| Objective: As a source of growth factor, Platelet-rich plasma(PRP) andbone marrow stromal stem cells(BMSCs) build bone tissue engineeringaround the implant together, which gets more and more people’s attention.BMSCs enter the process of osteoblast transformation, and can be used as seedcells in bone tissue engineering. Type I collagen is an organic matter secretedby osteoblast, it is the most basic matrix for composition of bone structure andformation of biomechanics of bone strength. In our experiment, BMSCs ofrabbit were cultured and treated with autologous PRP in vitro, in order toinvestigate the influence of PRP in proliferation and differentiation of BMSCsand the expression of type I collagen of osteoblast. The aim is to explore themechanism of PRP and to provide an experimental evidence about thereasonable application time of PRP for clinic.Methods:①Culture of BMSCs: Primary BMSCs of the rabbits isobtained from the whole bone marrow were cultured in vitro. The methods toobtain BMSCs are as below:2to3-month New Zealand white rabbits wereselected for experiment, and anesthetized by injecting sodium pentobarbitalinto the ear vein. The whole bone marrow cells were obtained by aspiratingwith16#bone marrow needle from bone marrow cavity of tibial plateau of therabbits. The extracted bone marrow (4-6ml) were seeded into flasks. Cellswere harvested and generated when they were attached up to70~80%. Then,the morphology of the primary cells and generated cells were observed everyday under inverted phase contrast microscope.②Drawing the growth curve ofcells: The third generation of BMSCs were seeded in24-well plates andcounted under the microscope during1to10days. Then, the cell growth curvewere drawn.③P reparation of PRP: PRP was isolated from the rabbit’s bloodby two-step centrifugation method. The amounts of platelet in the blood and PRP were compared by electronic equipment respectively. The steps in detailare as follows: Experimental animals were anesthetized as before andseparated carotid artery, then100ml whole blood were obtained via the carotidartery. At first, all the supernatant and2-3mm of the red blood cell surfacewere collected after centrifuging at1100r/min for10minutes, then, further2200r/min for10minutes, two-thirds of the supernatant were discarded, andthe remaining liquid is PRP, which was activated with bovine thrombin.④Identify 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 alizarin red staining.⑤Divided into groups: Treatmentgroup: The fourth generation of BMSCs were cultured with serum freecompletely DMEM including20%PRP and mineralized induced liquid;Control group: The fourth generation of BMSCs were cultured with serumfree completely DMEM including mineralized induced liquid; Blank controlgroup: The fourth generation of BMSCs were cultured with complete DMEMwithout serum. Each repeated three times.⑥Assay of Osteoblast proliferation:MTT assay of proliferation: At1,4,7,10day, all the wells of treatment groupand control group were collected, discarded medium, added MTT reagent, andincubated at37℃for4hours, then added DMSO. OD value of each specimenwere measured in the microplate reader after crystallization was uniformlydissolved.⑦A ssay of osteoblast alkaline phosphatase activity:To assay theactivity of ALP of treatment group and control group according to alkalinephosphatase kit steps at4,7,10,14d respectively.⑧A ssay oftype I collagenexpression of osteoblast: The expression of type I collagen of treatment groupand control group were observed with Western blot at4,9,14,19,24d.Results:①T he cellmorphology under inverted microscope: The shapeof the primary cell was polygon, ellipse or short-spindle, and80%cells werefused in12days, the typical cell was long-spindle, spindle or paralleluniformly at this moment, the cell body was smaller than before, processedmore apparent, increased extracellular matrix, cell boundary was not clear, bump was more apparent and arranged in cluster shape, school of fish orwhirlpool. What’s more, the subculture cells were more uniform in shape thanprevious generations. During passage culture, the incubation period isgenerally1-2days, cell proliferation is not obvious, the logarithmic growthphase is after3days that cell proliferate significantly faster, then cellproliferation becomes slowly in8days and enter the plateau period.②Identifyof osteoblast: The result of Alkaline phosphatase staining and Calciumnodules alizarin red staining were positive, which showed cells have thecapacity of osteogenic differentiation. In alkaline phosphatase staining, thenuclei are purple and beige or deep black granule or flake precipitated can beobserved in the cytoplasm. In calcium nodules alizarin red staining, theorange-red calcium nodules can be found clearly. All of aboves showed thatthe cultured BMSCs have osteogenetic differentiation ability, and can beinduced into osteoblast.③Detection of PRP: The amount of platelet in PRPgained by two-step centrifugation method was869±29×109/ml, which was4.2times of that(207±41×109/ml) in the blood.④MTT assay of proliferation:The test result in the treatment group is significantly higher than the controlgroup at each time point. The highest OD value was gained in treatment groupon4day, and there were significant differences (P<0.05). There was nosignificant differences among different times of control group. So the BMSCsproliferative effect was closely related to the time of PRP.⑤Assay of alkalinephosphatase activity: The test result in the treatment group is significantlyhigher than the control group at each time point. The highest OD value wasgained in treatment group on10day, and there were significant differences(P<0.05). There was no significant differences among different times ofcontrol group. So the BMSCs differentiation ability was closely related to timeof PRP.⑥Expression of type I collagen: the expression of treatment group arehigher than those in the control group, the peak expression of type I collagenwas found in treatment group on9day, and there were significant differences(P<0.05). So the type I collagen expression of osteoblast was closely related totime of PRP. Conclusions:①20%PRP could effectively promote the proliferation ofBMSCs, and the strongest effect of promote proliferation is on the7th day.②H igh concentrations of growth factor could be obtained in PRP by two-stepcentrifugation method.20%PRP could effectively promote the osteoblastdifferentiation, and the strongest effect of promote differentiation is on the10th days.③20%PRP could significantly improve the level of expression ofosteoblast type I collagen, and the highest expression level is on the9th day. |
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