Aims. Pellino1 (Peli1) has been reported to regulate various inflammatory diseases. This study aims to explore the role of Peli1 in the occurrence and development of osteoarthritis (OA), so as to find new targets for the treatment of OA. Methods. After inhibiting Peli1 expression in chondrocytes with small interfering RNA (siRNA), interleukin (IL)-1β was used to simulate inflammation, and OA-related indicators such as synthesis, decomposition, inflammation, and apoptosis were detected. Toll-like receptor (TLR) and nuclear factor-kappa B (NF-κB) signalling pathway were detected. After inhibiting the expression of Peli1 in macrophages Raw 264.7 with siRNA and intervening with lipopolysaccharide (LPS), the polarization index of macrophages was detected, and the supernatant of macrophage medium was extracted as conditioned medium to act on chondrocytes and detect the apoptosis index. The OA model of mice was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity to reduce the expression of Peli1. The degree of cartilage destruction and synovitis were evaluated by haematoxylin and eosin (H&E) staining, Safranin O/Fast Green staining, and immunohistochemistry. Results. In chondrocytes, knockdown of Peli1 produced anti-inflammatory and anti-apoptotic effects by targeting the TLR and NF-κB signalling pathways. We found that in macrophages, knockdown of Peli1 can inhibit M1-type polarization of macrophages. In addition, the corresponding conditioned culture medium of macrophages applied to chondrocytes can also produce an anti-apoptotic effect. During in vivo experiments, the results have also shown that knockdown Peli1 reduces cartilage destruction and synovial inflammation. Conclusion. Knockdown of Peli1 has a therapeutic effect on OA, which therefore makes it a potential therapeutic target for OA. Cite this article: Bone Joint Res 2023;12(2):121–132

Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Conclusion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing. Cite this article: Bone Joint Res 2023;12(3):219–230

Osteoarthritis, the most common degenerative joint disease, significantly impairs life quality and labor capability of patients. Synovial inflammation, initiated by HMGB1 (High mobility group box 1)-induced activation of macrophage, precedes other pathological changes. As an upstream regulator of NF-κB (nuclear factor-kappa B) and MAPK (mitogen-activated protein kinase) signaling pathway, TAK1 (TGF-β activated kinase 1) participates in macrophage activation, while its function in osteoarthritis remains unveiled. This study aims to investigate the role of TAK1 in the pathogenesis of osteoarthritis via both in vitro and in vivo approaches. We performed immunohistochemical staining for TAK1 in synovial tissue, both in osteoarthritis patients and healthy control. Besides, immunofluorescence staining for F4/80 as macrophage marker and TAK1 were conducted as well. TAK1 expression was examined in RAW264.7 macrophages stimulated by HMGB1 via qPCR (Quantitative polymerase chain reaction) and Western blotting, and the effect of TAK1 inhibitor (5z-7 oxozeaenol) on TNF-α production was evaluated by immunofluorescence staining. Further, we explored the influence of intra-articular shRNA (short hairpin RNA) targeting TAK1 on collagenase-induced osteoarthritis in mice. Immunohistochemical staining confirmed significant elevation of TAK1 in osteoarthritic synovium, and immunofluorescence staining suggested macrophages as predominant residence of TAK1. In HMGB1-stimulated RAW264.7 macrophages, TAK1 expression was up-regulated both in mRNA and protein level. Besides, TAK1 inhibitor significantly impairs the production of TNF-α by macrophages upon HMGB1 stimulation. Moreover, intra-articular injection of lentivirus loaded with shRNA targeting TAK1 (sh-TAK1) reduced peri-articular osteophyte formation in collagenase-induced osteoarthritis in mice. TAK1 exerts a potent role in the pathogenesis of osteoarthritis by mediating the activation of macrophages

Aseptic inflammation is the main factor causing aseptic loosening of artificial joints. Studies have shown that inflammatory cells can activate STING (stimulator of interferon genes, STING) after being stressed. This study aims to explore the specific mechanism of STING in aseptic loosening of artificial joints, and provide new strategies for disease prevention. Titanium particles with a diameter of 1.2-10 μm were prepared to stimulate macrophages (RAW 264.7) to simulate the periprosthetic microenvironment. A lentiviral vector targeting the STING gene was designed and transfected into macrophages to construct a cell line targeting STING knockdown. The expression and secretion levels of TNF-α were detected by qPCR and ELISA, the activation levels of inflammatory pathways (NF-κB, IRF3, etc.) were detected by western blot, and the nucleus translocation of P65 and IRF3 was observed by cellular immunofluorescence. After titanium particles stimulated macrophages, qPCR and ELISA showed that the transcription and secretion levels of TNF-α were significantly increased. Western blot showed that titanium particle stimulation could increase the phosphorylation levels of NF-κB and IRF3 pathways. While knockdown of STING can significantly reduce titanium particle-induced TNF production, attenuate the activation levels of NF-κB and IRF3 pathways as well as the nucleus translocation of P65 and IRF3. Conclusions: STING positively regulates the level of inflammation in macrophages induced by titanium particles, and targeted inhibition of STING can reduce inflammation, which may delay the progression of aseptic loosening of artificial joints

