Experimental Study Of Advanced Platelet-rich Fibrin On Adipogenic Differentiation Of Adipose Stem Cells

Background and purpose: Many reasons can lead to local skin tissue defects and body deformities,such as congenital malformations,trauma,tumor surgery and so on,especially breast and facial tumors often cause the entire organs to be defective.At present,clinical treatment methods are complicated,mainly filled or repaired with autologous tissue and artificial materials.Although this treatment method has a some effect,they still have many shortcomings.Recently,fat transplantation has attracted wide attention because of its own origin,wide selection of materials and convenience,and tissue-free rejection.Early fat block transplantation and later conversion to granular fat transplantation help improve survival rate,but it is still not ideal.Granulated fat transplantation is easily absorbed,the survival rate is not high,and the disadvantages of fat liquefaction necrosis and calcification still exist.Adipose stem cells(ASCs)are derived from the subcutaneous fat tissue in the body and have strong regeneration ability.We’ve been using platelet concentrate for a while.It has an effect on tissue regeneration by promoting angiogenesis at various stages in the healing process.PRF or A-PRF is a gel-like substance obtained from human venous blood after processing and centrifugation.It is rich in platelets and fibrin.It has been proved to have certain potential application value in soft tissue reconstruction therapy,but it has an effect on the proliferation and adipogenic differentiation of ASCs.uncertain.This study intends to explore the status of A-PRF secretion of growth factors and the effects of ASCs proliferation and adipogenic differentiation in vivo and in the external environment,as well as the practical role and significance of ASCs adipogenic differentiation in soft tissue reconstruction.Materials and methods: Pre-experiment: We often obtain the required ASCs from the subcutaneous fat tissue of healthy adults,isolate and culture,determine and expand,pass to the third generation cells,observe and record cell morphology and related biological characteristics detection,optimize and standardize extraction and culture And induce operation flow.Organization training: clarify the tasks of the members of the research group and train the members of the research group,and clarify and unify the research methods,methods and purposes of the research.The specific contents are: unified experimental observation indicators,cell and animal grouping methods,collection of test indicator results,data analysis,and work plans,requirements,completion time,etc.1.Isolation,cultivation and identification of human adipogenic differentiation augmenting of adipose-derived stromal cells(hASCs).Adipose tissue was obtained from 6 female patients who underwent liposuction surgery,and P0 hASCs were digested and separated.After three passages,the differentiation ability of P3 hASCs into adipocytes,Osteoblasts and chondrocytes were evaluated.We used immunofluorescence staining to identify the expression of hASCs specific cell surface markers,such as CD29,CD44,CD45,CD54,CD105,CD49,and CD106.2.In vitro experiments to detect the effect of A-PRF on the proliferation function of hASCs:(1)P3 hASCs were cultured in the following six groups of medium conditions(Note: except for the addition of 10% fetal bovine serum in the complete medium of the BM group,the other groups were completely cultured Added 2% fetal bovine serum),20% A-PRFe group: 20% A-PRFe BM culture,10% A-PRFe group: 10% A-PRFe BM culture,5% A-PRFe group: 5BM culture of% A-PRFe,2.5% A-PRFe group: BM culture of 2.5% A-PRFe,negative control 0% A-PRFe group: BM culture of 0% A-PRFe,positive control group BM group: basic culture Basal medium(BM),each group of cells contains 3 mediums,cultured under corresponding conditions for 7 days,the absorbance value(OD value)of each group of medium is detected by CCK-8test,and the daily The average number of absorbance values was analyzed statistically.(2)Cultivate hASCs in the following five groups of medium conditions,Control group and 0% A-PRFe group: Adipogenic induction medium(AIM)only,2.5% A-PRFe group: 2.5% A-PRIM AIM culture,5% A-PRFe group: 5% A-PRFe AIM culture,10% A-PRFe group: 10% A-PRFe AIM culture,20% A-PRFe group: 20% A-PRFe After AIM culture,the cells of each group were cultured under the corresponding conditions for 14 days.The oil red O staining experiment,cell density,and lipid concentration test proved the effect of A-PRFe on the adipogenic differentiation ability of hASCs.3.In vitro experiments explore the function of A-PRF secreting growth factors.15 ml of venous blood will be drawn from healthy adults,divided into three disposable sterile blood collection tubes,5ml per tube,placed in sterile negative pressure blood collection tubes,centrifuged for 14 min at a speed of1500 rpm / min,it can be seen that autologous whole blood is divided into three layers: The top layer is: Platelet-poor plasma(PPP);middle layer: PRF membrane;lowest layer: red blood cell debris(RBC).Remove the uppermost layer and the lowermost layer as the primary A-PRF gel,and mark it,and detect VEGF,b-FGF,EGF,PDGF-at the same time every day on the 1/3/7/14/21 day.The secretion level of AB,PDGF-BB,IGF-1,TGF-β,IL-1β and IL-4 should be recorded.4.Animal transplantation experiment to explore the effect of A-PRF on the adipogenic differentiation function of hASCs.We set up a treatment group and a control group,group A: nanofat + A-PRF + granular fat,group B: nanofat +granular fat,group C: A-PRF + granular fat,group D control group: simple granulated fat.The four groups of materials were implanted into the right back,left back,right front,and left front of the back of 8 nude mice respectively,and they were marked and fed normally,and photographs were taken during the period.Four months later all the animals were put to death and the tissue at the transplant site was dissected.Observe the general condition and weigh it.The retention rate was measured.