The Study On Suppression Effects Of Long-term Cigarette Smoking In Early Stages Of Fracture Healing And Its Mechanism

OBJECTIVESLong-term cigarette smoking is associated with a higher risk of nonunion and delayed union.Few specific interventions are available for prevention and treatment as the molecular mechanisms of cigarette smoking that result in these negative sequelae are poorly understood.Nowadays,various methods have been developed and used to investigate the mechanisms of smoking-related impaired fracture healing,including in vitro cell contact and in vivo animals treated with individual compounds such as cigarette smoke extract or nicotine.However,the exposure and resorption route of smoke in these ways is significantly different to the actual route in humans,which limits the generalizability of some conclusions.Murine models exposed to cigarette smoke have been widely used to investigate smoking-related lung diseases and demonstrate efficacy and reliability because they recapitulate the physiological absorption that occurs in humans with minimal alterations of the chemicals in the whole cigarette smoke aerosol.Nonetheless,studies of fracture healing with such models are limited.The determination of a suitable murine model that recapitulates the compromised fracture healing in smokers is crucial to further understand the related pathogenesis and potential interventions.Smoking harms nearly every organ of the body and affects a person's overall health.It is challenging to characterize the detailed mechanisms responsible for the impaired fracture healing capacity noted in smokers under such complicated systemic changes.Nevertheless,the fracture healing process has been widely investigated and characterized with three partially overlapping phases — inflammation,repair,and remodeling — regulated by a precise sequence of growth factors,cytokines,and cells.However,knowledge of fracture healing in the context of cigarette smoking is limited,and greater comprehension of the cellular and molecular changes during fracture healing in cigarette smoking may be critical for combating its negative effects.Insight into the local changes of the natural fracture healing process in cigarette smoking may help in finding critical local pathways and further enable the development of therapies to restore the reduced fracture healing capacity found in smokers.Thus,the aims of this investigation are(1)to construct long-term smoking murine models and determine a proper one that best recapitulates the depressed fracture healing phenotype and(2)to investigate the critical cellular and molecular characteristics in early stages of fracture healing in cigarette smoking and(3)preliminarily explore the potential biomolecular mechanisms for cigarette smoke-induced delayed fracture healing and further enable the development of therapies to restore the reduced fracture healing capacity found in smokers.METHODSConstruction of compromised fracture-healing murine models induced by long-term cigarette smoking1.C57BL/6,129 Sv J,and Balb/c mice were divided into sham-exposed control groups(NS)and cigarette smoke-exposed groups(CS).The smoke-exposed animals were subjected for 6 days a week for 3 months prior to surgery.The smoking regimen consisted of exposure to two standard research nonfiltered cigarettes generated by a modified whole-body cigarette smoke exposure system per day.The body weights,complete blood count,plasma cotinine levels,and multiplex cytokines levels of all mice were measured to check the effects of smoke exposure.2.Mice were subjected to surgical femoral fracture and continued the smoking regimen until the sacrifice at 4 weeks after surgery.Healing of fracture was assessed by radiography,Micro-CT,and biomechanical testing.Susceptibilities to the compromised fracture healing after exposure to cigarette smoke of three strains were confirmed and compared to determine the strain that best recapitulates the depressed fracture healing phenotype in smokers.The molecular and cellular characteristics in early stages of fracture healing in cigarette smoking1.The fractured femurs were harvested at day 7 and stained with H&E and Safranin O stains.Callus and cartilage sizes were quantified.2.Uninjured femurs from uninjured mice and dissected fracture calluses of post-fracture day 3,7 were harvested.SSCs(CD45-Ter-119-Tie2-Alpha V+Thy-6C3-CD105-)and BCSPs(CD45-TER119-Tie2-Alpha V+Thy-6C3-CD105+)were detected and analyzed by flow cytometry.3.Paraffin-embedded fractured femurs of day 7 post fracture were stained by multiplex immunofluorescence.Immunofluorescence with Ki67-and CD105-specific antibodies was used to detect actively proliferating BCSPs in fracture callus.4.Brd U was administered by intraperitoneal injection 12 hours prior to harvest on post-fracture day 3.Cells were processed as described above and were permeabilized,subjected to DNase,stained with Brd U antibody,and analyzed by flow cytometry.5.Uninjured femurs from NS and CS mice were harvested and used to isolate SSCs with FACS.The proliferation of SSCs isolated from NS or CS mice were detected and compared with CCK-8 assay.6.Fracture hematoma on 6 h and 24 h post fracture was carefully collected,respectively.Concentrations of total 32 inflammatory mediators were determined by multiplex cytokine/chemokine