Monocyte-macrophage Response And Polarization Behavior To RhBMP-2 Induced Inflammation And Ectopic Osteogenesis Related Study In Mice

Objective: To validate and clarify the relationship between different doses of exogenous BMP-2 application with inflammatory response and monocyte-macrophage polarization behavior after biomaterial implantation in vivo.To elucidate the effect of perioperatively locally using of dexamethasone on high dose of BMP-2 induced inflammatory response and osteogenesis.To provide experimental evidence for the more effective use of rhBMP-2 growth factor in clinic.Methods: In this study,without and with different doses of BMP-2 loading absorbable gelatin sponge biomaterials were implanted into the mouse dorsal subcutaneous area to produce aseptic inflammatory response and ectopic osteogenesis animal model.88 male mice of 8 weeks old were randomly assigned into following four experimental groups.Blank absorbable gelatin sponges without loading BMP-2(0μg BMP-2),low dose of BMP-2(20μg BMP-2),high dose of BMP-2(50μg BMP-2)and high dose of BMP-2 + 40μg dexamethasone locally using group(50μg BMP-2 + 40μg dexamethasone)respectively.There were 19 mice in Each group.Mice were anesthetized with 1% pentobarbital sodium0.15 ml intraperitoneally injection and killed by cervical dislocation at 2 days,4 days,1week,2 weeks and 4 weeks after implantation.Different doses of BMP-2 loaded material implantation induced local inflammatory and osteogenetic reactions were investigated by diverse characterization approachs including flow cytometry,ELISA,RT-PCR,histologic--al HE staining,immunofluorescence staining,living body imaging,general observationof explanted samples,micro-CT scanning and histological Masson trichrome staining.Results: When compared to the blank control the inflammatory response induced by different doses of BMP-2 was dose-dependent.The statistical data of inflammatory response obtained by flow cytometry,ELISA and RT-PCR showed significant statistical differences(P <0.05 or P <0.01)at different time points after implantation.Low dose of BMP-2 induced relatively gentle inflammatory response while high dose of BMP-2remarkably led accumulation of more inflammatory cells to the implantation area,more monocyte-macrophages polarizing into M1 and M2 subtypes,higher production of pro-inflammatory(IL-1β,IL-6,TNF-α)and anti-inflammatory(IL-10,VEGF-α)cytokines on the implanted region and higher gene expression of both types of inflammatory cytokines so that caused stronger cellular immune responses.These were obvious at the early stage of implantation(2,4 days after implantation).At the same time,this study found that locally using of dexamethasone almost has no inhibitory or preventional effect on the high dose of BMP-2 induced chemotactic efficacy to macrophages.However,in the early phase(2 days after implantation)of high dose of BMP-2 induced inflammation locally using of dexamethasone significantly reduced the production and expression of pro-inflammatory cytokines(IL-1β,IL-6,TNF-α)that indicated characteristcs of M1macrophage’s response.On the contrary,the production and expression of anti-inflammatory cytokines(IL-10,VEGF-a)that of M2 macrophage response were not negatively affected at different time points studied(2,4 and 7 days after implantation).In addition,ectopic osteogenesis related results showed that BMP-2 induced osteogenesis is also dose-dependent.However,along with the time passing,high dose of BMP-2 induced more obvious bone destruction and bone absorption of newly formed bone.Intraoperatively locally application of appropriate dose of dexamethasone significantly inhibited the bone destruction and bone absorption of newly formed bone induced by the high dose of BMP-2 at the later osteogenesis.In contrast,low-dose of BMP-2 induced osteogenesis were showed more stablity.Conclusion: Different doses of BMP-2 can cause different degrees of inflammatory reaction,can induce both M1 and M2 phenotypic macrophage polarization and related cytokine’s expression and production so that pro-inflammatory and anti-inflammatory effects are coexisted.showing a double-edged blade like characteristics.These are related with the application doses of BMP-2,the greater the dose the more obvious this phenome--non.Lower doses of BMP-2 application may be more beneficial to reduce the aseptic in--flammatory response,more conducive to bone regeneration and repair after bone tissue engineering biomaterial implantation.Intraoperatively locally administration of dexamethasone with proper dosage is not only beneficial to reduce the strong inflammatory response caused by high dose of BMP-2 use but also beneficial to reduce the degrees of bone destruction and bone absorption at the later stage of osteogenesis,can be a eligible option.The more precise mechanism associated with this study needs to be further investigated in the future.