BMP-2 Peptide Combined With Immune Regulation Relieves Osteolysis Induced By Titanium Particles

Part Ⅰ:Effect of BMP-2 peptide on osteolysis induced by titanium particlesObjective:Bone morphogenetic protein-2(BMP-2)is currently one of the strongest osteoinductive factors,But it is not easy to be stored and inactivated in vitro.Our study used BMP-2 peptides to see if it could promote osteoblast differentiation and inhibit osteoclast activation under the induction of titanium particles,thereby alleviating osteolysis induced by titanium particles.Methods:(1)Establish a mouse osteolysis model induced by titanium particles and give BMP-2 peptide local treatment.After 2 weeks,the skull of each experimental group was removed and analyzed by Micro-CT.(2)Human umbilical cord mesenchymal stem cells(UMSCs)and bone marrow-derived macrophages(BMMs)were intervened with titanium particles of different concentrations(0,0.025mg/ml,0.05mg/ml,0.1mg/ml,0.2mg/ml).Cell Counting Kit-8(CCK-8)measures the proliferation efficiency of two cells.(3)Seven days after osteogenesis induction,alkaline phosphatase staining(ALP)and real-time fluorescent quantitative PCR(qRT-PCR)was performed.Alizarin red staining was performed 14 days after osteogenesis induction under titanium particulate environment.(4)BMMs were detected by PCR after 3 days of osteogenesis induced by the addition of titanium particles,macrophage colony-stimulating factor(M-CSF)(30ng/ml)and NF-kB ligand receptor activator(RANKL)(50ng/ml).Tartrate-resistant acid phosphatase staining(TRAP)was performed 5 days after the same induction to compare the effects of osteoclast differentiation.Results:(1)Micro-CT showed cranial pits formed by the destruction of titanium particles in the control group,while the BMP-2 peptide treatment group reduced significantly.Quantitative analysis showed the same results for bone density(BMD)and bone volume/total volume(BV/TV).(2)0.1 mg/ml titanium particles had no effect on the proliferation efficiency of UMSCs and BMMs,and had no toxicity to cells,and could be used in subsequent experiments.(3)Compared with normal UMSCs,ALP staining and alizarin red staining and quantitative results showed that the osteogenesis of cells was weakened in the presence of titanium particles,but BMP-2 peptide could significantly inhibit the effects of titanium particles.(4)TRAP showed that titanium particles could promote osteoclast formation,BMP-2 peptide could inhibit to a certain extent,and osteoclast-related genes showed the same trend.Conclusion:BMP-2 peptide can promote the differentiation of osteoblasts,inhibit the activation of osteoclasts stimulated by titanium particles,and directly relieve osteolysis induced by titanium particles.Part Ⅱ:Effect of BMP-2 peptide on osteogenic differentiation of mesenchymal stem cells pretreated with titanium particlesObjective:The organization has number of mesenchymal stem cells involved in regeneration and tissue repair.The wear particles around the prosthesis can affect the activity of mesenchymal stem cells in this area,and then affect their osteogenic differentiation ability.This part explores the effect of BMP-2 peptide on the osteogenic differentiation of stem cells after pretreatment with titanium particles for a period of time.Methods:(1)Flow cytometry was used to detect the cell phenotype of normal UMSCs and UMSCs pretreated with titanium particles for 6 days,and CCK-8 was used to detect the cell proliferation efficiency.(2)Combined with BMP-2 peptide,ALP staining and PCR detection of osteogenesis-related genes were performed on UMSCs pretreated with titanium particles 7 days after osteogenesis induction.Alizarin red staining was performed 14 days after osteogenesis induction to compare the effects of osteogenic differentiation.Results:(1)There were no significant differences in the surface markers CD29,CD90,CD34,and CD45 between normal UMSCs and UMSCs pretreated with titanium particles.CCK-8 results show that UMSCs treated with titanium particles proliferate more slowly than normal UMSCs.(2)Compared with normal UMSCs cells,the osteogenic ability of UMSCs pretreated with titanium particles is reduced,and BMP-2 peptide can significantly improve this effect.Conclusion:Titanium particles have no effect on the surface dryness markers of UMSCs cells,but inhibit the cell’s proliferation and osteogenic differentiation ability.BMP-2 peptide can reduce the osteogenic differentiation ability of UMSCs induced by titanium particles.Part Ⅲ:Effect of BMP-2 peptide on macrophage polarization and its effect on osteoblast differentiationObjective:After being swallowed by macrophages,titanium particles will induce the release of a series of cytokines and inflammatory mediators related to osteolysis,and then affect osteoblast differentiation.This part explores the effects of BMP-2 peptides on the polarization of macrophages,and then explores the effects of polarized macrophages on the osteogenic differentiation of U-MSCs.Methods:(1)Mouse mononuclear macrophage leukemia cells(RAW264.7)were induced by titanium particles and BMP-2 peptide for one day,and the expression of polarization-associated genes was measured by real-time quantitative PCR.After induction for three days,flow cytometry was performed to detect relevant cell surface markers.(2)Collect RAW264.7 cell culture supernatant and prepare conditioned medium with osteogenic induction medium at a ratio of 1:2,UMSCs were subjected to osteogenic induction in conditioned medium for 7 days after ALP staining and PCR to detect osteogenesis related gene.Results:(1)Flow cytometry showed that the expression of M1 polarized molecule CD86 increased and the expression of M2 polarized molecule CD206 decreased in the surface of RAW264.7 cells treated with titanium particles.PCR showed that the BMP-2 peptide significantly inhibited the expression of inflammatory genes,while promoting the expression of anti-inflammatory genes,and promoted the polarization of RAW264.7 to M2.(2)The results of ALP staining and PCR showed that the osteogenic ability of UMSCs cells cultured in titanium particle conditioned medium was significantly weakened;conditioned medium added with BMP-2 peptide could promote osteogenic differentiation of UMSCs.Conclusion:BMP-2 peptide promotes the polarization of macrophages to M2 and indirectly relieves the osteogenic differentiation of UMSCs caused by titanium particles.