Aims. The use of 3D-printed titanium implant (DT) can effectively guide bone regeneration. DT triggers a continuous host immune reaction, including macrophage type 1 polarization, that resists osseointegration. Interleukin 4 (IL4) is a specific cytokine modulating osteogenic capability that switches macrophage polarization type 1 to type 2, and this switch favours bone regeneration. Methods. IL4 at concentrations of 0, 30, and 100 ng/ml was used at day 3 to create a biomimetic environment for bone marrow mesenchymal stromal cell (BMMSC) osteogenesis and macrophage polarization on the DT. The osteogenic and immune responses of BMMSCs and macrophages were evaluated respectively. Results. DT plus 30 ng/ml of IL4 (DT + 30 IL4) from day 3 to day 7 significantly (p < 0.01) enhanced macrophage type 2 polarization and BMMSC osteogenesis compared with the other groups. Local injection of IL4 enhanced new bone formation surrounding the DT. Conclusion. DT + 30 IL4 may switch macrophage polarization at the appropriate timepoints to promote bone regeneration. Cite this article: Bone Joint Res 2021;10(7):411–424

As peri-prosthetic aseptic loosening is one of the main causes of implant failure, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL to expand the lifespan of implant. Here, we aim at exploring the role of p110δ, a member of class IA PI3K family, and Krüppel-like factor 4 (KLF4) in titanium particles (TiPs) induced macrophages-inflammation and osteolysis. Firstly, IC87114, the inhibitor of p110δ and siRNA targeting p110δ were applied and experiments including ELISA and immunofluorescence assay were conducted to explore the role of p110δ. Sequentially, KLF4 was predicted as the transcription factor of p110δ and the relation was confirmed by dual luciferase reporter assay. Next, assays including RT-PCR, western blotting and flow cytometry were performed to ensure the specific role of KLF4. Finally, TiPs-induced mice cranial osteolysis model was established, and micro-CT scanning and immunohistochemistry assay were performed to reveal the role of p110δ and KLF4 in vivo. Here, we found that p110δ was upregulated in TiPs-stimulated macrophages. The inhibition of p110δ or knockdown of p110δ could significantly dampen the TiPs-induced secretion of TNFα and IL-6. Further mechanistic studies confirmed that p110δ was responsible for TNFα and IL-6 trafficking out of Golgi complex without affecting their expression in TiPs-treated macrophages. Additionally, we explored the upstream regulators and confirmed that Krüppel-like factor 4 (KLF4) was the transcription repressor of p110δ. Apart from that, KLF4, targeted by miR-92a, could also attenuate TiPs-induced inflammation by mediating NF-κB pathway and M1/M2 polarization. By the establishment of TiPs-induced mice cranial osteolysis model, we found that KLF4 knockdown exacerbated TiPs-induced osteolysis which was strikingly ameliorated by knockdown of p110δ. In summary, our study suggests the key role of miR-92a/KLF4/p110δ signal in TiPs-induced macrophages inflammation and osteolysis

Aims. Tert-butylhydroquinone (tBHQ) has been identified as an inhibitor of oxidative stress-induced injury and apoptosis in human neural stem cells. However, the role of tBHQ in osteoarthritis (OA) is unclear. This study was carried out to investigate the role of tBHQ in OA. Methods. OA animal model was induced by destabilization of the medial meniscus (DMM). Different concentrations of tBHQ (25 and 50 mg/kg) were intraperitoneally injected in ten-week-old female mice. Chondrocytes were isolated from articular cartilage of mice and treated with 5 ng/ml lipopolysaccharide (LPS) or 10 ng/ml interleukin 1 beta (IL-1β) for 24 hours, and then treated with different concentrations of tBHQ (10, 20, and 40 μM) for 12 hours. The expression levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured. The expression levels of interleukin 6 (IL-6), IL-1β, and tumour necrosis factor alpha (TNF-α) leptin in plasma were measured using enzyme-linked immunoabsorbent assay (ELISA) kits. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathway proteins, and macrophage repolarization-related markers, were detected by western blot. Results. Tert-butylhydroquinone significantly attenuated cartilage destruction in DMM-induced mice in vivo. It demonstrated clear evidence of inhibiting IL-1β-induced chondrocyte apoptosis, inflammation, and differentiation defect in vitro. Meanwhile, tBHQ inhibited LPS-induced activation of NF-κB and MAPK signalling pathways, and also inhibited LPS-induced reactive oxygen species production and macrophages repolarization in vitro. Conclusion. Taken together, tBHQ might be a potential therapeutic strategy for protecting against OA development. Cite this article: Bone Joint Res 2021;10(11):704–713