(Formula: retention rate=weight of residue/weight at the beginning of transplantation).Quality,take tissue for frozen section,oil red O staining,HE staining,measure fat density and lipid content.Result:1.After initial isolation and amplification(P1),homogeneous hASCs can be observed,and 80-90% fusion can be achieved after 7-14 days of cultivation.The cells grow in a single layer in a spindle-shaped morphology and show a strong proliferation ability.Positive staining by oil red O,alizarin red and Alcian blue confirmed that hASCs can induce differentiation into adipocytes,bone cells and chondrocytes.Immunofluorescence staining results showed that the expression of CD29,CD44,CD45,CD54 and CD105 was higher,while the expression of CD49 and CD106 was relatively low.2.A-PRF autocrine function test can secrete a variety of growth factors,including VEGF,b-FGF,PDGF-AB,PDG-BB,IGF-1,IL-4 factors increased from day 1 to day 7 The trend reached a peak on the 7th day and then declined;EGF,TGF-β,and IL-1β secretion showed an increasing trend from day 1 to day14,and reached a peak on the 14 th day,followed by a downward trend.3.The effect of A-PRF on the growth and proliferation of ASCs: CCK-8assay was used to evaluate the proliferation of the third-generation hASCs cells.The cell proliferation populations in all groups showed different growth trends.After reaching the 5th day,they all stabilized.Statistical analysis of the data measured every day,the difference between the 20% A-PRFe and 10% A-PRFe,5% A-PRFe,BM group was significant on the first day(P <0.05).2.5% A-PRFe,0% A-PRFe is meaningless(P> 0.05),the difference between the 10% A-PRFe group and 5% A-PRFe,2.5% A-PRFe,0% A-PRFe and BMA group is significant(P<0.05),the difference between 5% A-PRFe and 0% A-PRFe is significant(P <0.05),5% A-PRFe is not meaningful with 2.5% A-PRFe and BM group(P> 0.05),2.5% A The difference between-PRFe and 0% A-PRFe was significant(P<0.05),and the difference between 2.5% A-PRFe and BM group was not significant(P>0.05).The difference between the 2.5% A-PRFe and BM groups on the second day was not significant(P>0.05),and the differences between the other groups were significant(P<0.05).On day 3,the differences between the groups were significant(P<0.05).On day 4 there was no statistically significant difference between the 5% A-PRFe and BM groups(P>0.05),and there were significant differences between the remaining groups(P<0.05).There was no statistically significant difference between the 2.5%A-PRFe and BM groups on Day 5,Day 6,and Day 7(P>0.05),and there were significant differences between the other groups(P<0.05).A-PRFe can significantly promote the proliferation of hASCs in a concentration-dependent manner.The higher the stimulation concentration of A-PRFe within a certain range(2.5%-20%),the higher the proliferation ability of hASCs is,and the ANOVA is used to evaluate.After adipogenic induction of hASCs stimulated with A-PRFe,larger oil red O-positive lipid droplets were observed in the cytoplasm compared with cells cultured with AIM alone.The number of adipocytes and lipid concentration in the A-PRFe group at different concentrations were both It was higher than the control group(P<0.05),and showed a positive dose-dependent effect.The higher the A-PRFe concentration,the greater the number of adipocytes and lipid concentration.4.(1)There were four sites on the left front,left back,right front,and right back of the back of 8 nude mice.All animals showed that there were still grafts at the transplant site 4 months after transplantation.Visual inspection of the residual tissues of each group.Group A has the largest volume,followed by Group C,and Group D has the smallest volume.(2)The retention rate of group A is significantly higher than that of the other three groups.After statistical analysis,the difference between group A and the other three groups is significant(P<0.05).(P>0.05).Compared with group D,there was a significant difference between group B and group C(P<0.05).(3)H & E staining of residual tissues and observation under different magnifications(A1-D1 100 times,A2-D2 200 times,A3-D3 400 times).Group A has more mature adipocytes,less fibrosis than other groups,and more and larger adipocytes,BC is equivalent in two groups,group D has the fewest cells,sparse cells and small volume;D4),all four groups were positive,group A contained more oil red O-positive lipid droplets,group D had the least.(5)Density of mature adipocytes: group A is significantly more than the other three groups,group C is the second,group D is the least,and the difference between each group is significant(P<0.05);(6)lipid content: the fat cells in group A The lipid content was significantly higher than that of the other three groups(P<0.05).There was no statistically significant difference between the two groups of BC(P>0.05).Compared with the three groups B,C and D,the difference was significant(P<0.05).Conclusion:1.ASCs have strong proliferation ability.ASCs have the potential to differentiate into adipocytes,chondrocytes,and osteoblasts.2.A-PRF can secrete a variety of factors,including VEGF,b-FGF,PDGFAB,PDG-BB,IGF-1,IL-4 factors peak on the 7th day;EGF,TGF-β,IL-1β It peaked at 14 days.3.A-PRF can optimize the proliferation and adipogenic differentiation functions of hASCs in a concentration-dependent manner.The higher the stimulation concentration of A-PRF in a certain concentration range(2.5%-20%),the stronger the proliferation function of hASCs.4.A-PRF can optimize the adipogenic differentiation function of hASCs in vivo,increase the number of mature adipocytes and the concentration of lipid content in the new tissue,and obtain new tissue with larger volume and weight.