectively.The injection volume of a single suture ring was 0.2ml.The samples were collected at 4 weeks,8 weeks and 12 weeks after fat transplantation.The graft retention rates of chylous fat group and granular fat group were compared by wet weighing and volume measurement.Living tissue of whole-mount staining and confocal microscopy were used to intuitively observe and evaluate the survival adipocytes in graft profile;Hematoxylin-eosin staining was used to evaluate the microstructure of grafts at each time point;Perilipin staining was used to evaluate the adipocytes of grafts at each time point.a-SMA staining was used to evaluate the number of vessels in the grafts at each time point.Result1.The results of scanning electron microscope showed that the upper segment of PRF was three-dimensional spatial structure of crosslinked fibrin,and the cell composition was less.The middle segment of PRF was still a three-dimensional spatial structure of tightly crosslinked fibrin,in which a medium amount of cell components,including platelets,white blood cells and a small number of red blood cells,could be seen.A large number of cellular components were distributed in the lower segment of PRF,including platelets,white blood cells and red blood cells.2.The results of scanning electron microscope on(PRF)membrane showed that the spatial structure of fibrin was denser than that before pressing,and the cellular components were not easy to be observed The cross section could be observed that the structure of the upper segment was the densest,the middle part was the second,and the lower part was relatively loose.3.The results of suspension chip method showed that the seven selected factors were released in varying degrees with time.The seven selected factors had reached their release platform respectively in the 28-day release cycle.The reducing order of total release amount was CXCL8,PDGF-BB,Angiopoietin-2,TNF-alpha,VEGF-A,EGF and FGF-basic.4.From the observation of time points release,it was found that the release peaks of CXCL8,PDGF-BB,Angiopoietin-2,TNF-alpha,VEGF-A,EGF and FGF-basic were on the 3rd,1st,1st,2nd,1st,1st and 4th day,respectively.Based on the cumulative release curve,it was found that the time of CXCL8,PDGF-BB,Angiopoietin-2,TNF-alpha,VEGF-A,EGF and FGF-basic entering the release platform were on the 20th,20th,20th,8th,20th,12th and 12th day,respectively.5.The results of in vivo experiments showed that there was no significant difference in the weight,volume and volume retention rate of fat grafts between chylous NS group and granular NS group at 4 weeks,8 weeks and 12 weeks(p>0.05).At each time point,the chylous NS group was superior to the granular NS group in terms of confocal microscope observation,HE score,the number of pericyte positive adipocytes and the number of blood vessels.There was significant difference between the two groups(p<0.05).6.The results of in vivo experiments showed that the weight,volume and volume retention rate of fat grafts in chylous PRF group and granular PRF group were significantly different from those in chylous NS group and granular NS group at 4 weeks,8 weeks and 12 weeks(p<0.05).There were significant differences between the experimental groups and the control groups in terms of confocal microscope observation;HE score,the number of perilipin positive adipocytes and the number of blood vessels at each time point.The difference between the experimental groups and the control groups was statistically significant(p<0.05).7.The results of in vivo experiments showed that there was no significant difference in the weight,volume and volume retention rate of fat grafts between chylous PRF group and granular PRF group at 4 weeks,8 weeks and 12 weeks(p>0.05).At each time point,the chylous PRF group was superior to the granular PRF group in terms of confocal microscope observation,HE score,the number of pericyte positive adipocytes and the number of blood vessels.There was significant difference between the two groups(p<0.05).Conclusion1.PRF has dense fibrin structure and segmental distribution of cell components.From the upper segment,the middle segment to the lower segment,the number of cells increase gradually.2.The structure of PRF is denser after pressing and the cellular components are not easily observed.The upper segment is the densest,the middle segment is the second,and the lower segment is relatively loose.3.PRF contains a variety of angiogenesis related factors,including VEGF-A,PDGF-BB,FGF-basic,and has the effect of sustained release growth factors in vitro.4.In vitro,release of selected factors of PRF all show peak release within 1 week and enter the release platform phase within 3 weeks.the release peak time and entry time vary with factors.5.Mechanical refinement of liposuction mixture can improve histological changes,but do not affect weight,volume and volume retention after fat grafting.6.PRF combined with different forms of fat can effectively improve the histological morphology after fat transplantation and increase the weight,volume and volume retention rate of fat in vivo.7.PRF combined with chylous fat grafting improve histological morphology better than PRF combined with granular fat,but there is no difference in the weight,volume and volume retention rates between the two groups. |
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