Sarcopenia is an age-related geriatric syndrome which is associated with subsequent disability and morbidity. Currently there is no promising therapy approved for the treatment of sarcopenia. The receptor activator of nuclear factor NF-κB ligand (RANKL) and its receptor (RANK) are expressed in bone and skeletal muscle. Activation of the NF-κB pathway mainly inhibits myogenic differentiation, which leads to skeletal muscle dysfunction and loss. LYVE1 and CD206 positive macrophage has been reported to be associated with progressive impairment of skeletal muscle function with aging. The study aims to investigate the effects of an anti-RANKL treatment on sarcopenic skeletal muscle and explore the related mechanisms on muscle inflammation and the polarization status of macrophages. Sarcopenic senescence-accelerated mouse P8 (SAMP8) mice at month 8 were treated intraperitoneally with 5mg/kg anti-RANKL (IK22/5) or isotype control (2A3; Bio X Cell) antibody every 4 weeks and harvested at month 10. Senescence accelerated mouse resistant-1 (SAMR1) were collected at month 10 as the age-matched non-sarcopenic group. Ex-vivo functional assessment, grip strength and immunostaining of C/EBPa, CD206, F4/80, LYVE1 and PAX7 were performed. Data analysis was done with one-way ANOVA, and the significant level was set at p≤0.05. At month 10, tetanic force/specific tetanic force, twitch force/specific twitch force in anti-RANKL group were significantly higher than control group (all p<0.01). The mice in the anti-RANKL treatment group also showed significantly higher grip strength than Con group (p<0.001). The SAMP8 mice at month 10 expressed significantly more C/EBPa, CD206 and LYVE1 positive area than in SAMR1, while anti-RANKL treatment significantly decreased C/EBPa, CD206 and LYVE1 positive area. The anti-RANKL treatment protected against skeletal muscle dysfunctions through suppressing muscle inflammation and modulating M2 macrophages, which may represent a novel therapeutic approach for sarcopenia. Acknowledgment: Collaborative Research Fund (CRF, Ref: C4032-21GF)

The Masquelet technique is a variable method for treating critical-sized bone defects, but there is a need to develop a technique for promoting bone regeneration. In recent studies of bone fracture healing promotion, macrophage-mesenchymal stem cell (MSC) cross-talk has drawn attention. This study aimed to investigate macrophage expression in the induced membrane (IM) of the Masquelet technique using a mouse critical-sized bone defect model. The study involved a 3-mm bone defect created in the femur of mice and fixed with a mouse locking plate. The Masquelet (M) group, in which a spacer was inserted, and the Control (C) group, in which the defect was left intact, were established. Additionally, a spacer was inserted under the fascia of the back (B group) to form a membrane due to the foreign body reaction. Tissues were collected at 1, 2, and 4 weeks after surgery (n=5 in each group), and immunostaining (CD68, CD163: M1, M2 macrophage markers) and RT-qPCR were performed to investigate macrophage localization and expression in the tissues. The study found that CD68-positive cells were present in the IM of the M group at all weeks, and RT-qPCR showed the highest CD68 expression at 1 week. In addition, there was similar localization and expression of CD163. The C group showed lower expression of CD68 and CD163 than the M group at all weeks. The B group exhibited CD68-positive cells in the fibrous capsule and CD163-positive cells in the connective tissue outside the capsule, with lower expression of both markers compared to the M group at all weeks. Macrophage expression in IM in M group had different characteristics compared to C group and B group. These results suggest that the IM differs from the fibrous capsules due to the foreign body reaction, and the macrophage-MSC cross-talk may be involved in Masquelet technique

