Effect Of Age-related Changes In Macrophage Polarization On Bone Regeneration

Part ⅠLipopolysaccharide-induced systemic inflammation affects bone healing in a murine tibia fracture modelObjective:The early stages of fracture repair require close coordination between the immune system and skeletal system. Conditions such as diabetes, rheumatoid arthritis, smoking, obesity and aging adversely affect bone healing. Common to all these conditions is a sustained inflammation. It has been shown that lipopolysaccharide-induced systemic inflammation results in fracture callus of inferior mechanical characteristics in a rat femur fracture model. The aim of this study was.to compare fracture healing in a murine tibia fracture model in the setting of LPS-induced systemic inflammation to that of control animalsMethods:Sustained inflammation was created by the injection of lipopolysaccharide intra-peritoneally (3μg/mouse/day).Control animals received equivalent volume of phosphate-buffered saline vehicle.Concurrently, diaphyseal tibial fractures were created and stabilized with intramedullary pins. Plasma and tibiae were collected on D1, D3, D7, D10, D14and D21.Plasma was isolated from whole blood by cadiac puncture and centrifuge at2,000xg for20minutes. All plasma samples were immediately stored at-20℃after isolation. IL-6levels were detected by ELISA. Prepared ELISA plates were read as suggested by the manufacturer. Concentrations were expressed in pg/mL. All samples were run in triplicate. Harvested limbs were fixed in4%paraformaldehyde and decalcified in EDTA for14days after which they were paraffin embedded and sectioned at5pm. Every tenth section was stained using a Trichrome stain. Adjacent sections were stained using the method of Hall and Bryant. Sections were then examined and tissue types were quantified by stereology. Calculated volumes were expressed in mm3. Mean volumes were compared using the student’s T-test with a significance level of p<0.05employed.Cab2T3and ADTC5cell lines were plated in standard12well plates.100μg/ml ascobic acid2-phosphate and5mM β-glycerophosphate were added into2%FBS a-MEM to induce osteogenesis.25μg/ml ascobic acid2-phosphate and1%ITS mixture were added into5%FBS a-MEM to induce chondrogenesis. Alizarin red and Alician blue were applied for access ostegenesis and chondrogenesis.Results:During the seven day injection period no behavioral differences were noted between treated and control animals. No symptoms of systemic illness were noted in either group. For the LPS group, the IL-6level rose shapely to concentration of209.9pg/mL24hours after fracture whereas the maximum IL-6level reached by controls was23.5pg/mL IL-6levels decreased in both groups to near-basal levels by post-fracture day3in controls and day7in the LPS-treated animals. At Day7the treatment group trended towards a smaller mean callus volume (Mean difference:15.5mm3; p=0.21). By Day10and14there was no difference in callus size between groups. The difference in callus size was primarily related to an increased volume of cartilage and undifferentiated tissue in control animals. Hall-Bryant quadruple stain confirmed the larger callus in control animals (Mean difference:17.4; p<0.076). The size difference noted was present in all tissue types suggesting an overall smaller callus, rather than a difference in one cell type. LPS inhibit both osteogenesis and chondrogenesis in vitro and related to LPS concentration.Conclusion:Sustained inflammation delayed the bone healing process, both quantity and quality of new bone was impaired. Part ⅡMacrophage plasticity and polarization plays an important role in bone healing processObjective:After a fracture occurs, the architecture and vascular supply of the bone are disrupted. This results in a loss of mechanical stability, a decrease in tissue oxygenation and nutrient supply, and the release of biological factors into the site of injury. During the inflammatory stage of repair, cells, including macrophages, neutrophils, and degranulating platelets, infiltrate to the fracture site and release cytokines. Macrophages exhibit varying phenotypes depending on the inflammatory environment in which they are located. In this study we use the CCR2-/-mice which could block macrophages immigrate to fracture site and Yet40and Yarg mice which is to understand the polarization during bone healing process according to the inflammation macroenvironment changes.Methods:Male,10week old Ccr2-/-mice were used in this study. Age-and gender-matched, congenic C57BL/6L mice were used as wild-type controls. Two lines of mice YET40and YARG is given as a gift by Dr.Richard Locksley.Tibial fractures were created by three-point bending. Harvested limbs were fixed in4%paraformaldehyde and decalcified in EDTA for14days after which they were paraffin embedded and sectioned at10μm. Every tenth section was stained using the method of Hall and Bryant. Sections were then examined and tissue types were quantified by stereology. Calculated volumes were expressed in mm3. Mean volumes were compared using the student’s T-test with a significance level of p<0.05employed.Total RNA were extracted from the fractured tibiae by D1,D3,D5,D7,D10and