The Effect Of Choukroun’s PRF On The Proliferation And Osteogenetic Differentiation Of Human Adipose-derived Stem Cells In Vitro

BackgroundBasic research have been demonstrated that the platelets which activated by a stimulus can release various growth factors, such as platelet-derived growth factor AB (PDGF-AB), endothelial cell growth factor (ECGF), epidermal growth factor (EGF), transforming growth factor β (TGF-β) and insulin-like growth factor (IGF). These growth factors can effectively effect on cell growth differentiation and tissue repair regeneration. In1984, Assion first extracted platelet-rich plasma from human plasma. Platelet-rich plasma (PRP) first used in cardiothoracic surgery by Ferrari in1987. Following, due to PRP might enhance tissue repair and regeneration through release of growth factors. It has become a popular additive in orthopaedic surgery and trauma surgery. But because of PRP was added heterogeneity species thrombin and anticoagulants in preparation process, there were some immune rejection and infectious disease risk. So its application has been controversial.In2000, French scholar Choukroun first developed a new generation platelet concentrate products (Choukroun’s platelet-rich fibrin, Choukroun’s PRF). It had the advantages of the preceding generation platelet concentrate products PRP, at the same time, and had the characteristic of strong osteogenesis ability. Unlike other platelet concentrates, PRF production process is completely natural, with no use of anticoagulant during blood harvest nor bovine thrombine and calcium chloride for platelet activation and fibrin polymerisation. PRF is often simply considered as a natural optimised blood clot. This open protocol is very simple and inexpensive: Blood is collected in dry glass tubes or glass-coated plastic tubes and immediately softly centrifuged. Three layers are formed:a red blood cell (RBC) base at the bottom, acellular plasma (platelet-poor plasma [PPP]) as a super-natant, and a PRF clot in the middle. This clot combines many healing and immunity promoters present in the initial blood harvest. It can be used directly as a clot or after compression as a strong membrane. A lot of studies have been demonstrated that potential clinical indications of PRF in oral and maxillofacial surgery are numerous, including, for example, the improvement of soft tissue healing and bone graft protection and remodeling.In clinic, bone defects which are caused by trauma, infection, tumor and deformity are very common. But the bone tissue repair and regeneration is difficult. In recent decades, with the development of tissue engineering technology and update of biological materials, the development of bone tissue engineering has become a new and ideal thought for large bone defect repair. And the source of seed cell has restricted the further application of bone tissue engineering. Ideal sources of seed cells:source is rich, conveniently and little damage to the donor; have strong ability of proliferation and extend; from autologous tissue, no immune rejection after transplantation; After implanted it can repair bone defect, and have high quality and keep a good forward curative effect observation. Bone marrow mesenchymal stem cells (BMSC) have the ability of proliferation and multi-directional differentiation, and have widely used in the research. But which have hard to access and are limited for osteogenetic differentiation, have limitations as bone tissue engineering seed cells. But so far, Choukroun’s PRF research is still in the primary stage, and needs further research and discussion.Since Zuk had first isolated and extracted adipose-derived stem cells from liposuction, adipose-derived stem cells have similar differentiation ability with bone marrow mesenchymal stem cells. In vitro differentiation experiment, to join in different chemical reagents or cytokines, adipose-derived stem cells can be formed into fat cells, osteoblasts, cartilage cells, nerve cells, endothelial cells, muscle cells, myocardial cells and liver cells. And adipose-derived stem cells can easily access, rapidly expanded, and have the characteristics of the multi-directional differentiation potential, which have broad application prospect in adult stem cells and provide a good source of cells for tissue engineering technology.Our experimental researches were observed that the effect of Choukroun’s PRF on the proliferation and osteogenetic differentiation of human adipose-derived stem cells in vitro, and the results provided a new prospect for bone tissue engineering.ObjectiveThe experimental researches were observed the proliferation and osteogenetic differentiation of human adipose-derived stem cells in vitro. Choukroun’s platelet-rich fibrin join the culture medium after the cultivation of human adipose stem cells in vitro,to investigate the effect of Choukroun’s PRF on the proliferation and osteogenetic differentiation of human adipose-derived stem cells in vitro. For bone tissue engineering this provides a new technology and ideas.Method1. Experimental tissue sources:Fat tissues were taken from eight patients who underwent abdominal and high fat liposuction and surgical excision fatty