Particle-induced Bone Research Articles

Matrix-bound nanovesicles alleviate particulate-induced periprosthetic osteolysis.

Aseptic loosening of orthopedic implants is an inflammatory disease characterized by immune cell activation, chronic inflammation, and destruction of periprosthetic bone, and is one of the leading reasons for prosthetic failure, affecting 12% of total joint arthroplasty patients. Matrix-bound nanovesicles (MBVs) are a subclass of extracellular vesicle recently shown to mitigate inflammation in preclinical models of rheumatoid arthritis and influenza-mediated "cytokine storm." The molecular mechanism of these anti-inflammatory properties is only partially understood. The objective of the present study was to investigate the effects of MBV on RANKL-induced osteoclast formation in vitro and particulate-induced osteolysis in vivo. Results showed that MBV attenuated osteoclast differentiation and activity by suppressing the NF-κB signaling pathway and downstream NFATc1, DC-STAMP, c-Src, and cathepsin K expression. In vivo, local administration of MBV attenuated ultrahigh molecular weight polyethylene particle-induced osteolysis, bone reconstruction, and periosteal inflammation. The results suggest that MBV may be a therapeutic option for preventing periprosthetic loosening.

Oxysophocarpine attenuates inflammatory osteolysis by modulating the NF-κb pathway and the reactive oxygen species-related Nrf2 signaling pathway.

Inflammatory diseases often result in bone loss due to persistent inflammation, which activates osteoclasts and increases bone resorption. Oxysophocarpine (OSC), a bioalkaloid extracted from the roots of Sophora japonica and other leguminous plants, has neuroprotective and anti-tumor properties. However, it is still uncertain whether OSC can effectively inhibit the differentiation of osteoclasts and bone resorption. Therefore, this study explored the potential role of OSC in osteoclast formation and inflammatory osteolysis and its underlying mechanisms. This study involved inducing primary mouse bone marrow macrophages (BMMs) into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) and examined the effects of OSC on osteoclast (OC) differentiation, function, and intracellular reactive oxygen species (ROS) production. The impact of OSC on the expression of osteoclast-specific genes and inflammation-related factors was assessed using real-time quantitative PCR. Additionally, changes in oxidative stress-related factors, NF-κB, and MAPK signaling pathways were examined using western blotting. Finally, this study investigated the influence of OSC on a mouse cranial bone resorption model induced by titanium (Ti) particles in vivo. OSC inhibited OC differentiation and resorption and reduces intracellular ROS levels. Moreover, OSC suppressed IL-1β, TNF-α, IL-6, and osteoclast-specific gene transcription while increasing Nrf2 and HO-1 protein expression. Furthermore, OSC inhibited the expression and autoregulation of the NFATc1 gene, ultimately leading to a reduction in Ti particle-induced bone resorption in mice. OSC could be regarded as an innovative medication for the treatment of osteoclast-associated inflammatory osteolytic diseases.

Inhibitory effects of norcantharidin on titanium particle-induced osteolysis, osteoclast activation and bone resorption via MAPK pathways

Wear particles generated from the surface of implanted prostheses can lead to peri-implant osteolysis and subsequent aseptic loosening. In the inflammatory environment, extensive formation and activation of osteoclasts are considered the underlying cause of peri-implant osteolysis. Current medications targeting osteoclasts for the treatment of particle-induced bone resorption are not ideal due to significant side effects. Therefore, there is an urgent need to develop more effective drugs with fewer side effects. Norcantharidin (NCTD), a derivative of cantharidin extracted from blister beetles, is currently primarily used for the treatment of solid tumors in clinical settings. However, the potential role of NCTD in treating aseptic loosening of the prosthesis has not been reported. In this study, the in vitro results demonstrated that NCTD could effectively inhibit the formation of osteoclasts and bone resorption induced by the RANKL. Consistently, NCTD strongly inhibited RANKL-induced mRNA and protein levels of c-Fos and NFATc1, concomitant with reduced expression of osteoclast specific genes including TRAP, CTR and CTSK. The in vivo data showed that NCTD exerted significant protective actions against titanium particle-induced inflammation and subsequent osteolysis. The molecular mechanism investigation revealed that NCTD could suppress the activations of RANKL-induced MAPK (p38, ERK). Overall, these findings support the potential use of NCTD for the treatment of aseptic loosening following total joint arthroplasty.

