Mechanism Of N-MWCNTs Enhancing Bone Remodeling Through Immunomodulatory Functions

Carbon nanotubes(CNTs)are one-dimensional tubular nanomaterials with graphite sheets coiled into cylinders.CNTs can be divided into single-walled carbon nanotubes(SWCNTs)and multi-walled carbon nanotubes(MWCNTs)according to the number of coiled graphite lamellae.Compared with SWCNTs,MWCNTs have several advantages,such as no catalyst required for synthesis,accumulate at high purity,exhibit greater stability than single walled CNTs,and are relatively easy to synthesize in bulk.MWCNTs can reportedly positively affect growth and differentiation of bone-related cells and therefore offer great potential in biomedical applications.However,MWCNTs are prone to induce moderate to high biotoxicity due to their large diameters,which poses a substantial obstacle for their clinical application.In order to overcome the biotoxicity limiting the application of CNTs,proper surface functionalization modification and the doping of biocompatible composite materials can reduce the toxicity,thereby enhancing the water solubility and biocompatibility of CNTs.Recent studies have brought attention to modified MWCNTs for their therapeutic potential in immunomodulation and tissue engineering.Functionalized MWCNTs can be internalized into cells primarily via phagocytosis,resulting in demonstrably enhanced immune responses and immunomodulation.In addition,modified MWCNTs may serve as an attractive strategy for use in bone tissue engineering.However,to incorporate nanocarbons into bone tissue engineering,it is first essential to systematically understand their effects on bone remodeling associated cells.Although several kinds of MWCNTs have been reported to exert regulatory effects on growth or differentiation of bone-related cells and new bone regeneration,the underlying mechanisms remain to be further clarified.Further investigation of their underlying mechanisms is important to support N-MWCNTs as potential biomaterials for the treatment of metabolic bone diseases in animals.The main research contents are as follows:1.Characterization and safety evaluation of nitrogen-doped carboxy-functionalized multiwalled carbon nanotubes(N-MWCNTs)To obtain MWCNTs with lower toxicity,better water solubility and better biocompatibility,N-MWCNTs were prepared.Scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray photoelectron spectroscopy(XPS)and Ultraviolet-visible spectroscopy(UV-Vis)were used to characterize the morphology,microstructure,element composition and other physical and chemical properties of N-MWCNTs.The biocompatibility of N-MWCNTs in vitro and in vivo was also verified by flow cytometry and mouse models.The results showed that N-MWCNTs prepared in this study showed clear lattice fringes and unique nozzle structure characteristics,uniform morphology,good dispersion,high purity.Moreover,it showed good biocompatibility in both human cell lines and mouse models,which can be used for subsequent studies on the biological function of N-MWCNTs.2.N-MWCNTs enhance bone remodeling through immunomodulatory functionTo investigate the regulatory effects of N-MWCNTs on Bone remodeling,we used Bone Marrow Stromal Cells(BMSCs)as cell models to investigate the effects of N-MWCNTs on osteoclast and osteoblast activities in BMSCs.The results showed that,0.2 μg/m L N-MWCNTs significantly increased the activities of tartrate-resistant acid phosphatase(TRAP)and alkaline phosphatase(AKP)in BMSCs.Alizarin red S(ARS)staining and quantitative detection of ARS content further confirmed the positive regulation of N-MWCNTs on the mineralization of BMSCs in vitro.To systematically explore which genes and signaling pathways are affected by N-MWCNTs,RNA-Seq was performed on BMSCs incubated with N-MWCNTs at 0.2 μg/m L for 8 days.N-MWCNTs promoted m RNA expression of osteoclast differentiation and functionrelated genes(RANKL,NFATc1,PU.1,MITF,TRAP,and β3-intergrin)and osteoblast differentiation and function-related genes(BMP2,BMP4,Runx1,Runx3,OPN,and BSP).Further studies by RNA interference assay(RNAi)showed that NFATc1 and BMP2 were involved in the regulation of osteoclast and osteoblast activity of BMSCs mediated by NMWCNTs.RNA-Seq data showed that in addition to promoting the m RNA expression of bone remodeling related genes in BMSCs,N-MWCNTs also triggered significant changes in the m RNA expression levels of