Objectives. Up to 10% of fractures result in undesirable outcomes, for which female sex is a risk factor. Cellular sex differences have been implicated in these different healing processes. Better understanding of the mechanisms underlying bone healing and sex differences in this process is key to improved clinical outcomes. This study utilized a macrophage–mesenchymal stem cell (MSC) coculture system to determine: 1) the precise timing of proinflammatory (M1) to anti-inflammatory (M2) macrophage transition for optimal bone formation; and 2) how such immunomodulation was affected by male versus female cocultures. Methods. A primary murine macrophage-MSC coculture system was used to demonstrate the optimal transition time from M1 to M2 (polarized from M1 with interleukin (IL)-4) macrophages to maximize matrix mineralization in male and female MSCs. Outcome variables included Alizarin Red staining, alkaline phosphatase (ALP) activity, and osteocalcin protein secretion. Results. We found that 96 hours of M1 phenotype in male cocultures allowed for maximum matrix mineralization versus 72 hours in female cocultures. ALP activity and osteocalcin secretion were also enhanced with the addition of IL-4 later in male versus female groups. The sex of the cells had a statistically significant effect on the optimal IL-4 addition time to maximize osteogenesis. Conclusion. These results suggest that: 1) a 72- to 96-hour proinflammatory environment is critical for optimal matrix mineralization; and 2) there are immunological differences in this coculture environment due to sex. Optimizing immunomodulation during fracture healing may enhance and expedite the bone regeneration response. These findings provide insight into precise immunomodulation for enhanced bone healing that is sex-specific. Cite this article: K. Nathan, L. Y. Lu, T. Lin, J. Pajarinen, E. Jämsen, J-F. Huang, M. Romero-Lopez, M. Maruyama, Y. Kohno, Z. Yao, S. B. Goodman. Precise immunomodulation of the M1 to M2 macrophage transition enhances mesenchymal stem cell osteogenesis and differs by sex. Bone Joint Res 2019;8:481–488. DOI: 10.1302/2046-3758.810.BJR-2018-0231.R2

Background. Mechanisms underlying implant failure remain incompletely described, though the presence of macrophage-mediated inflammatory reactions is well documented. Hypoxia has a critical role in many diseases and is known to be interdependent with inflammation. Metals used for joint replacements have also been reported to provoke hypoxia-like conditions. In view of this, we aim to investigate hypoxia-associated factors in aseptic loosening and osteoarthritis with a focus on macrophages. Methods. Western blotting, calorimetric assay, haematoxylin-eosin staining, immunohistochemistry, double-immunofluorescence and transmission electron microscopy were performed on capsular tissue obtained from patients undergoing primary implantation of a total hip replacement for osteoarthritis and from patients undergoing revision surgery for aseptic loosening to investigate the presence of hypoxia-associated factors. Results. Tissues from patients with osteoarthritis and aseptic loosening showed the presence of inflammatory cells, many of which were macrophages as confirmed with CD68 immunostaining. In aseptic loosening, macrophages containing metal particles were present in clusters. This was observed both at the light and electron microscopic levels. Under the electron microscope, endothelial cells appeared to be hypertrophied and some showed signs of degeneration. The presence of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and nitric oxide was demonstrated by western blotting and colorimetric assay. Macrophages were the predominant cell type to release HIF-1α, VEGF, inducible nitric oxide synthase (iNOS). This was confirmed by double-immunofluorescence showing co-localization of HIF-1α, VEGF, iNOS with the macrophage marker CD68. Endothelial cells were stained for endothelial nitric oxide synthase as assessed by immunohistochemistry. Conclusion. This study demonstrates the release of hypoxia-associated factors by macrophages. The presence of hypoxia-associated factors in both, osteoarthritis and aseptic loosening suggest that hypoxia may be a factor underlying both pathologic conditions. This study was supported by research grant (NMRC/CNIG/1147/2016) from National Medical Research Council (NMRC), Singapore

Human synovium harbours macrophages and T-cells that secrete inflammatory cytokines, stimulating chondrocytes to release proteinases like aggrecanases and matrix metalloproteinases (MMPs) during the development of Osteoarthritis (OA). Inflammation of the synovium is a key feature of OA, linked to several clinical symptoms and the disease progression. As a prelude to testing in an OA mouse model, we have used the tetracycline system (Tet) to modify mouse mesenchymal stem cells (mMSCs) to over-express viral interleukin 10 (vIL10), an anti-inflammatory cytokine, to modulate the osteoarthritic environment and prevent disease development. MSCs isolated from the marrow of C57BL/6J mice expressed CD90.2, SCA-1, CD105, CD140a, and were negative for CD34, CD45 and CD11b by flow cytometry. Adenoviral transduction of MSCs carrying CMVIL10 and TetON as test, and untransduced, AdNull and TetOFF as negative controls was successful and tightly controlled vIL10 production was demonstrated by CMVIL10 and TetON MSCs using a vIL10 ELISA kit. Co-incubation of vIL10MSC CM with lipopolysaccharide activated bone-marrow derived murine macrophages (BMDMs) resulted in reduction of TNF-α, IL-6 levels and elevated production of IL-10 by ELISA and high iNOS release by Griess assay. Co-culture of active macrophages with TetON MSCs, resulted in polarisation of macrophage cell population from M1 to M2 phase, with decrease in pro-inflammatory MHC-II (M1 marker) and increase in regulatory CD206 (M2 marker) expression over time. The PCR profiler array on MSC CM treated BMDMs, also showed changes in gene expression of critical pro-inflammatory cytokines and receptors involved in the TLR4 pathway. The biscistronic TetON transduced MSCs proved to be most immuno-suppressive and therefore feasible as efficient anti-inflammatory therapy that can utilised in vivo