Caffeic acid phenethyl ester ameliorates titanium particle-induced bone loss and inflammatory reaction in a mouse acute model

With the increasing clinical application of dental and orthopedic implants, the problem of peri-implant osteolysis has attracted attention. The inflammatory response and osteoclast differentiation induced by wear particles play an important role in peri-implant bone loss. However, the treatment of peri-implant osteolysis is still lacking. In the present study, we investigated the effect of caffeic acid phenethyl ester (CAPE) on titanium particles induced bone loss in a mouse model. We found that CAPE significantly suppressed titanium particle-induced bone loss in vivo. CAPE treatment decreased ratio of nuclear factor kappa B receptor activator ligand (RANKL)/osteoprotegerin (OPG) and subsequently reduced osteoclastogenesis in the mouse model. In addition, CAPE downregulated the expression and secretion of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) stimulated by titanium particles in vivo. In summary, we conclude that CAPE prevent the titanium particles-induced bone loss.

Particle-induced osteolysis is mediated by endoplasmic reticulum stress-associated osteoblast apoptosis

Osteoblast dysfunction plays a crucial role in periprosthetic osteolysis and aseptic loosening, and endoplasmic reticulum (ER) stress is recognized as an important causal factor of wear particle-induced osteolysis. However, the influence of ER stress on osteoblast activity during osteolysis and its underlying mechanisms remain elusive. This study aims to investigate whether ER stress is involved in the detrimental effects of wear particles on osteoblasts. Through our investigation, we observed elevated expression levels of ER stress and apoptosis markers in particle-stimulated bone specimens and osteoblasts. To probe further, we employed the ER stress inhibitor, 4-PBA, to treat particle-stimulated osteoblasts. The results revealed that 4-PBA effectively alleviated particle-induced osteoblast apoptosis and mitigated osteogenic reduction. Furthermore, our study revealed that wear particle-induced ER stress in osteoblasts coincided with mitochondrial damage, calcium overload, and oxidative stress, all of which were effectively alleviated by 4-PBA treatment. Encouragingly, 4-PBA administration also improved bone formation and attenuated osteolysis in a mouse calvarial model. In conclusion, our results demonstrate that ER stress plays a crucial role in mediating wear particle-induced osteoblast apoptosis and impaired osteogenic function. These findings underscore the critical involvement of ER stress in wear particle-induced osteolysis and highlight ER stress as a potential therapeutic target for ameliorating wear particle-induced osteogenic reduction and bone destruction.

Kaempferol attenuates wear particle-induced inflammatory osteolysis via JNK and p38-MAPK signaling pathways

Ethnopharmacological relevanceWear particle-induced inflammatory osteoclast activation is a master contributor to periprosthetic osteolysis, which can cause pathological bone loss and destruction. Hence, inhibiting inflammation and osteoclastogenesis is an important strategy for preventing wear particle-induced osteolysis. To date, there are no FDA-approved non-surgical pharmacotherapies for arresting periprosthetic osteolysis. Kaempferol (KAE), a natural flavonol abundant in many traditional Chinese herbal medicines, has been shown to have protective effects against inflammatory bone diseases such as rheumatoid arthritis, but no previous study has evaluated the effects of KAE on wear particle-induced osteolysis. Aim of the studyThe study aimed to investigate the effects of KAE on wear particle-induced inflammatory osteolysis and osteoclast activation, and further explore the underlying mechanisms. Materials and methodsTiAl6V4 metal particles (TiPs) were retrieved from the prosthesis of patients who underwent revision hip arthroplasty due to aseptic loosening. A mouse calvarial osteolysis model was used to investigate the effects of KAE on wear particle-induced inflammatory osteolysis in vivo. Primary bone marrow-derived macrophages (BMMs) were used to explore the effects of KAE on osteoclast differentiation and bone-resorbing activity as well as the underlying mechanisms in vitro. ResultsIn the present study, we found that KAE alleviated wear particle-induced inflammatory bone loss in vivo and inhibited osteoclast differentiation and function in vitro. Furthermore, we revealed that KAE exerted anti-osteoclastogenic effects by downregulating JNK and p38-MAPK signaling as well as the downstream NFATc1 expression. ConclusionsKAE is an alternative therapeutic agent for preventing and treating periprosthetic osteolysis and aseptic loosening.

