The Effects Of Autologous PRP On Bone Sialoprotein Expression Of Osteoblasts

Objective: With the improvement of people’s living standard, Dentalimplant has been accepted by more and more people. The theory ofOsseointegration on the interface between implant and bone was proposed byProfessor Branemark in the last century60’s. Since then, Oral implanttechnology and theory research have been developed rapidly. At present,unable to form a good osseointegration and lack bone tissue in the dentalimplant area are common problems in oral implant research. In order to solvethe problems, many scientists have done a lot of research. Bone marrowstromal cells is ideal seed cell for tissue engineering, and it can be inducedinto osteoblast. Platelet-rich plasma can release large amounts of growthfactors after stimulated, which can promote the repair and reconstruction ofbone defect. Bone sialoprotein is a sign of osteoblast maturation and thesymbol of the osteoblast mineralization stage. In this study, rabbit BMSC wasinduced into osteoblast in vitro. And after a period of time, we use the blood ofthe same rabbit to extract PRP. Then add PRP into the osteoblast in order toobserve the growth and proliferation and mineralization situation. Through theobservation to the effect of PRP on proliferation, differentiation andmineralization of osteoblast in different periods, in order to further reveal themechanism of PRP and provide experimental basis for clinical rational use.Methods:①Culture of BMSCs: Get ready a three months old NewZealand white rabbit with pentobarbital sodium intravenous anesthesia, extractbone marrow4-6ml in tibial of the rabbit with16bone marrow punctureneedle, Centrifugal the cells and inoculate the cells in culture medium. Whenthe cells reach70~80%adherent and fusion, make it go down to the futuregeneration. In this period, we use inverted microscope morphology to observethe primary and passage cells.②Drawing the growth curve of cells: Take the BMSCs in the third generation and prepare BMSCs cell suspension withdensity1.0×104/ml.Then inoculate the cells into24hole culture plate. Inevery group, prepare three hole to observe and count, and record for10dayscontinuously. At last, draw the growth curve of cells.③Preparation of PRP:At first, get the same rabbit with pentobarbital sodium intravenous anesthesia,via ear marginal vein. Then separate the common carotid artery and extract thewhole of the blood. PRP was isolated from the rabbit’s blood by two-stepcentrifugation method. The amounts of platelet in the blood and PRP werecompared by electronic equipment respectively. The PRP activated withbovine thrombin must be immediately added into the cell culture system.④Divided into groups: Treatment group: The fourth generation of BMSCswere cultured with serum free completely DMEM including20%PRP andmineralized induced liquid; Control group: The fourth generation of BMSCswere cultured with serum free completely DMEM including mineralizedinduced liquid.⑤Identify of osteoblast: The third generation of BMSCs werecollected and induced to osteoblasts with osteogenic induced media. Throughthe method of alkaline phosphatase staining and alizarin red staining, we candetermine BMSCs differentiating into osteoblasts is successful or not.⑥Assay of Osteoblast proliferation: Apply MTT method to detect theproliferation of osteoblasts in treatment group and control group selecting timeappoint of1,4,7,10day, each group set3side holes.⑦Assay of osteoblastalkaline phosphatase activity: Using the alkaline phosphatase detectionkit to assay the content of alkaline phosphatase at4,7,10,14day. every groupset3side holes.⑧Assay of type bone sialoprotein expression of oste-oblast:The expression of bone sialoprotein of treatment group and control group wereobserved with Western blot at4,9,14,19,24d.Result:①The cell morphology under inverted microscope: the primaryBMSCs cultured for five days was observed under inverted micaroscope. Wecan see the cells is in the shape of spindle, triangle or polygon, some cellsgathered into a colony which distribute diffusely. with the prolongation ofculture time, cell number increased significantly. about11-12d, the cells covered with the bottom of the80-90%, the adherent cells were spindle,spindle arrangement, the whole cells arranged closely resemble fasciculate, theshape of swirl. cell proliferation rate was accelerated markedly During passageculture. after the third generation, the cell morphology is more uniform, thecell number increased fast, and the adherent cells look like fusiform orspindle, cells showed an overall fish like, whorled or parallel arrangement.Cells growth curve show: the cells cultured for the first day and second day isincubation, cell proliferation is not obvious, after3days cell proliferationwas accelerated, entered the logarithmic growth phase,7days after theproliferation slow down and enter the flat period, Cell proliferation tend tobe stable.②Identify of osteoblast: After osteogenic induction, alkalinephosphatase staining and alizarin red staining showed positive expression,the results show that the BMSCs cultured in vitro could be induced intoosteoblast and has the ability of mineralization.③Detection of PRP: Thenumber of platelets in PRP is855±27×109/L and the number of platelets inwhole blood was203±33×109/L. the platelet count in PRP is4.2times of thewhole blood. The result meet the requirements in this experiment.④MTTassay of proliferation: The experimental group at each time point issignificantly higher than that in the control group, the difference is statisticallysignificant (P<0.05), and the experiment in the7day group received thehighest OD values.⑤Assay of alkaline phosphatase activity: Theexperimental group at each time point was significantly higher than that in thecontrol group, the difference was statistically significant (P <0.05), and theexperiment in the10day group received the highest OD values.⑥Expressionof bone sialoprotein: the expression of treatment group are higher than thosein the control group at each time point, and there were significant differences(P<0.05), the peak expression of bone sialoprotein was found in treatmentgroup on19day.Conclusion:①Rabbit BMSCs have the potential of multilineagedifferentiation, which can be differentiated into osteoblasts in vitro.②PRPcan be obtained by two-step centrifugation method, it can effectively promote the rabbit osteoblast proliferation and differentiation in vitro. The strongesteffect of promote proliferation is on the7th day, and the strongest effect ofpromote differentiation is on the9th day.③Autologous PRP can promote theexpression of bone sialoprotein, and the highest expression level is on the19th day. PRP can promote bone sialoprotein expression and promote boneformation.