analysis.In addition,inflammatory cells in the fracture hematoma on 6 h after fracture and bone marrow from unfractured femurs were analyzed by flow cytometry.The cells and markers were as follows: neutrophils(Ly-6G+,F4-80-),inflammatory monocytes(Ly-6G+,F4-80+),macrophages(Ly-6G-,F4-80+),B cells(CD3-,CD19+),and T cells(CD3+,CD19+).7.The hematomas were collected in both smoking and non-smoking mice 24 hours post-fracture,digested,and centrifuged.The supernatants were labeled with NS-Hematoma and CS-Hematoma,and used to culture SSCs isolated from the entire femur.The proliferation of SSCs cultured in NS-Hematoma or CS-Hematoma were detected and compared with CCK-8 assay.Investigation of the potential biomolecular mechanisms for the suppressed SSC proliferation caused by cigarette smoking in the early stage of fracture healing1.Role of IL-6 Trans signaling in regulating SSC proliferation: Selectively inhibit IL-6 Trans signaling with sgp130 Fc and analyze SSC proliferation with CCK-8 assay.2.Role of STAT3 in regulating SSC proliferation:(1)Isolate SSCs from the calluses of post-fracture day 3 in NS and CS mice,and detect STAT3 levels with RT-PCR.(2)Culture the SSCs from uninjured femurs of NS mice in NS-Hematoma or CS-Hematoma,and detect STAT3 expressions with RT-PCR.(3)Specifically inhibit STAT3 in SSCs with AG490 or Ig G,and analyze their proliferations with CCK-8 assay.3.Role of IL-6 Trans signaling in regulating STAT3: Selectively inhibit IL-6 Trans signaling with sgp130 Fc and analyze STAT3 expressions with RT-PCR.4.Classify the mice into CS,NS,and CS+sgp130Fc groups.To selectively inhibit IL-6 Trans signaling,mice in CS+sgp130Fc group received 0.5 mg/kg sgp130 Fc 30 min and 48 h after osteotomy.Mice in other two groups received same amount of Ig G antibodies.5.Evaluate fracture healing 4 weeks following osteotomy with X-Ray,Micro-CT,and biomechanical analysis.6.The fractured femurs were harvested at day 7 and stained with Safranin O stains.Callus and cartilage sizes were quantified.7.Dissected fracture calluses of post-fracture day3,7 were harvested.SSCs and BCSPs were detected and analyzed by flow cytometry.Brd U was administered by intraperitoneal injection 12 hours prior to harvest on post-fracture day 3.Cells were processed as described above and were permeabilized,subjected to DNase,stained with Brd U antibody,and analyzed by flow cytometry.8.Isolate SSCs from the calluses of post-fracture day 3 in different groups,and detect STAT3 levels with RT-PCR.RESULTSSusceptibilities to the compromised fracture healing after exposure to cigarette smoke of three strains are variable,and 129X1/Sv J is close to the phenotype in smokers1.Following three months of active smoking,body weight in smokers was lower than in nonsmokers after 3 months of smoke exposure in both C57BL6/J and 129X1/Sv J mice.Smoking caused increased WBC in all three strains.Smoking mice showed a significant increase in the plasma cotinine levels in comparison non-smoking mice.The inflammatory marker CRP was not significantly changed between smoking and control mice in all three strains.But most proinflammatory cytokines and chemokines were increased in each smoking strain when compared to the corresponding non-smoking mice,indicating increased systemic inflammation in all three strains which is consistent with direct smoke exposure studies in human smokers.2.On inspection of X-ray imaging of both smoking and non-smoking mice,smoking 129X1/Sv J mice showed visible fracture line and discontinuous callus.RUST scoring of 129X1/Sv J determined a significant decrease in fracture healing capacity in smoking mice.Micro-CT results showed robust fracture healing with invisible fracture gap and continuous cortical bones in all non-smoking mice while impaired fracture healing with clear fracture gap and extensive trabecular bones is noted in all murine models of smoking.Flexural rigidity of fractured femurs from all cigarette smoking mice were significantly lower as compared to nonsmoking controls.Torsional testing demonstrates decreased rigidity of bones from cigarette smoking mice of all three strains and reduced maximal torque in only 129X1/Sv J smoking mice.Long-term smoking leads to enhanced initial inflammatory response that inhibits the proliferation and activity of skeletal stem / progenitor cells,which further inducing impaired chondrogenesis during fracture healing1.Fracture callus size did not significantly change on day 7 post-fracture,indicating that the fibrous tissues formation was not influenced by smoking.However,the cartilage area and its percentage in callus of femurs from smoking mice were markedly decreased as compared to non-smoking mice.2.Fracture stimulated the expansion of SSC and BCSP.The analysis of cells in uninjured femurs indicated that no difference in either SSC number or BCSP number was observed between smoking and non-smoking mice.However,as to the numbers of SSCs and BCSPs in callus at day 7 post-fracture,both were notably reduced in smoking mice compared to non-smoking mice.3.Multi-immunofluorescence analysis showed that the number of proliferating BCSP(double CD105+Ki67+ cells)decreased significantly in smoking mice.4.The proliferating SSCs in calluses at day 3 post-fracture were significantly reduced in smoking mice compared with non-smoking mice.5.SSCs