Inhibitory effects of Formononetin on CoCrMo particle-induced osteoclast activation and bone loss through downregulating NF-κB and MAPK signaling

Wear particle-induced osteoclast over-activation is a major contributor to periprosthetic osteolysis and aseptic loosening, which can cause pathological bone loss and destruction. Hence, inhibiting excessive osteoclast-resorbing activity is an important strategy for preventing periprosthetic osteolysis. Formononetin (FMN) has been shown to have protective effects against osteoporosis, but no previous study has evaluated the effects of FMN on wear particle-induced osteolysis. In this study, we found that FMN alleviated CoCrMo alloy particles (CoPs)-induced bone loss in vivo and inhibited the formation and bone-resorptive function of osteoclasts in vitro. Moreover, we revealed that FMN exerted inhibitory effects on the expression of osteoclast-specific genes via the classical NF-κB and MAPK signaling pathways in vitro. Collectively, FMN is a potential therapeutic agent for the prevention and treatment of periprosthetic osteolysis and other osteolytic bone diseases.

NRF2 is a critical regulator and therapeutic target of metal implant particle-incurred bone damage

Aseptic metal implant loosening due to wear particle-induced bone damage is a major complication of total joint arthroplasty often leading to revision surgery, of which the key regulators mediating the processes are not clearly defined. Here we reported that in a mouse model of calvarial osteolysis, titanium particles (TiPs) and cobalt–chromium–molybdenum particles induced severe osteolysis accompanied by marked suppression of a master redox transcriptional factor NRF2 (Nuclear factor erythroid derived 2-related factor 2). Nfe2l2 knockout mice treated with TiPs developed worse osteolytic alterations compared with wild-type mice. On the contrary, NRF2 restoration by an NRF2 agonist TBHQ (tert-butylhydroquinone) effectively alleviated the osteolysis and the abnormal expression of NRF2 downstream antioxidant enzymes, inflammatory cytokines and osteogenic factors. Further, TiPs induced adverse osteoblastogenesis and osteoclastogenesis in cultured bone cells, which were substantially blocked by TBHQ in an NRF2 inhibition-sensitive manner. Consistently, the osteoprotective effects of TBHQ observed in wild-type mice were largely limited in Nfe2l2 knockout mice. Collectively, our data suggest that NRF2 suppression is a critical causal event of metal wear particle-incurred osteolysis, and the strategies reinstating NRF2 are effective to lessen the bone damage and potentially reduce the incidence of metal implant loosening.

Chrysin Protects Against Titanium Particle-Induced Osteolysis by Attenuating Osteoclast Formation and Function by Inhibiting NF-κB and MAPK Signaling.

