Effect Of Parathyroid Hormone-related Protein/polylactide-co-glycolide(PTHrP/PLGA) Composite Microspheres On Proliferation And Differentiation Of Osteoclasts

Objective: To explore the effect of parathyroid hormone-related peptide/polylactide-co-glycolide(PTHrP/PLGA)composite microspheres on the proliferation and differentiation of osteoclasts,and to further elucidate the theoretical basis for the proliferation and differentiation of osteoclasts by PTHrP,it provides a new idea for promoting the degradation of calcium phosphate bone repair materials.Methods: This experiment consists of two parts:1.Preparation of PTHrP-loaded PLGA microsphere complex and in vitro release of PTHrPThe PLGA blank microspheres were prepared by double emulsion-solvent evaporation method.The surface morphology of PLGA microspheres was observed by SEM scanning electron microscopy.The average particle size was calculated.The PLGA empty microspheres were extracted by different concentrations of PTHrP solution,and the surface was prepared by freeze drying.PLGA microspheres with effectiveconcentration of PTHrP were prepared by BCA kit method,and the protein standard curve of protein standard was detected by microplate reader.The protein standard curve of different groups of microspheres was calculated according to the standard curve,and the release curve was drawn and the optimal was selected.The concentration of PTHrP released was proceeded to the next experiment.2.Study on Proliferation and differentiation of osteoclastst promoting effect of PTHrP in vitroOsteoclasts were isolated and cultured from rat bone marrow whole bone marrow cells by differential adherence method.Cell morphology and TRAP staining were used to identify osteoclasts.The effect of PTHrP protein on the osteogenic activity of osteoclast precursor cells was investigated by CCK-8 method.The effect of PTHrP protein on osteoclasts of osteoclasts was studied.RT-PCR was used to detect the concentration of different PTHrP for 7d and 9d.Differential expression of RANK mRNA,CK mRNA,Acp5 mRNA and NFATC1 mRNA in osteoclasts.The expression levels of osteoclastin in OC precursor cells were detected by Western blot(WB)assay after different concentrations of PTHrP to induce osteoclast precursor cells for 7d and 9d.Statistical analysis was performed using SPSS 20.0 statistical software.Results: 1.The PLGA empty microspheres prepared by the W/O/W double emulsion solvent volatilization method are spherical or ellipsoidal with a round surface and an average particle size of 56.7±7.2 um.2.The in vitro release of PTHrP showed that the release rate of protein was faster in the first 100 h,then PTHrP entered a slow release period,and the sustained release of PLGA microspheres encapsulatingPTHrP could reach more than 300 h.The percentage of PTHrP released in the first 100 h was about 63.1% of the total release.By the end of the release,the percentage of PTHrP released was about 87.3% of the total release.3.The results of anti-tartaric acid acid phosphatase staining showed that TRAP-positive osteoclasts were observed in both groups after induction culture for 7 days.The morphology of osteoclasts was observed under light microscope;the cell body was large,the shape was irregular,and there were red or deep red stained particles in the cytoplasm,multinuclear,pseudopodia and protuberance.The number of osteoclasts promoted by the PTHrP culture group(experimental group)was significantly higher than that of the blank group(P <0.01).4.The results of CCK-8 showed that the blank control group,PLGA empty group and PLGA carrier protein group had latency(0-1 days)after inoculation,exponential growth period(3-7 days),and gradual period(7-9).There was no statistically significant difference in cell proliferation between the three groups at different time points(P>0.05).5.The results of RT-PCR showed that the relative expression levels of the same osteoclast marker proteins RANK,CK,Acp5 and NFATc1 mRNA were consistently and stably expressed after adding different concentrations of PTHrP for 7 and 9 days,which was dose-dependent with PTHrP concentration.All the patients were higher than the blank group(P<0.05).At the same time point,the difference between the two groups in the PTHrP group was statistically significant(P<0.05).The 9d group was slightly more than the 7d group.Raise.6.The results of WB showed that RANK protein expression wasincreased at the same time point(P<0.05).There was an increasing trend in expression,but it was not statistically significant,P > 0.05.CK protein expression: At the same time point,the expression of RANK protein was increased in the PTHrP-added group compared with the blank group(P<0.05).There was no significant difference between the two groups in different doses of PTHrP-added group(P>0.05)..Conclusion: 1.The PTHrP-PLGA sustained-release system prepared by freeze-drying method has a sustained release effect and the sustained-release micro-microspheres can be successfully prepared by W/O/W double emulsion solvent evaporation method.2.In the scope of this study,PTHrP promotes The ability of OC precursor cells to OC into differentiation,and the promotion effect is positively correlated with the concentration of PTHrP.