isolated from uninjured femurs of NS or CS mice showed no significant difference in proliferation.6.Within the bone fracture–induced hematoma,smoking mice showed high concentrations of the proinflammatory cytokines IL-1?,IL-1?,IL-2,IL-6,IL-9,IL-15,and TNF-?,and KC,LIX,MIP-1?,MIP-1?,MIP-2,MCP-1,and RANTES 6 hours post fracture.Meanwhile,VEGF-A and G-CSF were significantly suppressed.Moreover,most of the increased mediators including IL-1?,IL-6,IL-9,IL-15,KC,LIX,MIP-1?,and MIP-2 remained at higher concentrations while the G-CSF and VEGF-A levels were still lower 24 hours post fracture in smoking mice compared to non-smoking mice.In addition,some other proinflammatory cytokines like IFN-? and IL-12p40,and proinflammatory chemokine IP-10 also showed increased levels while several mediators with anti-inflammatory functions including LIF and IL-13 were inhibited in the smoking group.Six hours after fracture,in the fracture hematoma,the number of infiltrated neutrophil granulocytes,macrophages,B cells,and T cells was significantly increased in smoking mice compared with non-smoking mice as revealed by flow cytometry.Additionally,the proportions of neutrophils,B cells,and T cells were also elevated even the monocytes showed a reduced occupation.7.CCK-8 test showed that the SSCs cultured in CS-Hematoma had suppressed proliferation.The activation of IL-6 Trans signaling upregulated the expression of STAT3 is the potential mechanism of cigarette smoking-inhibited SSC proliferation1.IL-6 Trans signaling regulated the proliferation of SSCs: sgp130Fc-pretreated SSCs showed significantly improved proliferation.2.STAT3 regulated the proliferation of SSCs:(1)SSCs isolated from callus of CS mice at day 3 post-fracture expressed higher levels of STAT3 in comparison with those isolated from callus of NS mice at day 3 post-fracture.(2)SSCs cultured in CS-Hematoma supernatant culture showed higher expression of STAT3 compared with those cultured in NS-Hematoma supernatant culture.(3)After the specific inhibition of STAT3,SSCs showed enhanced proliferation in CS-Hematoma supernatant culture.3.IL-6 Trans signaling regulated the expression of STAT3: After the selective inhibition of IL-6 Trans signaling,SSCs cultured in CS-Hematoma supernatant culture expressed decreased levels of STAT3.4.Selective inhibition of IL-6 Trans signaling rescued cigarette smoking-induced compromised fracture healing: On inspection of X-ray imaging of mice in three groups,mice in CS+sgp130Fc group showed significant callus formation and bridge,invisible fracture lines.RUST scoring of mice in CS-sgp130 Fc group determined a significant increase in fracture healing capacity compared with that in CS group.Micro-CT results showed robust fracture healing with invisible fracture gap and continuous cortical bones in both NS and CS+sgp130Fc mice while impaired fracture healing with clear fracture gap and extensive trabecular bones is noted in CS mice.Flexural rigidity of fractured femurs from CS+sagp130 Fc mice were significantly higher as compared to smoking controls but lower than normal controls.Torsional testing demonstrated same trend as showed in bending testing.5.Fracture callus size did not significantly change on day 7 post-fracture among three groups.However,the cartilage area and its percentage in callus of femurs from smoking mice were markedly decreased as compared to mice in other groups.6.The analysis of cells in calluses at day 3 post-fracture indicated that no difference in either SSC number or BCSP number was observed among three groups.However,as to the numbers of SSCs and BCSPs in callus at day 7 post-fracture,both were notably reduced in CS mice compared to mice in other groups.The proliferating SSCs in calluses at day 3 post-fracture were significantly reduced in CS mice compared with mice in other groups.7.SSCs isolated from callus of CS+sgp130Fc mice at day 3 post-fracture expressed lower levels of STAT3 in comparison with those isolated from callus of CS mice at day 3 post-fracture.CONCLUSIONS1.The way of constructing long-term smoking murine models in our study is valid and reliable.Smoking mice of three strains all showed delayed callus remodeling of fracture healing while 129 Sv J exhibited higher susceptibility to the compromised fracture healing and best recapitulated the depressed phenotype in smokers.2.With the 129 Sv J compromised fracture healing models induced by cigarette smoking,we first reported the critical cellular and molecular characteristics of fracture healing in cigarette smoking.cigarette smoking triggered enhanced initial inflammatory response that inhibited the proliferation and activity of skeletal stem / progenitor cells,and further causing deficient expansions of reparative cells in callus and impaired chondrogenesis,finally leading to poor fracture healing.3.In vitro and in vivo studies demonstrated that IL-6 Trans signaling was the critical molecular pathway in the cigarette smoke-induced inflammatory local response,which activated STAT3 in SSCs and inhibited the proliferation of these cells,further compromising chondrogenesis and fracture healing.This conclusion provides a potential molecular mechanism in explaining cigarette smoking-induced compromised fracture healing,and also a promising target for future studies as well as clinical interventions.