| Objective: Platelet-rich plasma (PRP) has been used in oral implantologyand tissue engineering for recent years and now is becoming an interestingrealm for clinicians and researchers. It is a storage vehicle of growth factors,such as transforming growth factor (TGF-β), platelet-derived growth factor(PDGF), platelet-derived epidermal growth factor(PDEGF),insulin growthfactor-1(IGF-1), which were known to influent bone regeneration. TGF-β1isthe most widespread used and remarkable one. However， the optimumconcentreation of TGF-β1is still debatable now. The aim is to explore theeffect of different concentrations of autologous PRP on the osteoblast ontitanium and the change of TGF-β1concentration. We hope to provide areasonable reference about application concentration&time of TGF-β1andPRP for clinic.Methods:1Culture of BMSCs: About4-6ml bone marrow were obtained from tibialplateau cavity of New Zealand white rabbits aged2-3months after anesthesia.Primary BMSCs were isolated from whole bone marrow and cultured inincubator at37℃,5%CO2and saturated humidity.2Drawing growth curves of cell：The third generation of BMSCs wereseeded in24-well plates and counted under the microscope during1to10days.Then, cell growth curve were drawn.3Treatment of titanium: Commercially pure titanium plate were cut intodiscs10mm in diameter and1mm thick after polished step by step with siliconcarbide abrasive papers600、800and1000grit. Then, rinsed by ultrasound for20minutes with acetone, pure alcohol,75%alcohol in turn, and vaporsterilized.4Preparation of PRP: PRP was isolated from the rabbit’s whole blood by two-steps centrifugation method. The steps are as follows: About120ml thewhole blood of rabbit was drawed via the carotid artery into tubes containing3.8%sodium citrate after anesthesia. After the first centrifugation at1100r/minfor10minutes，all the supernatant and2-3mm of the red blood cell below theinterface were obsorbed, then two thirds supernatant was discarded after thesecond centrifugation at2200r/min for10minutes，and the rest was PRP. PRPwas activated with calcium chloride solution and bovine thrombin. All of theabove steps must be finished in2hours.5Identify of osteoblast: The third generation of BMSCs were collected andinduced to osteoblasts with osteogenic induced media. The capacity ofosteogenic differentiation was examined by Alkaline phosphatase staining andCalcium nodules staining.6Divided into groups:⑴Treatment groups: Generation4BMSCs werecultured with various PRP concentration (10％、20％、30％) and titanium diskand complete DMEM without serum.⑵Control group: Generation4BMSCswere cultured with titanium disk and complete DMEM without serum.⑶Blank control group: Generation4BMSCs were cultured with completeDMEM without serum. Trituplicate were done in all groups.7ELISA assay of ALP activity：To assay the activity of ALP of every groupaccording to ELSIA kit steps at4d、7d、10d、14d respectively.8ELISA assay of TGF-β1concentration: The concentration of TGF-β1intitanium plate groups were assayed by ELISA at1d、2d、3d、4d.9Fluorescent staining of cell morphology on titanium disks: The titaniumdisks with osteoblast cells in experimental groups were gained at4d、7d、10d,cytoskeleton was stained with F-actin fluorescent dye and the nucleus werestained with DAPI finally. The cell morphology was observed with afluorescence microscope.Results:1The cell morphology under inverted microscope: The morphology of theprimary passage was short-spindle, and80％of BMSCs were confluenced in12days, when the cells were uniform long-spindle, and arranged in circinate. The subculture cells were more various in shape than before. In subcultureincubation period, the incubation phase is generally2-3days, and4-7day isthe logarithmic growth phase, and in8-10d cell proliferation become slowly,which means the plateau phase.2Identify of osteoblast: Both Alkaline phosphatase staining and Calciumnodules staining were all positive. Alkaline phosphatase staining showedbrown grain in the cytoplasm. In Calcium nodules staining staining, red-brownor the orange-red calcium nodules can be observed clearly. The results showedcells have the capacity of osteogenic differentiation.3Detection of PRP: The amount of platelet in PRP gained by two-stepcentrifugation method was878±87×109/L, which was4.2times than that in thewhole blood217±43×109/L．4Assay of ALP activity: OD value of all the treatment groups were higherthan that of control group(P <0.01), OD value in treatment group was20%PRP group＞10%PRP group＞30%PRP group, and there was significantdifferences, too（P＜0.01）. The highest OD value was gained in20％PRPgroup on10day. The optimal concentration was20%PRP. The BMSCsdifferentiation was related intimately with the concentration of PRP. So theBMSCs differentiation effect was closely related to time and concentration ofPRP.5Concentration of TGF-β1: According to the standard curve, the conc-entration of TGF-β1in PRP was188.358ng/ml, which showed highconcentration of TGF-β1could be obtained by two-step centrifugation methodand activated by activator. Comparation results of TGF-β1concentration ineach treatment group in different times are as follows:0d＜1d,2d,3d（p＜0.05）;1d,2d,3d＞4d（p＜0.05）;1d＜2d,3d（p＜0.05）, and there weresignificant differences among them. The basic trend was that most of TGF-β1was released in the very first day. A high concentration was still maintained forthe first three days, however, it started to decline sharply after the third day.Concentration of TGF-β1was in direct proportion with the concentration ofPRP. 6Fluorescence staining results: Nucleus fluorescence staining was blue, andcytoskeleton showed green with irregular shape around nucleus, which wascorresponded with the osteoblast morpology.Conclusion:1We conclude that PRP contains osteo-inductive growth factors. AutologousPRP could stimulate the initial growth and differentiation of rabbit osteoblastsin vitro.2The optimal concentration range of TGF-β1is from81.725±19.374to96.571±15.916ng/ml, which is contributed to the osteoblast differentiation andexpression of ALP.3The majority of TGF-β1were released on the first day and its concentrat-ion still maintain a high period for the first three days. The half-life of TGF-β1is short. |
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