| Objective：Bone marrow stromal cells(BMSCs) of rabbit were culturedin vitro to establish the composite culture model of cell-Ti. BMSCs weretreated with Autologous platelet-rich plasma(PRP), in order to observe theinfluence of autologous PRP in different concentrations in proliferation ofBMSCs on titanium and the concentration change of platelet-derived growthfactor (PDGF) in PRP. The aim is to explore the mechanism of PRP and toprovide an experimental evidence about the available concentration of PDGFand reasonable time of application for clinic.Methods：①Culture of BMSCs: Primary BMSCs of the rabbits isolatedfrom the whole bone marrow were cultured in vitro. The methods to obtainBMSCs are as below.2to3-month New Zealand white rabbits were selectedfor experiment, and anesthetized by injecting sodium pentobarbital into theear vein. The whole bone marrow cells were obtained by aspirating with16~#bone marrow needle from bone marrow cavity of tibial plateau of the rabbits.The extracted bone marroe(4-6ml) were seeded into flasks. Cells wereharvested with Trypsin/EDTA and generated when they were attached up to90％. Then,the morphology of the primary cells and generated cells wereobserved every day with inverted phase contrast microscope.②Drawing thegrowth curve of cells：The third generation of BMSCs were seeded in24-wellplates and counted under the microscope during1to10days. Then, cellgrowth curve were drawn.③Treatment of titanium: The experimentaltitanium plate (Diameter10mm, thickness1mm) was cleaned with ultrasound,followed by acetone, ethanol,75%ethanol and double distilled water for20minute, then sterilized after autoclaving for30minute and stored.④Preparation of PRP: PRP was isolated from the rabbit's blood by two-stepcentrifugation method．The amounts of platelet in the blood and PRP were compared by electronic equipment. The steps in detail are as follows:Experimental animals were anesthetized as before and about120ml the wholeblood were obtained via the carotid artery. At first, all the supernatant and2-3mm of the red blood cell surface were collected after centrifugating at1100r/min for10minutes, then, further2200r/min for10minutes, two-thirdsof the supernatant were discarded, and the remaining liquid is PRP, which wasactivated with bovine thrombin. All the up steps were finished and PRP wasadded to cells in2hours.⑤Role of PRP in BMSCs: The fourth generationof BMSCs were seeded on titanium disks and cultured with PRP in variousconcentrations(10％,20％,30％). Further more, Control group without PRPand blank control group without titanium were set. Trituplicate were donein each.⑥Identify of osteoblast: The third generation of BMSCs werecollected and induced to osteoblasts with osteogenic induced media．Thecapacity of osteogenic differentiation was examined by Alkaline phosphatasestaining and Calcium nodules staining.⑦MTT assay of proliferation: At1,4,7,10day, all the wells of experimental group were collected, discardedmedium, added MTT reagent after washing the titanium plate, and incubatedat37℃for4hours, then add DMSO. OD value of each specimen weremeasured in the microplate reader after crystallization was uniformlydissolved.⑧ELISA assay of the concentration of PDGF-AB: Theconcentration of PDGF-AB in titanium plate groups were assayed byenzyme-1inked immunoassay(ELISA)at1,2,3,4day.⑨Fluorescentstaining of cell morphology on titanium disks: The titanium disks withosteoblast cells in experimental groups were gained at4,7,10day,cytoskeleton were stained by fluorescent dye F-actin and the nucleus werestained by DAPI finally. The cell morphology was observed with afluorescence microscope.Result:①The cell morphology under inverted microscope: The shape ofthe primary cell was short-spindle, and80％cells confluenced in12days, thecell was long-spindle uniformly at this moment, and arranged in circinate.What's more, the subculture cells were more variform in shape than pre vious generations. During passage culture, the incubation period is generally2-3days, the logarithmic growth phase is4-8days, then cell proliferation becomesslowly in9-10days and enter the plateau period.②Identify of osteoblast: Theresult of Alkaline phosphatase staining and Calcium nodules staining werepositive, which showed cells have the capacity of osteogenic differentiation.In alkaline phosphatase staining, red-brown or brown grain can be observed inthe cytoplasm. In calcium nodules staining, the orange-red calcium nodulescan be found clearly.③Detection of PRP: The amount of platelet in PRPgained by two-step centrifugation method was864±37×109/ml, which was4.3times of that(201±26×109/ml)in the blood．According to the standard curve,the result of ELISA showed that the corresponding value of PDGF-AB was95.537ng/ml. High concentrations of growth factor PDGF-AB were obtainedby two-step centrifugation method and activated by activator.④MTT assayof proliferation: The BMSCs proliferation was correlated intimatly with theconcentration of PRP. The highest OD value was gained in20％PRP group on7day, during the first6days,20%＞30%＞10%, and there was significantdifferences(P＜0.05). between7-10days,20%＞10%＞30%, and there wassignificant differences too(P＜0.05). There was no significant differencesamong different times of0%group, So the BMSCs proliferative effect wasclosely related to time and concentration of PRP, and the optimalconcentration was20%.⑤Concentration of PDGF-AB: The value ofPDGF-AB was reduced gradually, and little fluctuations were shown by theinfluence of autocrine and paracrine. However, a high concentration was stillmaitained in the first three days.⑥Fluorescence staining results: Nucleusfluorescence staining was blue, and cytoskeleton showed green with irregularshape around nucleus, which was corresponded with the osteoblastmorpology.Conclusion：①All of PRP in different concentrations(10％,20％and30％)could promote the proliferation of BMSCs effectively. The bestconcentration is20％.②High concentrations of growth factor PDGF-ABcould be obtained by two-step centrifugation method. The optimal concentration of growth factor PDGF-AB was18.66-24.30ng/ml, which wascorresponded to20%PRP.③The half-life of the growth factor PDGF-AB isshort, and the actions in the first three days were the best. |
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