Bone homeostasis only exists when the physical function of osteoblast and osteoclast stays in the balance between bone formation and resorption. Bone resorption occurs when the two processes are uncoupled, shifting the balance in favour of bone resorption. Excessive activation of osteoclasts leads to a range of osteolytic bone diseases including osteoporosis, aseptic prosthesis loosening, rheumatoid arthritis, and osteoarthritis. Receptor activator of nuclear factor kappa-B ligand (RANKL) and its downstream signaling pathways are recognized as key mediators that drive the formation and activation of osteoclastic function. Hence, osteoclast formation and/or its function remain as dominant targets for research and development of agents reaching the treatment towards osteolytic diseases. Chrysin (CHR) is a flavonoid with a wide range of anti-inflammatory and anti-tumor effects. However, its effect on osteoclasts remains unknown. In this study, we found the effects of CHR on inhibiting osteoclast differentiation which were assessed in terms of the number and size of TRAcP positive multinucleated osteoclasts (OCs). Further, the inhibitory effects of CHR on bone resorption and osteoclast fusion of pre-OC were assessed by hydroxyapatite resorption pit assay and F-actin belts staining; respectively. Western blotting analysis of RANKL-induced signaling pathways and immunofluorescence analysis for p65 nuclear translocation in response to RANKL-induced osteoclasts were used to analyze the mechanism of action of CHR affecting osteoclasts. Lastly, the murine calvarial osteolysis model revealed that CHR could protect against particle-induced bone destruction in vivo. Collectively, our data strongly suggested that CHR with its promising anti-tumor effects would also be a potential therapeutic agent for osteolytic diseases.

Piperlongumine Inhibits Titanium Particles-Induced Osteolysis, Osteoclast Formation, and RANKL-Induced Signaling Pathways.

Wear particle-induced aseptic loosening is the most common complication of total joint arthroplasty (TJA). Excessive osteoclast formation and bone resorptive activation have been considered to be responsible for extensive bone destruction and prosthesis failure. Therefore, identification of anti-osteoclastogenesis agents is a potential therapy strategy for the treatment of aseptic loosening and other osteoclast-related osteolysis diseases. In the present study, we reported, for the first time, that piperlongumine (PL), a key alkaloid compound from Piper longum fruits, could significantly suppress the formation and activation of osteoclasts. Furthermore, PL effectively decreased the mRNA expressions of osteoclastic marker genes such as tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), and cathepsin K (CTSK). In addition, PL suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced activations of MAPKs (ERK, JNK and p38) and NF-κB, which down-regulated the protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Using a titanium (Ti) particle-induced calvarial osteolysis model, we demonstrated that PL could ameliorate Ti particle-induced bone loss in vivo. These data provide strong evidence that PL has the potential to treat osteoclast-related diseases including periprosthetic osteolysis (PPO) and aseptic loosening.

Cimifugin Suppresses NF-κB Signaling to Prevent Osteoclastogenesis and Periprosthetic Osteolysis.

Background: Aseptic loosening of prosthesis (ALP) is one of the most common long-term complications of knee and hip arthroplasty. Wear particle-induced osteoclastogenesis and subsequent periprosthetic osteolysis account for the morbidity of ALP. Here, we investigate the potential of cimifugin (CIM), a natural extract from Cimicifuga racemosa and Saposhnikovia divaricata, as a bone-protective drug in the treatment of ALP. Method: First, we performed cell viability and osteoclast formation assays to assess the effect of noncytotoxic CIM on osteoclast differentiation in vitro. Bone slice resorption and F-actin ring immunofluorescence assays were adopted to assess the effects of CIM on bone-resorption function. Then, quantitative real-time polymerase chain reaction (qRT–PCR) analysis was performed to further assess the repressive effects of CIM on osteoclastogenesis at the gene expression level. To elucidate the mechanisms underlying the above findings, Western blot and luciferase reporter gene assays were used to assess the regulatory effects of CIM on the NF-κB and MAPK signaling pathways. Moreover, a Ti particle-induced murine calvarial osteolysis model and subsequent histomorphometric analysis via micro-CT and immunohistochemical staining were used to elucidate the effect of CIM on periprosthetic osteolysis in vivo. Result: CIM dose-dependently inhibited both bone marrow-derived macrophage (BMM)- and RAW264.7 cell-derived osteoclastogenesis and bone resorption pit formation in vitro, which was further supported by the reduced expression of F-actin and osteoclast-specific genes. According to the Western blot analysis, inhibition of IκBα phosphorylation in the NF-κB signaling pathway, not the phosphorylation of MAPKs, was responsible for the suppressive effect of CIM on osteoclastogenesis. Animal experiments demonstrated that CIM alleviated Ti particle-induced bone erosion and osteoclast accumulation in murine calvaria. Conclusion: The current study suggested for the first time that CIM can inhibit RANKL-induced osetoclastogenesis by suppressing the NF-κB signaling pathway in vitro and prevent periprosthetic osteolysis in vivo. These findings suggest the potential of CIM as a therapeutic in ALP.