Introduction. The combined incidence of anatomic (aTSA) and reverse total shoulder arthroplasties (rTSA) in the US is 90,000 per annum and rising. There has been little attention given to potential long-term complications due to periprosthetic tissue reactions to implant debris. The shoulder has been felt to be relatively immune to these complications due to lower acting loads compared to other joint arthroplasties. In this study, retrieved aTSAs and rTSAs were examined to determine the extent of implant damage and to characterize the nature of the corresponding periprosthetic tissue responses. Methods. TSA components and periprosthetic tissues were retrieved from 23 (eleven aTSA, twelve rTSA). Damage to the implants was characterized using light microscopy. Head/stem taper junction damage was graded 1–4 as minimal, mild, moderate or marked. Damage on polyethylene (PE) and metal bearing surfaces was graded 1–3 (mild, moderate, marked). H&E stained sections of periprosthetic soft tissues were evaluated for the extent and type of cellular response. A semi-quantitative system was used to score (1=rare to 4=marked) the overall number of particle-laden macrophages, foreign body giant cells, lymphocytes, plasma cells, eosinophils, and neutrophils. Implant damage and histopathological patterns were compared between the two TSA groups using the Mann-Whitney and Spearman tests. Results. The PE bearing surfaces of aTSAs were dominated by three-body wear and plastic deformation, whereas the rTSA PE components exhibited mainly polishing and scratching. Metal surface damage occurred in a few cases of both groups. Only one aTSA case exhibited marked taper corrosion. In both groups the primary nature of the inflammatory response was a moderate to marked macrophage response to wear particles (78% of cases). The particle-laden macrophages tended to occur in broad sheets and contained metal, PE, bone cement and suture debris. The extent of macrophage and foreign body giant cell responses was greater in the aTSA group (p≤0.001). Metal particles were seen in 63% of aTSAs and 83% of rTSAs. In the aTSA group, bone cement was seen in all cases and suture was observed in 9 cases, and their presence was larger compared to the rTSA group (p≤0.022). There was no difference in the number of other cell types between the groups. A mild lymphocyte response and chromium-phosphate debris was present within the tissue of the aTSA case with marked corrosion, which may be indicative of an early stage adverse local tissue reaction (ALTR) analog to total hip replacements with taper corrosion. Conclusion. Both groups exhibited a strong macrophage response to a combination of different types of implant debris—PE, metal, bone cement and suture. The prevalence of a marked macrophage response was larger in the aTSA group which may be explained by the larger overall presence of cement and suture within this group. PE particles may differ in size between groups due to different acting wear mechanisms which may also affect the extent of the macrophage response. Although corrosion within modular junctions was overall rare, the presence of one case with marked corrosion shows that taper corrosion and subsequent ALTRs are possible in TSAs. For any figures or tables, please contact authors directly

All types of regenerative materials, including metal implants, porous scaffolds and cell-laden hydrogels, interact with the living tissue and cells. Such interaction is key to the settlement and regenerative outcomes of the biomaterials. Notably, the immune reactions from the host body crucially mediate the tissue-biomaterials interactions. Macrophages (as well as monocytes and neutrophils), traditionally best known as defenders, accumulate at the tissue-biomaterials interface and secrete abundant cytokines to create a microenvironment that benefits or inhibits regeneration. Because the phenotype of these cells is highly plastic in response to varying stimuli, it may be feasible to manipulate their activity at the interface and harness their power to mediate bone regeneration. Towards this goal, our team have been working on macrophage-driven bone regeneration in two aspects. First, targeting the abundant, glucan/mannan-recognising receptors on macrophages, we have devised a series of glucomannan polymers that can stimulate macrophages to secrete pro-osteogenic cytokines, and applied them as coating polymer of mesenchymal stem cells-laden hydrogels. Second, targeting the toll-like receptors (TLRs) on macrophages, we have screened TLR-activating polysaccharides and picked up zymosan (beta-glucan) to be modified onto titanium and glass implants. We evaluated both the efficacy of integration and safety of immune stimulation in both in vitro and in vivo models. Our future exploration lies in further elaborating the different roles and mechanisms of macrophages of various types and origins in the regenerative process