inflammation-related factors.Since phagocytosis of macrophages initiates the innate immune response and induces the transcription of various pro-inflammatory factors(such as TNF-α,IL1,IL6,IL12,etc.)through toll-like receptors(TLRs),it is speculated that N-MWCNTs may trigger phagocytosis of macrophages and further activate the bone immune effect of macrophages to enhance bone remodeling.In order to further clarify that the above changes in immune response and m RNA expression levels of several inflammatory cytokines induced by N-MWCNTs were initiated by macrophage phagocytosis,the phagosome localization of N-MWCNTs in macrophages was confirmed by confocal laser scanning microscopy(CLSM)imaging and TEM imaging.To explore whether N-MWCNTs can also induce macrophage bone immune effect to enhance bone remodeling in vivo,the effect of intramuscular injection of N-MWCNTs at a dose of 1.0 mg/kg body weight on bone metabolism in vivo was studied.Animal tests showed that the femoral and tibial bone indexes were significantly increased after the administration of NMWCNTs.Dual-energy X-ray absorptimeter imaging showed that the femurs and tibia of mice treated with N-MWCNTs were stronger than those of mice treated with Vehicle.Similarly,NMWCNTs increased bone mineral density(BMD)and bone mineral content(BMC)in mice.The activities of TRAP and AKP were enhanced in the protein extracts of femur and tibia in the N-MWCNTs group.In addition,N-MWCNTs induced enhanced TRAP and ARS positive staining of mouse femur.Fluorescence quantitative results showed that N-MWCNTs significantly increased the m RNA expression levels of the genes related to bone remodeling in the femur and tibia(BMP2,Runx2,Runx3,COLⅡ,OCN,NFATc1 and TRAP).TEM imaging of the mouse injection site revealed macrophage-like cells with internalized N-MWCNTs,confirming the internalized uptake of N-MWCNTs by macrophages in muscle tissue.Fluorescence quantitative results showed that N-MWCNTs significantly increased the m RNA expression levels of inflammatory cytokines in the femur and tibia of mice.In addition,the serum levels of IL-1α,IL10 and IL16 were significantly increased after N-MWCNT administration.To explore whether N-MWCNT can relieve the symptoms of osteoporosis,the osteoporosis model of female Kunming white adult mice was established by bilateral ovarectomy,and the mice were treated with N-MWCNTs 8 weeks after the operation.The effects of N-MWCNTs on the morphological characteristics of cancellous bone of proximal femur in osteoporosis model were observed by Micro CT analysis.The results showed that,compared with the blank group(sham operation group),the cancellous bone of proximal femur in the operating Vehicle group showed significant degenerative changes,such as decreased bone mass,decreased bone density,decreased number,thinning and spacing of bone trabeculae,and the transformation from plate to rod.Compared with the Vehicle group,the symptoms of osteoporosis were relieved after the intervention of N-MWCNTs,the number of Trabecular bone number(Tb.N),Trabecular bone thickness(Tb.Th)and BMD were significantly increased,while Trabecular bone pattern factor(Tb.Pf)was significantly decreased.In conclusion,this study revealed several major findings surrounding the effects of NMWCNTs on bone remodeling and development.First,N-MWCNTs exhibit good biocompatibility both in vitro and in vivo,supporting their safety as a potential therapeutic nanomaterial.Second,N-MWCNTs are recognized as foreign agents by macrophages in the innate immune system and induce phagocytosis,thereby enhancing the immune response.These cytokines are accompanied by secretion of pro-inflammatory and anti-inflammatory cytokines,which together induce m RNA expression of genes associated with differentiation and function of osteoclasts and osteoblasts.Thus,the activity of osteoclasts and osteoblasts is up-regulated,bone resorption and bone formation are stimulated,and bone remodeling is enhanced in vitro and in vivo.Finally,a mouse model of osteoporosis was successfully established,which confirmed that the administration of N-MWCNTs could alleviate the changes in the morphological characteristics of cancellous bone.These results reveal that NMWCNTs enhanced the regulatory role of bone remodeling through the bone immune effect of macrophages.Therefore,N-MWCNTs may be bone tissue engineering biomaterials for metabolic bone diseases in animals.