Prednisone prevents particle induced bone loss in the calvaria mouse model

IntroductionGlucocorticoids are essential in the treatment of many chronic inflammatory and malignant diseases but are known to have detrimental effects on bone. This study aimed to investigate the effects of prednisone on osteoclast functioning in vivo in the calvaria particle-induced bone loss mouse model. Methods12-week-old male C57BL6/J mice received subcutaneously implanted prednisone (2.5 mg/d, 60 day release (n = 14)) or placebo pellets (n = 10). Osteolysis of the calvaria bone was induced two weeks later by application of ultra-high-molecular-weight polyethylene- (UHMWPE) particles to the dome (vs sham operation). The extent of osteolysis was determined histologically and by micro-computer tomography. ResultsPrednisone significantly inhibited particle-induced osteolysis in the skull. No significant difference in osteoclast numbers was seen in mice with prednisone vs placebo treatment. Prednisone treatment alone without particle application did not reduce bone mineral density or deterioration in bone microarchitecture parameters. ConclusionsThe calvaria particle-induced bone loss mouse model can be adapted to investigate osteoclast activity in vivo and the effect of prednisone on osteoclasts. In this preventive experimental design, the application of short-term low-dose prednisone has osteoprotective effects without measurable systemic side effects on bone parameters.

Celastrol Attenuates RANKL-Induced Osteoclastogenesis in vitro and Reduces Titanium Particle-Induced Osteolysis and Ovariectomy-Induced Bone Loss in vivo.

Excessive bone resorption by osteoclasts contributes significantly to osteoclast-related diseases such as periprosthetic osteolysis and osteoporosis. Osteolysis in a titanium particle-induced calvarial model and bone loss in an ovariectomized mice model occurred similarly to those in humans; thus, these models can be used to evaluate potential therapies for aseptic prosthetic loosening and osteoporosis. Celastrol, which is extracted from the seeds of the genus Tripterygium, has been thoroughly investigated for its anti-inflammatory and anti-cancer pharmacological effects. However, the mechanisms involving bone metabolism by which celastrol inhibits osteoclastogenesis are not yet fully understood. We demonstrated that celastrol inhibited the receptor activator of nuclear factor κB ligand-induced osteoclastogenesis and the bone resorptive function of osteoclasts in vitro by inhibiting the activation of transforming growth factor β-activated kinase 1-mediated NF-κB and mitogen-activated protein kinase signaling pathways and downregulating osteoclastogenesis marker-related genes. Furthermore, celastrol was also shown to be beneficial in both the titanium particle-induced osteolysis calvarial and the murine ovariectomy-induced bone loss. Collectively, our results suggested that celastrol is promising for the prevention of aseptic prosthetic loosening and osteoporosis in the treatment of osteolytic diseases induced by disrupted osteoclast formation and function.

Harmine Alleviates Titanium Particle-Induced Inflammatory Bone Destruction by Immunomodulatory Effect on the Macrophage Polarization and Subsequent Osteogenic Differentiation.