It has been previously shown that Low-Magnitude High-Frequency Vibration (LMHFV) is able to enhance ovariectomy-induced osteoporotic fracture healing in rats. Fracture healing begins with the inflammatory stage, and all subsequent stages are regulated by the infiltration of immune cells such as macrophages and the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10). Therefore, the aim of this study was to investigate the effect of LMFHV treatment on the inflammatory response in osteoporotic fracture healing. In this study, ovariectomy-induced osteoporotic and sham-operated closed-femoral fracture SD-rats were randomized into three groups: sham control (SHAM), ovariectomized control (OVX-C) or ovariectomized vibration (OVX-V) (n=36, n=6 per group per time point). LMHFV (35Hz, 0.3g) was given 20 min/day and 5 days/week to OVX-V group. SHAM operation and ovariectomy were performed at 6-month and closed femoral fracture was performed at 9-month. Callus morphometry was determined by callus width from weekly radiography. Local expressions of inducible nitric oxide synthase (iNOS) (macrophage M1 marker), CD206 (macrophage M2 marker), TNF-α, IL-6 and IL-10 were detected by immunohistochemistry and quantified by colour threshold in ImageJ, assessed at weeks 1 and 2 post-fracture. Significant difference between groups was considered at p≤0.05 by one-way ANOVA. Callus formation was higher in OVX-V than that of OVX-C as shown by callus width at weeks 1 and 2 (p=0.054 and 0.028, respectively). Immunohistochemistry results showed that CD206 positive signal and the M2/M1 ratio which indicates the progression of macrophage polarization were significantly higher in OVX-V rats (p=0.053 and 0.049, respectively) when compared to OVX-C at week 1. Area fraction of TNF-α positive signal was significantly higher in SHAM and OVX-V rats at week 1 (p=0.01 and 0.033, respectively). IL-6 signal was also significantly higher in SHAM and OVX-V groups at week 1 (p=0.004 and 0.029, respectively). IL-10 expression was significantly lower in SHAM and OVX-V groups at week 1 (p=0.013 and 0.05, respectively). Here we have shown that LMHFV treatment promoted the shift from pro-inflammatory stage towards anti-inflammatory stage earlier. It has been reported that the polarization of pro-inflammatory macrophages M1 to anti-inflammatory macrophages M2 was indicative of the endochondral ossification process in the long bone fracture model. Besides, we found that LMHFV treatment enhanced pro-inflammatory markers of TNF-α and IL-6 and suppressed anti-inflammatory marker of IL-10 at week 1, showing that inflammatory response was enhanced at week 1 post-fracture. These inflammatory cytokines involved in fracture healing were shown to coordinate different fracture healing processes such as mesenchymal stem cell recruitment and angiogenesis. Our previous study has demonstrated that ovariectomized rats exhibit lower levels of inflammatory response after fracture creation. Therefore, we report that LMHFV treatment can modulate macrophage polarization from M1 to M2 at an earlier time-point and partly restore the impaired inflammatory response in OVX bones at the early stage of fracture healing that may lead to accelerated healing of osteoporotic fracture as shown by promoted callus formation

Although osteoarthritis (OA) is characterized by articular cartilage damage, synovial inflammation is a prominent feature contributing to disease progression. In addition to synovial tissue resident macrophages, infiltrating macrophages and monocytes, their lineage precursors, may also contribute to pathological processes. In mice, peripheral blood monocytes may be categorized according to pro-inflammatory/classical and patrolling/non-classical subsets. The aim of this study was to identify profiles of peripheral blood monocyte subsets as well as different synovial macrophage phenotypes during disease development. OA was induced in knees of C57BL/6 mice by destabilization of the medial meniscus (DMM). Blood was harvested from the facial vein 7 days prior to and 1, 7, 14, 28, and 56 days post induction of OA. Separate mice were sham-operated as a control. Monocyte subsets and synovial macrophage populations were identified by flow cytometry. Levels of classical monocytes were significantly higher at day 14 (p<0.001) and day 28 (p=0.031) in peripheral blood of DMM-operated mice compared to control. Furthermore, the percentage of non-classical monocytes was significantly lower in DMM-mice at day 14 (p=0.026). At day 56 post OA-induction, an increase in total synovial macrophages (CD11b+F4/80+ cells) was observed between DMM and sham operated knees (p=0.021). The ratio between pro-inflammatory (CD11b+F4/80+CD86+) and tissue repair (CD11b+F4/80+CD206+) synovial macrophage subsets tended to be higher in DMM knees, however this finding was not statistically significant (p>0.05). In light of the present findings, further investigation is required to elucidate the relationship of peripheral blood monocyte subsets to synovial inflammation and features of OA pathogenesis

