Nicotine Impairs Fracture Healing By Inhibiting The Recruitment Of Bmscs And Promoting IL-22 Secretion

BackgroundCigarettes was formed by about 4,000 compounds.Nicotine was an important ingredient and the main addictive substance of cigarettes.It was also the major component of cigarette substitutes such as nicotine patches,nicotine gum,and ecigarettes.There wa a large amount of reports about the effects of nicotine on osteoblasts,osteoclasts,bone homeostasis,etc.It was interesting that although in vivo experiments had confirmed that nicotine could led to fracture healing disorders,bone density declines,but some In vitro experiments showed that nicotine had no effect or even promoted the differentiation and osteogenic function of osteoblasts at low concentrations.Therefore,the mechanism by which nicotine interferes with the normal healing of fractures was still a worthy topic for further discussion.Bone marrow mesenchymal stem cells(BMSCs)was a group of cells with various differentiation potentials in the bone marrow.When the fracture was happened,BMSCs was recruited and differentiated into osteoblasts at the fracture site,which was one of the most important sources of osteoblasts during the fracture healing process.When the homing of BMSCs was disturbed,fracture healing could be suppressed.Whether nicotine could interfere with the homing of BMSCs to the fracture site and the mechanism of tha was still unclear.Interleukin-22(IL-22)was a member of the IL-10 cytokine subfamily and highly expressed in ectopic bone formation,erosive arthritis,ankylosing spondyloarthropathy,and ossified fibrous dysplasia.The progression of the disease could be slowed by IL-22 neutralizing.The high expression of IL-22 around the implant in the oral cavity was closely related to implant osseointegration.These studies suggested that IL-22 may play an important role in bone formation and reconstruction.Bone formation and bone remodeling were the key to fracture healing,but there was little research on the effects of nicotine on IL-22.This study was intended to research the effects of nicotine on the proliferation and differentiation of BMSCs,the mechanism of nicotine affecting the ability of BMSCs recruitment,the expression of IL-22 in bone,and its role in osteogenic osteoclast interaction,and the effect of nicotine on IL-22 by the three experiments bellow.Experiment Ⅰ:Effect of nicotine on proliferation,differentiation and migration of bone marrow mesenchymal stem cells in vitroOBJECTIVE:To explore the effects of low concentration nicotine on the proliferation,differentiation and migration of BMSCs in vitro.METHODS:BMSCs were stimulated with different concentrations of nicotine,and cell proliferation was measured by CCK-8 method.The osteogenic induction medium containing different concentrations of nicotine was cultured for 21 days,and the amount of mineralized nodules was semi-quantitatively detected by alizarin red staining to evaluate the effect of nicotine on the osteogenic differentiation of BMSCs.Scratch test and Transwell chamber migration test were used to examine the effect of nicotine on the migration ability of BMSCs.RESULTS:BMSCs proliferation was not affected by nicotine at low concentrations,but promoted at a concentration of 1 m M,BMSCs proliferation strongly inhibited at 10 m M and caused cell death.The osteogenic differentiation of BMSCs was not affected by nicotine at low concentrations but only at high concentrations(1 m M).Nicotine inhibited the migration of BMSCs whitch was positively correlated with concentration.In the concentration range from 1 μM to 100 μM nicotine did not affect the osteogenic capacity of BMSCs but significantly inhibited migration.Conclusion:There was a bimodal effect on the proliferative capacity in vitro by nocotine.It did not affect the proliferation and the osteogenic differentiation of BMSCs at low concentrations.Osteogenesis was inhibited and leads to cell death at high concentrations.The migration of BMSCs was inhibited by nicotine whitch was positively correlated with concentration.Experiment Ⅱ:The mechanism of recruitment of BMSCs affected by nicotineOBJECTIVE:To explore the pathological mechanism of BMSCs homing to fracture sites,the molecular mechanism of nicotine on the migration ability of BMSCs,and the effect of nicotine on the ability of osteoblasts to secrete chemokines.METHODS:Activated or inactivated bone fragments were used in co-culture systerm with BMSCs,and the recruitment of BMSCs was observed.Nicotine was added to observe the effect of nicotine.MC3T3-E1 cells and BMSCs were stimulated with different concentrations of nicotine to detect the m RNA expression levels of CXCR4,CXCR7,MMP-8 and MMP-13 