Peri-prosthetic osteolysis (PPO) and following aseptic loosening are regarded as the prime reasons for implant failure after joint replacement. Increasing evidence indicated that wear-debris-irritated inflammatory response and macrophage polarization state play essential roles in this osteolytic process. Harmine, a β-carboline alkaloid primitively extracted from the Peganum harmala seeds, has been reported to have various pharmacological effects on monoamine oxidase action, insulin intake, vasodilatation and central nervous systems. However, the impact of harmine on debris-induced osteolysis has not been demonstrated, and whether harmine participates in regulating macrophage polarization and subsequent osteogenic differentiation in particle-irritated osteolysis remains unknown. In the present study, we investigated the effect of harmine on titanium (Ti) particle-induced osteolysis in vivo and in vitro. The results suggested harmine notably alleviated Ti particle-induced bone resorption in a murine PPO model. Harmine was also found to suppress the particle-induced inflammatory response and shift the polarization of macrophages from M1 phenotypes to M2 phenotypes in vivo and in vitro, which improved anti-inflammatory and bone-related cytokines levels. In the conditioned medium from Ti particle-stimulated murine macrophage RAW264.7 cells treated with harmine, the osteoblast differentiation ability of mouse pre-osteoblastic MC3T3-E1 cells was greatly increased. And we also provided evidences that the immunomodulatory capacity of harmine might be attributed to the inhibition of the c-Jun N-terminal kinase (JNK) in wear particle-treated macrophages. All the results strongly show that harmine might be a promising therapeutic agent to treat PPO.

Inhibition of osteoclastogenesis by histone deacetylase inhibitor Quisinostat protects mice against titanium particle-induced bone loss

Periprosthetic osteolysis (PPO) and subsequent aseptic loosening are major long-term complications after total joint arthroplasty and have become the first causes for further revision surgery. Since PPO is primarily caused by excessive bone resorption stimulated by released wear particles, osteoclast-targeted therapy is considered to be of great potential for PPO prevention and treatment. Accumulating evidences indicated that inhibition of histone deacetylases (HDACs) may represent a novel approach to suppress osteoclast differentiation. However, different inhibitors of HDACs were shown to exhibit distinct safety profiles and efficacy in inhibiting osteoclastogenesis. Quisinostat (Qst) is a hydroxamate-based histone deacetylase inhibitor, and exerts potent anti-cancer activity. However, its effect on osteoclastogenesis and its therapeutic potential in preventing PPO are still unclear. In this study, we found that Qst suppressed RANKL-induced production of TRAP-positive mature osteoclasts, expression of osteoclast-specific genes, formation of F-actin rings, and bone resorption activity at a nanomolar concentration as low as 2 nM in vitro. Furthermore, we found that as low as 30 μg/kg of Qst was sufficient to exert preventive effect on titanium particle-induced osteolysis in the murine calvarial osteolysis model. Mechanistically, we found that Qst suppressed osteoclastogenesis by interfering with NF-κB and c-Fos/NFATc1 pathways. Thus, our study revealed that Qst may serve as a potential therapeutic agent for prevention and treatment of PPO and other osteoclast-mediated diseases.

Inhibitory effect of acetyl-11-keto-β-boswellic acid on titanium particle-induced bone loss by abrogating osteoclast formation and downregulating the ERK signaling pathway

Wear debris-induced osteoclast accumulation around implants plays a crucial role during the progression of periprosthetic osteolysis (PPO). We have confirmed that acetyl-11-keto-β-boswellic acid (AKBA) promotes bone formation and protects against particle-induced bone destruction in vivo. However, the effect of AKBA on titanium-induced bone resorption is unknown. In this study, we detected the inhibitory effect of AKBA on titanium-induced bone erosion in vivo and used RAW264.7 cells and bone marrow macrophages (BMMs) to investigate the effect and underlying mechanism of AKBA on the differentiation and resorptive function of osteoclasts. Our findings revealed that AKBA inhibited particle-induced bone loss and osteoclast formation in vivo. Furthermore, AKBA exerted inhibitory effects on RANKL-induced osteoclastogenesis, osteoclastic ring-dependent resorption and the expression of osteoclast marker genes via the ERK signaling pathway in vitro. Our data further established the protective effect of AKBA on titanium particle-induced bone erosion from a new perspective of bone erosion prevention, strongly confirming that AKBA is an appropriate agent for protection against PPO.

