Research Of Modified Platelet-rich Plasma On The Proliferation And Mineralization Of Bone Marrow Mesenchymal Stem Cells In Vitro

Background and objectiveHood first proposed the concept of platelet-rich plasma (PRP) in1993. PRP was platelet concentrate that obtained by centrifugation of whole blood, which contain high concentrations of growth factors. PRP compared to the recombinant human growth factor,which was expensive, high-dose, short-term and can not be effectively delivered to the target cells, was with a series of advantages like autologous biomaterial,easily obtain and cheap, high biological security, reduce implant material the series of complications, such as the spread of disease and immune response causing by implant material,the preparation process is simple, easy operation, etc, was believed by most researchers and clinicians that has a broad prospects of development.Activated PRP contained a large number of growth factors such as platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) and transforming growth factor-P (TGF-β).Those growth factor played an important role of in osteoblast chemotaxis, proliferation, differentiation and promote the formation and regeneration of bone tissue which has been successfully applied to the jaw, the skull and spinal bone graft surgery, cardiovascular surgery and associated metabolic bone disease.Seeking full potential scaffolds for tissue regeneration and establishing of biological micro-environment is the core of the research of bone tissue engineering. Research from Xie, X, etc. indicated that PRP can form a mesh-like3D scaffolds composed ultrastructure and release of endogenous growth factors,and transplanted cells can rapidly binding to the scaffolds. Mesenchymal stem cells that implanted in the PRP scaffold showed a higher proliferation rate and expression of cartilage-related genes and proteins. PRP scaffold enhanced the appearance and histological aspects of cartilage and the expression of immune-histochemical characteristics related gene and protein and promote the subchondral bone tissue regeneration of BMSCs seeded in a rabbit bone defect modelPeriosteum played a crucial role in bone formation and repair by forming blood vessels and providing bone progenitor cells. Elbackly RM proposed to use PRP gel film and autologous bone marrow-derived mesenchymal stem cells (PRP/BMSC gel film) as an alternative to the periosteum embraced osteoconductive scaffold to guide the regeneration of bone defects.The study found that PRP/BMSC gel film can significantly induced migration of human endothelial cells, and increased the expression of bone morphogenetic protein-2(BMP-2) of cultured bone marrow stromal cells(BMSCs) in vitro. These cells also secrete large amounts of the soluble pro-angiogenic factors such as PDGF-BB, VEGF, IL-8, etc. to promote the formation of blood vessels. Ultimately, PRP/BMSCs gel film were proved to enhance bone regeneration the periosteum of bionic as a substitute of periosteum in ectopic mouse models and partial bone defect rabbit model in vivo.Liu X found that platelet-rich plasma was observed to promote alkaline phosphatase activity and increase the total cellular protein content of autologous Bone Marrow Stromal Cells(BMSCs)in vitro.Which improved that PRP can induce differentiation of bone marrow stromal cells into bone cells, and improve the differentiation capability of bone marrow stromal cells.However, there are several opponents inference that PRP has rarely beneficial effect. Aghaloo suggested that after adding PRP to autologous bone graft in a rabbit skull defect modle, assessed by tissue morphology showed that only slightly larger than a single autologous bone graft, significant differences can not be found。 Some research even suggested that PRP play a negative inhibition.PRP reduced the osteoinductive of demineralized bone matrix, and inhibits the activity of bone matrix demineralized in nologic inadequacy mice modles.Bone marrow mesenchymal stem cells (BMSCs)was derived from the mesoderm, with the capability of strong self-renew and horizontal differentiation potential.A large number of studies have shown that BMSCs can differentiate into bone, cartilage, nerve, fat, muscle and liver cells in vitro by specific induction conditions.BMSCs also play an important role in regeneration of interstitial tissue, such as bone, cartilage, tendon, fat and bone marrow stromal regeneration.Maniatopoulos first reported the separation and culture of BMSCs from adult rats,which can form calcified bone tissue in vitro. Testing by X-ray diffraction fold, was confirmed as hydroxyapatite-like structure. Results proved that BMSCs can differentiate into osteoblast and form bone-like tissue. Ouyang observed that after adding ascorbic acid in the culture medium, BMSCs was tightly packed flaky growth. Combined BMSCs onto bone graft chips and implanted into the site of injury, morphological and histological results showed that the implants structure was similar to normal periosteum and differented into osteogenic after three weeks.BMSCs was consider the seed cell of gene therapy and with great clinical value Because of its relativly lack of immunity, easily culture technique, rapidly proliferation and cryopreservation property. Nowadays BMSCs cultured in vitro are generally required to use products containing fetal bovine serum or fetal calf serum, the use of exogenous serum, leading to the risk of spread zoonosis throught bone tissue engineering and clinical potential applications. Generally, PRP was activated by calcium chloride or thrombin