| Objective:The biological effects of injectable platelet rich fibrin(i-PRF)on human adipose mesenchymal stem cells(hADSCs)were studied.Clinical study on the treatment of facial depression with i-PRF combined with fat particles.Study on the possible mechanism of i-PRF promoting the survival of fat graft fillers.Methods:a.Human peripheral venous blood was collected.I-PRF was prepared by centrifuging at 700 rpm for 3 min at room temperature.The white blood cells and platelets of human peripheral venous blood were counted with an automatic blood cell counter.Manually count the number of white blood cells and platelets in i-PRF using a human blood cell counting board.Place i-PRF in an incubator for 1.5 h and solidify into an i-PRF film.The ultrastructure of i-PRF was observed by scanning electron microscopy.b.Collagenase digestion centrifugation was used to isolate clinical fat aspiration samples and cultivate primary hADSCs.Purify the extracted primary hADSCs and pass them on to the P3 generation.The morphology of the cells after HE staining was observed under an inverted phase contrast microscope and differentiated using adipogenic,osteogenic,and chondrogenic differentiation media.The differentiation ability of stem cells was analyzed by staining.The surface antigens of hADSCs were detected by flow cytometry.c.DMEM low sugar medium was added to the i-PRF membrane and soaked for 3 days.Conditional media with different concentrations were prepared for subsequent experiments.The effects of i-PRF on the proliferation and migration of hADSCs were detected by CCK-8 cell proliferation test and Trans well chamber cell migration test.The effects of i-PRF on the proliferation and adipogenic differentiation of hADSCs were detected by Real-Time PCR assay.d.The patients’ venous blood was drawn to prepare i-PRF.The fat particles obtained by liposuction are mixed with i-PRF.Injection filling treatment was performed on 23 patients undergoing facial depression treatment in hospitals between March 2021 and May 2022.Follow up was conducted after 6 months.Observe the postoperative complications and treatment effects of the patients.Understand patient satisfaction with treatment.Results:a.The white blood cell and platelet count in i-PRF is 2-3 times higher than that in human peripheral venous blood.After film formation,i-PRF has a complex three-dimensional stent network structure.Scanning electron microscopy(SEM)observation showed that fibrin agglomerated into bundles and interwoven with each other.And there are white blood cells attached to it.A bracket structure with a backbone and branch interconnected.b.The isolated and cultured cells were observed under microscope to identify their cell morphology.After three line differentiation,P3 cells were stained with oil red O,alizarin red,and Alcian blue to identify their ability to differentiate into three lines.The expression of stem cell surface markers was analyzed using flow cytometry(positive for CD29,CD73,CD90,CD105,and CD166,and negative for CDllb,CD32,CD34,CD45,and HLA-DR).All experimental results showed that the cultured cells had the ability to differentiate into stem cells.The cells used in this study are adipose mesenchymal stem cells.The experiment can continue.c.Set six concentration groups within the concentration range of 1%-30%of i-PRF:1%,5%,10%,15%,20%,and 30%.The control group was a complete culture medium group without adding i-PRF extract.The results of cell proliferation experiments showed that the proliferation ability of hADSCs cells was positively correlated with the concentration of i-PRF.Higher concentrations of i-PRF(15%,20%,30%)showed significant statistical differences compared to the control group on the 3rd,5th,and 7th days(p<0.05).This means that it can greatly support the proliferation of hADSCs.However,the difference between i-PRF at 20%and 30%concentrations was not statistically significant(p>0.05).RT-PCR results showed that adding i-PRF to the culture medium could significantly increase the expression of PCNA.Starting from a concentration of 10%i-PRF,it has statistical significance(P<0.01).The concentrations of 15%,20%,and 30%were the most significant(P<0.0001),but there was no statistical difference among the three(P>0.05).Transwell experiment showed that the cell migration ability of hADSCs was positively correlated with the concentration of i-PRF.Medium and high concentrations of i-PRF can significantly improve the migration ability of hADSCs.The effects of i-PRF at concentrations of 10%,15%,20%,and 30%on the migration ability of hADSCs were statistically significant(P<0.0001).The results of experiments related to adipogenic differentiation showed that there was a positive correlation between the adipogenic differentiation ability of hADSCs cells and the concentration of i-PRF.Use imageJ software to count and detect the cells stained with oil red O after adipogenic differentiation.The number of cells stained with oil red O from the concentration of 10%was statistically different from that of the control group(P<0.01).RT-PCR results showed that after adding i-PRF configured at different concentrations to the adipogenic differentiation medium,PPAR-γ、The expression of adhilin and LPL were significantly increased.PPAR-γ And LPL have statistical differences from the concentration of 15%(p<0.01).From the beginning of the 5%concentration of i-PRF,there was a statistically significant expression of the adenophilin gene(p<0.05).Therefore,the promotion effect of high concentrations of i-PRF on the expression of the above genes is relatively obvious,and there is no statistical difference in the expression of the above genes in the high concentration group of i-PRF(p>0.05).The above experiments indicate that adding i-PRF to the adipogenic differentiation medium can promote the adipogenic differentiation ability of hADSCs.Among them,the concentrations of 15%,20%,and 30%can better promote the adipogenic differentiation of hADSCs(p<0.001),and there is no statistical difference among the three concentrations(p>0.05).In summary,15%concentration of i-PRF can effectively promote the proliferation,migration,and adipogenic differentiation of hADSCs.d.This study followed up 23 patients for a total of 6 months.21 cases(91.30%)were followed up effectively.The main complications were hematoma and ecchymosis.The second is itching.Less abnormal facial sensations.There were no serious complications such as fat embolism,fat liquefaction,infection,and uneven donor site.The recovery time of patients decreased after surgery.After surgery,it can be seen that the patient’s concave filling area is flat and rich,with a natural expression.Seventeen patients(80.95%)were satisfied with the treatment.Seven patients(14.29%)were more satisfied.One patient(4.76%)was dissatisfied.According to the formula,the patient’s satisfaction rate with the filling effect can be obtained as 95.24%.Patients who expressed dissatisfaction were given two fat fills six months after surgery.After 6 months of follow-up,all patients expressed satisfaction with the refilling effect.Conclusion:a.i-PRF is rich in white blood cells and platelets,has a complex fibrin three-dimensional network scaffold structure,and i-PRF has a unique liquid-solid phase conversion ability.b.The biological effects of different concentrations of i-PRF on hADSCs are significantly different.High concentrations of i-PRF can promote cell expansion,migration,and adipogenic differentiation in vitro.c.The addition of i-PRF to fat grafts showed good clinical efficacy and high overall satisfaction of patients,which provided certain clinical reference value for the clinical application of i-PRF for fat grafting. |
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