Inhibition of Sirtuin 3 prevents titanium particle-induced bone resorption and osteoclastsogenesis via suppressing ERK and JNK signaling.

Implant-derived wear particles can be phagocytosed by local macrophages, triggering an inflammatory cascade that can drive the activation and recruitment of osteoclasts, thereby inducing peri-prosthetic osteolysis. Efforts to suppress pro-inflammatory cytokine release and osteoclastsogenesis thus represent primary approaches to treating and preventing such osteolysis. Sirtuin 3 (SIRT3) is a NAD+-dependent deacetylases that control diverse metabolic processes. However, whether SIRT3 could mitigate wear debris-induced osteolysis has not been reported. Herein we explored the impact of the SIRT3 on titanium particle-induced osteolysis. Tartrate resistant acid phosphatase (TRAP) staining revealed that the inhibition of SIRT3 suppressed nuclear factor-κB ligand (RANKL)-mediated osteoclasts activation in a dose-dependent fashion. Notably, inhibition of SIRT3 also suppressed matrix metallopeptidase 9 (MMP9) and nuclear factor of activated T‐cell cytoplasmic 1 (NFATc1) expression at the mRNA and protein levels, while also inhibiting the mRNA expression of dendritic cell-specific transmembrane protein (DC-STAMP), ATPase H+ Transporting V0 Subunit D2 (Atp6v0d2), TRAP and Cathepsin K (CTSK) . In addition, inhibition of SIRT3 suppressed titanium particle-induced tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) expression and prevented titanium particle-induced osteolysis and bone loss in vivo. This inhibition of osteoclasts differentiation was found to be linked to the downregulation and reduced phosphorylation of JNK and ERK. Taken together, inhibition of SIRT3 may be a potential target for titanium particle-induced bone loss.

Interleukin-4 repairs wear particle induced osteolysis by modulating macrophage polarization and bone turnover.

Periprosthetic osteolysis remains as a major complication of total joint replacement surgery. Modulation of macrophage polarization with interleukin-4 (IL-4) has emerged as an effective means to limit wear particle-induced osteolysis. The aim of this study was to evaluate the efficacy of local IL-4 delivery in treating preexisting particle-induced osteolysis. To this end, recently established 8week modification of murine continuous femoral intramedullary particle infusion model was utilized. Subcutaneous infusion pumps were used to deliver polyethylene (PE) particles into mouse distal femur for 4 weeks to induce osteolysis. IL-4 was then added to the particle infusion for another 4 weeks. This delayed IL-4 treatment (IL-4 Del) was compared to IL-4 delivered continuously (IL-4 Cont) with PE particles from the beginning and to the infusion of particles alone for 8 weeks. Both IL-4 treatments were highly effective in preventing and repairing preexisting particle-induced bone loss as assessed by μCT. Immunofluorescence indicated a significant reduction in the number of F4/80 + iNOS + M1 macrophages and increase in the number of F4/80 + CD206 + M2 macrophages with both IL-4 treatments. Reduction in the number of tartrate resistant acid phosphatase + osteoclasts and increase in the amount of alkaline phosphatase (ALP) + osteoblasts was also observed with both IL-4 treatments likely explaining the regeneration of bone in these samples. Interesting, slightly more bone formation and ALP + osteoblasts were seen in the IL-4 Del group than in the IL-4 Cont group although these differences were not statistically significant. The study is a proof of principle that osteolytic lesions can be repaired via modulation of macrophage polarization.

Genetic and pharmacological activation of Hedgehog signaling inhibits osteoclastogenesis and attenuates titanium particle-induced osteolysis partly through suppressing the JNK/c-Fos-NFATc1 cascade.