It is reported that more than 10 million Japanese suffer from arthrosis. To cure these cartilage defects, total joint replacements, which are the most popular treatment methods for severe disease situation, have been operated as about two hundred thousand cases a year in Japan. Although the implants made of either ceramics, metals or plastics have high wear resistance quality, it becomes apparent that the endurance life of the artificial joints in considerable cases is limited by aseptic loosening to between 10–15 years. Here we focused on a poly(vinyl alcohol) (PVA) hydrogel as an artificial cartilage tissue to make an improvement of friction surface of the artificial joints. In this paper, we observed morphology of wear particles and assessed immune responses of wear particles from the hydrogel for confirming the validity of the gel as a biotribological material. We prepared 20 w/w% of PVA hydrogel by repeated freezing-thawing method. The number of the freezing-thawing cycles was five times. Polymerization degree and saponification degree of PVA (Kishida Chemical Co. Ltd., Japan) were 2000 and 98.4–99.8 mol%, respectively. To collect the wear particles of PVA hydrogel, we processed wear testing by using a purpose-build wear test machine of reciprocating pin-on-plate tribometer as shown in figure 1. We installed a Co-Cr-Mo ball of 26 mm in diameter as a stationary upper specimen and a PVA hydrogel plate of 2 mm in thickness as reciprocating lower specimen in a water bath. The lubricant was a distilled water containing eluted PVA which PVA-FT gel had been soaked in, filtered by 0.22 μm and autoclaved, subsequently. Siding speed was 50 mm/s and the total sliding distance was 3 km. We observed the wear particles which had been dried in a desiccator, by scanning electron microscope (SEM; SU8000, Hitachi High-Technologies). Additionally, to investigate the effect of the wear particles on response of phagocytosis of macrophages, here we used THP-1 cell line from Human acute monocytic leukemia as a macrophage, which was purchased from JCRB Cell Bank, and attached the macrophages on a dish after stimulating THP-1 by phorbol 12-myristate 13-acetate (PMA; Wako). After the wear test, we harvested the lubricant in aseptic clean hood and applied the particles to the macrophages to clarify the effect of wear particles of PVA hydrogel on immune response of the cell. To assess cytokine biosynthesis as immune responses, we assayed IL-1β and TNF-α biosynthesis in culture medium by ELISA (Thermo scientific), respectively. Figure 2 shows an SEM image of PVA hydrogel after wear test under 4.9 N loading. We observed the wear particles of varied sizes. When applying the wear particles to the macrophages in RPMI-1640 supplement with 10 v/v% fetal bovine serum, it seemed that there were not enough change on cytokine synthesis in culture medium between with/without the particles

Introduction. Periprosthetic joint infection (PJI) and particle-induced osteolysis are closely related to peri-implant local immunity and macrophage function. We previously demonstrated that titanium particles attenuate the immune response of macrophages caused by chronic inflammation [1]. In a separate study, we have determined that UHMWPE wear particles containing vitamin E (VE) induce less osteolysis compared to HXL UHMWPE wear particles in a murine calvarium model [2]. For this study we hypothesized that macrophages exposed to HXL UHMWPE particles containing VE would better maintain their ability to respond to S. aureus compared to HXL UHMWPE without VE. Methods. A gamma-sterilized, HXL UHMWPE tibial bearing containing VE (E1, Biomet, “VE-PE”) and 100kGy irradiated and melted UHMWPE (“CISM 100”) were cryomilled to particles by Bioengineering Solutions (Oak Park, IL). In the first in vitro study, RAW 264.7 mouse macrophages were exposed (inverted co-culture) to either VE-PE particles or CISM100 particles and lipopolysaccharide (LPS) for 1–7 days. Macrophage viability was measured using a cell counting kit (CCK-8). Control group with no particles and a LPS group were also included. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to determine macrophage apoptosis rate in response to particle exposure over time. In the second study, macrophages were exposed to VE-PE or CISM100 particles for 48h, then exposed to LPS for 30 min. Subsequently, reactive oxygen species (ROS) generation and extracellular regulated protein kinase (ERK) phosphorylation were measured. In a third study, after exposure to particles for 48h, fatigued macrophages were co-cultured with bioluminescent S. aureus strain Xen29 for 3h and 6h. Bioluminescence signal was determined to measure the total amount of bacteria. Bacterial live/dead staining and optical density at 600 nm (OD 600) were also performed to determine S. aureus viability. Statistical analysis was performed using one-way or two-way ANOVA with a post hoc examination. *indicates p<0.05. Results. CISM100 particles significantly decreased macrophage viability at day 5 and day 7 (p<0.05, Fig. 1A), while the viability of macrophages exposed to VE-PE particles was similar to controls (macrophages not exposed to particles). After 48h, macrophages exposed to VE-PE particles showed a lower TUNEL-positive rate (less apoptosis) compared to CISM100 particles (Fig. 1B, C). 48h-exposure to VE-PE particles increased ROS generation and ERK phosphorylation in 30 min-LPS-activated macrophages when compared to CISM100 particles (Fig. 2). This immune response caused by VE-PE particles resembles that of macrophages without particles. Furthermore, 48h exposure to E1 particles showed less S. aureus at 6h (Fig. 3). Conclusions. These results suggest that VE-PE particles cause reduced macrophage apoptosis and protect the macrophages' immune response. VE-PE particles also preserved the innate immunity of macrophages, unlike CISM100, as evidenced by the S. aureus co-culture study. Thus, patients with vitamin-E containing implants may be less likely to develop PJI