after preparation to produce and release of large amounts of bioactive protein. In this study, we use liquid nitrogen to freezing and thawing PRP to active platelet activity to gain modified PRP. Compared with exogenous substances activation, using liquid nitrogen freezing and thawing can reduce the potential risk of infection caused by exogenous substances, and to avoid infectious diseases and immune reactions.Meanwhile a lot of preliminary studies from our department show that mPRP promoted the proliferation of human dental pulp cells and mouse osteoblast,with concentration-dependent manner. Further experiments and clinical studies is needed to clarify the effect mechanism and the optimal concentration for different king of cells of improved PRP.The present study was aims to use the same kind of improvement of autologous platelet-rich plasma-derived exogenous serum replacement products for human BMSCs cultured in vitro, in order to avoid the risk of disease transmission.And getting the best used concentration of modified PRP in BMSCs proliferation and differentiation for further bone tissue engineering research application.Materials and methods1Preparation of modified PRPPlatelet was collected from the cubital vein of the same bone marrow donor and counted by blood cells analyzer. Dispensed the PRP into the vials,then liquid nitrogen was used to freezing and thawing for three times to activated PRP. Then centrifugated the liquid and the filterable supernatant was used in the experiment. 2Isolation and culture of human BMSCs5mL of bone marrow blood was gained from healthy volunter, density gradient centrifugation was used to separated the bone marrow mesenchymal stem cells and then passaged for3generations to pure the cell culture.3Identification of human BMSCsThird-generation cell at the density of1×105cells/well was seeded in a6-well plate. Nutrient solution was changed to adipogenic and osteogenic induction after the cells growth into the logarithmic phase. Oil red O staining and alizarin red staining were used to identify the cells respectively after14and21days cultured in the induction solution.Flow cell cytometry was used to detect the cell surface antigen CD44, CD105, CD34,STOR-1.4Effect of human BMSCs proliferation cultured by mPRPWell growth human bone marrow mesenchymal stem cells were seeded onto a96-well plates and cultured with PRP solution at the concentration of2%,5%,10%and20%.10%fetal bovine serum culture medium were used as control. CCK-8cell counting kits was used to test four holes of each group for10days.Used time as the horizontal axis and OD value for the vertical axis to draw the cell growth curve.5Effect of human BMSCs mineralization cultured by mPRPCell at passage3were culture in the mineralization induced medium at different concentration of PRP for7days. Testing ALP activity as alkaline phosphatase kit requires. Cell at passage3were culture in the mineralization induced medium at different concentration of PRP for21days and used alizarin red staining to observe mineralization nodule formation.6Real-time PCR to detect the expression of gene OCN and RUNX-2Cell at passage3were culture in the mineralization induced medium at different concentration of PRP for7days. The cells were processed for OCN and RUNX-2detection by RT-PCR.7Statistical analysisStatistic analysis was performed using SPSS13.0software. The results were expressed as mean±standard deviation (x±s).A probability of P<0.05was considered statistically significant.Result1Modified PRP platelet count resultThe concentration of modified PRP was1001×109/L which was about5.4times compared to the concentration of187×109/L of Whole blood.2Growth and purification conditions of human BMSCsA small amount of human BMSCs that cultured for48h were adherent growth and present spindle shape. After10days of culturing cell become fibroblast-like with irregular synapses. The culture was in clusters. The cells reached approximately80%of fusion after two weeks culturing.3Identification of human BMSCsOsteogenesis differentiation of human BMSCs after21days induction, Alizarin red staining of mineralized nodules was positive. Adipocytes differentiation of human BMSCs after14days induction,oil red O staining was positive.The expression of CD44, CD105and STOR-1in human BMSCs was positive, but the expression of CD34was negative, in line with the general BMSCs surface marker phenotype.4Effect of human BMSCs proliferation cultured by mPRPModified PRP at the concentration of5～10%evidently promoted the proliferation of BMSCs on the6th and8th days. Modified PRP at the concentration of10%still promoted the proliferation of BMSCs on the10th day. 5Effect of human BMSCs minerlization cultured by mPRPModified PRP at the concentration of5%evidently promoted the alkain phosphatase activity and the formation of mineralized nodules in BMSCs.6Real-time PCR to detect the expression of gene OCN and RUNX-2The expression of gene OCN and RUNX-2were evidently enhanced at5%concentration of mPRP mineralization induction group by RT-PCR.ConclusionThis experiment successful gained the separation of human BMSCs, which expressed the surface markers of stem cells, differentiated into osteogenesis and fat.5-10%of the modified PRP significantly promoted the proliferation of BMSCs, especially with10%concentration group.5%of the modified PRP promoted the differentiation of BMSCs osteogenesis. The results of the experiment suggest that modified PRP combined human BMSCs in bone tissue engineering not only promoted the proliferation and differentiation but also has high biological safety.