Rationale: Wear particle-induced periprosthetic osteolysis (PPO) is a common long-term complication of total joint arthroplasty, and represents the major cause of aseptic loosening and subsequent implant failure. Previous studies have identified the central role of osteoclast-mediated bone resorption in the pathogenesis of PPO. Thus, therapeutic approaches of inhibiting osteoclast formation and activity are considered to be of great potential to prevent and treat this osteolytic disease. Hedgehog (Hh) signaling has been shown to play an important role in promoting osteoblast differentiation and bone formation. While Hh signaling is also implicated in regulating osteoclastogenesis, whether it can directly inhibit osteoclast differentiation and bone resorption remains controversial. Moreover, its potential therapeutic effects on PPO have never been assessed. In this study, we explored the cell-autonomous role of Hh signaling in regulating osteoclastogenesis and its therapeutic potential in preventing wear particle-induced osteolysis.Methods: Hh signaling was activated in macrophages by genetically ablating Sufu in these cells using LysM-Cre or by treating them with purmorphamine (PM), a pharmacological activator of Smoothened (Smo). In vitro cell-autonomous effects of Hh pathway activation on RANKL-induced osteoclast differentiation and activity were evaluated by TRAP staining, phalloidin staining, qPCR analyses, and bone resorption assays. In vivo evaluation of its therapeutic efficacy against PPO was performed in a murine calvarial model of titanium particle-induced osteolysis by μCT and histological analyses. Mechanistic details were explored in RANKL-treated macrophages through Western blot analyses.Results: We found that Sufu deletion or PM treatment potently activated Hh signaling in macrophages, and strongly inhibited RANKL-induced TRAP+ osteoclast production, F-actin ring formation, osteoclast-specific gene expression, and osteoclast activity in vitro. Furthermore, we found that Sufu deletion or PM administration significantly attenuated titanium particle-induced osteoclast formation and bone loss in vivo. Our mechanistic study revealed that activation of Hh signaling suppressed RANKL-induced activation of JNK pathway and downregulated protein levels of two key osteoclastic transcriptional factors, c-Fos and its downstream target NFATc1.Conclusions: Both genetic and pharmacological activation of Hh signaling can cell-autonomously inhibit RANKL-induced osteoclast differentiation and activity in vitro and protect against titanium particle-induced osteolysis in vivo. Mechanistically, Hh signaling hinders osteoclastogenesis partly through suppressing the JNK/c-Fos-NFATc1 cascade. Thus, Hh signaling may serve as a promising therapeutic target for the prevention and treatment of PPO and other osteolytic diseases.

Inhibition of protein phosphatase 2A attenuates titanium-particle induced suppression of bone formation

Peri-prosthetic osteolysis (PPO) often generates after total joint arthroplasty, which can bring implant failure and following revision surgery. Wear debris shed from prostheses strongly enhances bone resorption and attenuates bone formation in osteolytic process. We previously proved that suppression of protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, inhibited wear-debris-induced osteoclastogenesis and alleviated local osteolysis. Whether PP2A inhibition facilitates osteoblastogenesis and bone formation in the osteolytic sites remains unclear. Here, we observed that PP2A inhibition with a selective inhibitor attenuated particle-induced bone destruction by accelerating osteoblast differentiation and promoting bone regeneration. Meanwhile, we proved inhibition of PP2A alleviated the inhibition of osteogenic differentiation by titanium particles in MC3T3-E1 cells. In addition, PP2A inhibition increased β-catenin expression and enhanced β-catenin nuclear translocation, compared with that in the vehicle group. ICG-001, a specific inhibitor of β-catenin, was further applied and was found to weaken the effect of PP2A inhibition on β-catenin expression and nuclear translocation. Therefore, we demonstrated PP2A inhibition exerts protective effects on osteogenic differentiation mainly by activating Wnt/β-catenin signaling pathway. Thus, all the results further revealed PP2A could be a promising target for treating PPO and other bone related diseases.