Mechanical load is crucial to maintaining skeletal homeostasis, but the pathways involved in mecha-notransduction are still unclear. The OPG/RANK/ RANKL triumvirate has recently been implicated in bone homeostasis. These molecules, which are produced by the osteoblast (OPG and RANKL) and the macrophage/osteoclast (RANK), modulate osteoclastogenesis. We have previously shown that cyclical hydrostatic pressure influenced synthesis of various molecules by cultured human macrophages. These factors are important in osteoclastogenesis and bone resorption and have been linked to the development of aseptic loosening. We have also demonstrated that 1,25-dihydroxyvitamin D3 (1,25D3) influences macrophage response to pressure. For this study human macrophages were co-cultured with osteoblasts and subjected to cyclical hydrostatic pressure (34.5x10–3MPa [5.0 psi]) for up to five days, with or without 1,25D3 supplementation. Cells were immunostained for RANK and culture media were assayed for sRANKL and OPG using specific ELISAs. Immunostaining for RANK showed that macrophages subjected to pressure or 1,25D3 supplementation synthesised more RANK than controls. In addition, when exogenous 1,25D3 and hydrostatic pressure were administered simultaneously, immunostaining for RANK was more intense. There was a reciprocal relationship between OPG and sRANKL in co-cultures subjected to pressure. If pressure increased synthesis of sRANKL, OPG was decreased. In cultures where pressure decreased sRANKL, a corresponding increase in OPG was seen. In addition, samples from different individuals responded differently to pressure. The majority of cell populations responded to pressure by increasing OPG synthesis, compared to non-pressurised controls. These results demonstrate for the first time that the OPG/RANK/RANKL complex is sensitive to hydrostatic pressure and that 1,25-dihydroxyvitamin D3 might be involved in this response. These findings suggest a possible transduction mechanism for mechanical load in the skeleton, which has implications for future therapies for aseptic loosening and for skeletal abnormalities such as osteoporosis

This study presents the use of precision surface machining on artificial joint bearing surfaces in order to inhibit macrophage activation. Ultra-high molecular weight polyethylene (UHMWPE) is widely used as a bearing material in polymer-on-hard joint prostheses. However, UHMWPE wear particles are considered to be a major factor in long-term osteolysis and implant loosening. Several studies report that wear particle size is a critical factor in macrophage activation, with particles in the size range of 0.1 – 1.0 μm being the most biological active. The surface for a conventional Co-Cr-Mo alloy joint implant generally has a 10.0 – 20.0 nm roughness. After precision machining, the Co-Cr-Mo alloy surface had a 1.0 – 2.0 nm roughness with scattered concave shapes up to 50 nm in depth. This precision surface machining method used a typical lapping method, but the relationship between the slurry and the machining surface was strictly controlled in order to emphasize the micro-erosion mechanism. A pin-on-disc wear tester capable of multidirectional motion was used to verify that the new surface was the most appropriate for joints. Tests were carried out in 25% (v/v) fetal calf serum with sodium azide to retard bacterial growth. UHMWPE pins, 12.0 mm in diameter with a mean molecular weight of 6.0 million, were placed on the Co-Cr-Mo alloy disc at a contact pressure of 6.0 MPa. A sliding speed of 12.1 mm/s, and a total sliding distance of 15.0 km were applied. The new surface reduced the amount of UHMWPE wear, which would ensure the long-term durability of joints. The new surface also enlarged the size of UHMWPE particles, but did not change their morphological aspect. Primary human peripheral blood mononuclear phagocytes were cultured with the particles. The wear particles generated on the new surface inhibited the production of IL-6, which indicates a reduction of induced tissue reaction and joint loosening