in BMSCs,SDF-1 in MC3T3-E1 cells,POSTN and Collagen I expression was detected by Western bolt in MC3T3-E1 cells.The mouse femur fracture model with subcutaneous injection of nicotine was constructed with special internal fixation stainless steel plate nails.The specimens were taken after surgery for 2 weeks and 5 weeks.The healing status of each group were analyzed by Micro CT.The unilateral mouse femur fracture model was constructed while BMSCs from EGFP mice were transplanted intracavitally.One week later,small animals imaged system was used to observe the homing of BMSCs to the fracture site.The immunofluorescence staining of the tissue sections was performed to observe the accumulation of BMSCs in the fracture area.Results:Bone fragments containing live osteoblasts have the ability to attract BMSCs to migrate.Nicotine inhibits the accumulation of BMSCs into the fracture fragments in vitro.Nicotine inhibited the secretion of SDF-1 and POSTN by osteoblasts,and inhibited the expression of chemokine receptors CXCR4 and CXCR7 in BMSCs,and the expression of MMP-8 and MMP-13 did not change significantly.Micro-CT threedimensional reconstruction images after 2 and 5 weeks of fracture model showed that the control group healed significantly better than the nicotine group 2 weeks after surgery.Quantitative data from Micro-CT showed that the new bone mass in the control group was significantly higher than that in the nicotine-stimulated group(P < 0.05).7-day small animal in vivo imaging and immunofluorescence staining results suggest.EGFP+BMSCs recruited by nicotine-treated mouse fracture ends were significantly reduced.Conclusion:The recruitment of BMSCs to osteoblasts was inhibited with nicotine by inhibiting the secretion of important chemokine SDF-1 and extracellular matrix POSTN from osteoblasts and inhibiting the expression of CXCR4 and CXCR7 in BMSCs.The healing of femoral fractures in mice was delayed by nicotine and the recruitment of BMSCs in the fracture area was inhibited.Experiment Ⅲ:Nicotine promoted the secretion of IL-22 by osteoblasts to regulate osteoblasts-osteoclast interaction.OBJECTIVE:To explore the expression of IL-22 in osteoblasts,the role and mechanism of IL-22 in osteogenic osteoclast interaction,and the effect of nicotine on IL-22 expression.METHODS:The expression of IL-22 in trabecular bone was determined by immunofluorescence,immunohistochemical staining and immunofluorescence double staining.After cultured in osteogenic induction medium for 13 days,the m RNA and protein in MC3T3-E1 cells were detected for ALP and IL-22,IL-22 BP expression level.IL-22 expression in osteoblasts with nicotine stimulation was exanimated.The function of IL-22 and nicoting in the Osteoblast-Osteoclast interaction was exanimated by Osteoblast-Osteoclast co-culture system.Neutralization IL-22 and nicotine stimulation was added in the system.RESULTS:Immunofluorescence and immunohistochemistry showed a large number of IL-22+ cells found in the epiphyseal plate and subchondral bone.Immunofluorescence double staining showed that there were a large number of ALP and IL-22 double positive cells on the trabecular bone surface.Real-time PCR results showed that IL-22 was expressed in MC3T3-E1 cells.ALP and IL-22 expression were increased by osteogenic induction.Western blot analysis showed that IL-22 secretion was increased while IL-22 BP secretion was reduced by osteogenic induction.The secretion of IL-22 in osteoblasts was promoted by nicotine.In the osteoblast-osteoclast co-culture system,osteoclast formation was promoted by nicotine,and IL-22 neutralization could significantly reduce the number of osteoclasts generated in the blank control group and nicotine stimulation group.Conclusion:IL-22 expression was increased as pre-osteoblast differentiation matures.In osteogenic osteoclast interactions,osteoclast formation was promoted by IL-22.Nicotine promotes the secretion of IL-22 by osteoblasts and promotes the formation of osteoclasts in the osteogenic osteoclast co-culture system.In summary:The migration of BMSCs was inhibited by low-concentration nicotine,and the ability of osteoblasts to recruit BMSCs was reduced,while little effect on the proliferation and osteogenesis of BMSCs was detected.The ability of BMSCs homing from the distal end to the fracture site was also inhibited by nicotine which led to bone healing delay.In this study,the secretion of IL-22 was found in osteoblasts,and IL-22 was found to be involved in osteoblastic-osteoclast interaction.Nicotine promoted the secretion of IL-22 by osteoblasts and destroyed the bone-forming osteoclast balance,which may be the effect of nicotine.Another important factor in fracture healing.