D14. cDNA synthesis and real-time PCR were proformed. Macrophages and neutrophils in the callus at D3after fracture were viasulized by immunostaining using macrophage-specific (F4/80or Mac-3) and neutrophile specific (MCA771G) antibodies.Ml and M2macrophage identified by anti-YFP antibody.Results:Results from this study prove that a lack of Ccr2affects early fracture healing. At3days after injury, Ccr2-/-mice had significantly fewer macrophages at the fracture site while a similar number of neutrophils were observed in both wild-type and mutant mice. At day7, the Callus in the Ccr2-/-mice were significantly smaller than the wild type mice (P=0.27),but there is no difference in total volume of new bone or cartilage in the callus.At day14,there was no difference in callus size but less new bone in Ccr2-/-mice.At day21,both callus size and volume of new bone remained large in the Ccr2-/-mice,but both were decrease in the wild type mice.For the Ml and M2profile study, we observed a large number of IL12p40-YFP positive cells and a lower number of Arg-1-YFP cells at the fracture site at day1.At Day7we observed a decrease in "M1"cells and an increase in "M2" cells.By using the real time PCR, we found that M2gene expression "disolves" rapidly and return back to normal level during the time course of bone fracture healing while the M1gene expression last relatively longer during the whole bone healing process.Conclusion:These results indicate that a deficiency of Ccr2reduces the infiltration of macrophages and impairs the bone fracture healing. The phenotype of macrophage shift from a pro-inflammation "M1" to an anti-inflammation "M2" during the bone healing process. Part ⅢAge-related changes in macrophage polarization affect osteogenesisObjective:Aging affects many cellular functions of macrophages, and macrophages exhibit various phenotypes depending on the inflammatory environment in which they are located. After injury macrophages exhibit a pro-inflammatory phenotype, termed M1, and then the macrophages polarize to an anti-inflammatory phenotype (M2). The M2phenotype is associated with stimulation of healing. We hypothesized that the phenotype and function of macrophages changes with age, and these changes would affect osteogenesis. Therefore, we developed a macrophage/pre-osteoblast co-culture system to assess the inter-reaction between these two cell types as a function of age.Methods:Bone marrow monocytes were isolated from mice of different age(4weeks,10weeks,6months,18months). Cells were differentiated into macrophages (BMM) in vitro. lOng/ml IFN-Y combined with lOng/ml LPS were introduced to the media to activate BMM to a pro-inflammatory phenotype (M1) and lng/ml IL-4was used to drive macrophages towards an anti-inflammatory phenotype (M2). Pro-inflammatory cytokines and anti-inflammatory cytokines were tested by ELISA from R&D Systems. M1marker Nitric oxide (NO) was assayed using the Giess reagent system and M2marker levels (YM1and FIZZ1) were quantified by Q-PCR.Macrophages of different phenotypes were then co-cultured with pre-osteoblast cell lines (Cab2t3) in three configurations.1) Standard co-culture2) macrophage-conditioned media and Cab2t3cells3) trans-well co-culture. We used qPCR to quantify ALP, Colla and osterix expression at different time points (D3, D7, D10) during osteoblast differentiation. Alizarin red staining was used to quantify mineralization and alkaline phosphatase was quantified to assess osteogenesisResults:1.BMM from aged mice secret higher levels of pro-inflammatory cytokines:IL-6levels in10weeks BMM and M2group were significance lower than18months BMM and M2group, while IL-10does not appear to be different. BMM from10weeks old mice secrete lower levels of NO and down-regulated gene expression of YM1and FIZZ1compared with BMM generated from18months old mice.2.Effect of different phenotype of macrophages on the bone cells differentiation:In10weeks and18months group, ALP, Coll a and osterix gene expression were up-regualted in M1groups by early time point but reduced in later time point. In M2group ALP, Colla and osterix gene expression were increased in10weeks group in all the time points but not significantly different in18groups.3.Effect of different phenotype of macrophages on the bone cells osteogenesis:In standard co-culture system:osteogenesis of Cab2T3cells did not show any difference between different age group. In condition media and Trans-well co-culture system, Cab2t3cells have more osteogenic nodules in M2group than Ml group from10weeks mice. However macrophages from18months mice, less nodules were detected in both M1and M2groups compare with BMM group.Conclusion:We demonstrate that macrophages from aged mice exhibit a more pro-inflammatory phenotype compare with macrophages from young adult animals. Our results suggest that osteogenesis may be impaired in inflammatory environments that do not resolve, while M2macrophages are beneficial for osteogenesis in adult animals. This is consistent with other conclusions that M2cells are good for tissue repair and regeneration. These data support our hypothesis and suggest that regulating macrophage polarity may be important for bone repair in aged patients.