tissue at Zhujiang Hospital, For all patients at the age of20to40.They were no infectious diseases,endocrine disorders and historyof long-term taking the medicine. Venous blood was from the corresponding source of fat for patients.2. Adipose-derived stem cells derived from fat tissues were maintained in vitro. Only cells from the third passages were analyzed in our research.3. Identification of human adipose stem cells:ADSCs at passage3were cultured in adipogenic, osteogenetic and neurospheres differentiation medium and underwent identification, flow cytometric analysis for cell surface antigen CD29, CD45and CD90were performed.4. Prepared platelet-rich fibrin (Choukroun’s PRF) using Choukroun’s method.5. Groups of our experiments:this experiment was divided into two groups, according to the ordinary cell cultures was join the Choukroun’s PRF or not, and selected the four time points (1th,3rd,5th and7th day), respectively observed cell proliferation of two groups. This experiment was divided into other two groups, according to the osteoinduction cell cultures was join the Choukroun’s PRFor not, and selected the four time points (7th、14th、21st and28th days), respectively observed osteogenesis induced of two groups in every time, and setting blank control in ordinary cell culture medium with not PRF. Sample sizes were3-4cases in each group.6. Making standard curve using CCK-8colorimetric mater.Using SPSS software to make standard curve, our tested the correlation between the OD value measured and number of adipose stem cells.7. CCK-8kits determined the effect of Choukroun’s PRF on the proliferation of human adipose-derived stem cells.8. Osteogenesis of two groups adipose stem cells were estimated detecting activity of alkaline phosphatase activity (ALP).9. Calcium nodules were observed using Von Kossa staining in each group osteogenesis cells.10. Spss13.0was used in statistical analysis. All data are expressed as mean±standard deviation (SD). Two independent sample tests were used to analyze a statistical significance of keloids volume. A P value<0.05was considered significant.Result1. Human ADSCs morphologic:the original generation of ADSCs were began to stick wall after eight hours cultivation. Adherent cells were colony growed as typical spindle cells. After the wedding cell were grown obviously faster than the original generation. Most of third-generation ADSCs were observed fusiform or polygon or triangle under inverted microscope.2. Characterization of human adipose-derived stem cells:Human ADSCs osteogenesis induction after14days, black calcium nodules were discoveryed with von Kossa staining. Oright red bright lipid droplets were formed adipogenic differentiation after14days with Oil red O staining. Typical red fluorescent ball was formed neurospheres differentiation after7days detecting nestin immunofluor -escence antibody. Flow cytometry detection of ADSCs surface antigens includin g CD29, CD90, CD45, the positive cells rate were respectively93.8%,99.3%an d0.4%.3. Standard curve was produced. R2=0.96, regression equation is Y=-2955.56+20809.46X (Y is the number of ADSCs, X is OD value). And this regression equation has statistics significance (F=347.65, P<0.001). So the OD value can reflect the actual numbers of ADSCs.4. Results of cell proliferation tested CCK-8:observed using inverted microscope, cell proliferation of the PRF group Ⅰ obviously accelerated growth than the control group Ⅰ, their shapes were no obvious difference with control group Ⅰ. OD value of PRF group Ⅰ and the control group Ⅰ are both increased gradually. But OD values of the PRF group Ⅰ at each time point were greater than control group Ⅰ. On the1th,3rd and5th day, absorbance value of PRF group Ⅰ and the control group Ⅰ were no significant difference (P>0.05). On the7th days, there was significant difference (P<0.05).5. ALP activity detection osteogenesis induction results of PRF group Ⅱ and control group Ⅱ:ALP activity detection showed PRF group Ⅱ were more compared with the control group Ⅱ on the7th、14th、21st and28th days. There was statistical significance (P<0.05).6. Calcium nodules of osteogenesis induction observed Von Kossa staining:after7days osteogenesis, von Kossa staining showed positive black calcium nodules. Calcium nodules were increased obviously. Control group II were not shown calcium nodules after7days.lt was observed a few of positive calcium nodules after14days. There were not observed any positive calcium nodule after14days in the blank group.ConclusionAdipose stem cells have made progress in research in recent years, and have demonstrated be good seed cells of tissue engineering. Our research have made a preliminary study about the effect of Choukroun’s PRF on the proliferation and osteogenetic differentiation of human adipose-derived stem cells in vitro,confirmed that it can promote the proliferation and osteogenetic differentiation of adipose stem cells. For adipose stem cells as seed cells of the bone tissue engineering provide a development thought. There were had great research value in adipose stem cells culture with Choukroun’s PRF. Current researches are still infancy at home and abroad, also deserving further research.