Metformin suppresses UHMWPE particle-induced osteolysis in the mouse calvaria by promoting polarization of macrophages to an anti-inflammatory phenotype
- Zhao Yan†1,
- Xiaoxi Tian†2,
- Jinyu Zhu†1,
- Zifan Lu3,
- Lifeng Yu1,
- Dawei Zhang1,
- Yanwu Liu1,
- Chongfei Yang1,
- Qingsheng Zhu1Email author and
- Xiaorui Cao1Email author
© The Author(s) 2018
Received: 10 January 2018
Accepted: 6 March 2018
Published: 9 May 2018
Implant failure remains a major obstacle to successful treatment via TJA. Periprosthetic osteolysis and aseptic loosening are considered as proof of wear debris-induced disruption of local regulatory mechanisms related to excessive bone resorption associated with osteolysis and the damage at the bone-prosthesis interface. Therefore, there is an immediate need to explore strategies for limiting and curing periprosthetic osteolysis and aseptic loosening.
We analyzed the in vitro cytokine production by primary mouse bone marrow macrophages (BMMs) that were exposed to ultra-high molecular weight polyethylene (UHMWPE) particles and treated with metformin at different concentrations with or without 5-aminoimidazole-4-carboxamide ribonucleoside to activate or inhibit AMPK. A mouse calvarial model was used to examine the in vivo effects of metformin on UHMWPE particle-induced osteolysis.
With particles, primary mouse BMMs secreted more pro-inflammatory cytokines tumor necrosis factor-α and interleukin (IL)-6. Treatment with metformin inhibited these variations and promoted the release of cytokine IL-10 with anti-inflammatory capability. In vivo, metformin reduced the production of pro-inflammatory cytokines, osteoclastogenesis, and osteolysis, increasing IL-10 production. Metformin also promoted the polarization of macrophages to an anti-inflammatory phenotype in vivo via AMPK activation.
A crucial point in limiting and correcting the periprosthetic osteolysis and aseptic loosening is the inhibition of inflammatory factor production and osteoclast activation induced by activated macrophages. The ability of metformin to attenuate osteolysis induced in mouse calvaria by the particles was related to a reduction in osteoclast number and polarization of macrophages to an anti-inflammatory functional phenotype.
Metformin could limit the osteolysis induced by implant debris. Therefore, we hypothesized that metformin could be a potential drug for osteolysis induced by implant debris.
Total joint arthroplasty (TJA) is a widely performed surgery for reducing the pain and restoring the mobility of patients with major joint damage or severe arthritis. Implant failure remains a major obstacle to successful treatment via TJA. Osteolysis induced by implant wear debris is the dominant cause of implant failure, causing ~ 50,000 revision surgeries per year in the United States alone (Kurtz et al. 2007). Long-range data regarding TJA outcomes are insufficient, and osteolysis continues to be a common complication. Therefore, there is an immediate need to explore strategies for limiting and curing periprosthetic osteolysis and aseptic loosening.
Osteolysis begins with the activation of macrophages in addition to foreign-body giant cells and the phagocytosis of particulate wear debris. These events stimulate the release of cytokines with pro-inflammatory capability and mediators and reduce the phosphorylation of AMP-activated protein kinase (AMPK) (Vasamsetti et al. 2015), promoting the production of osteoclasts that are differentiated from phagocyte precursors and in turn facilitating the periprosthetic osteolysis and corresponding implant loosening (Glant et al. 1993). Osteoblast activity is hampered by exposure to prosthetic particles. For example, osteoblasts expressed a catabolic phenotype upon exposure to polyethylene particles (Atkins et al. 2009). Periprosthetic osteolysis and aseptic loosening, thus, are considered as proof of wear debris-induced disruption of local regulatory mechanisms related to excessive bone resorption associated with osteolysis and the damage at the bone-prosthesis interface (Purdue et al. 2006). A crucial point in limiting and correcting the periprosthetic osteolysis and aseptic loosening, thus, is the inhibition of inflammatory factor production and osteoclast activation induced by activated macrophages.
As an anti-hyperglycemic agent, metformin is widely used in treating type II diabetes to improve insulin resistance (Inzucchi et al. 2014). Metformin has a bone-protective property (La Fontaine et al. 2016) and enhances the mineralization and differentiation of osteoblasts (Kanazawa et al. 2008), negatively regulating RANKL in osteoclast differentiation (Lee et al. 2010). The reason is related to the activation of a serine/threonine protein kinase of AMP-activated protein kinase (AMPK), which is critical in maintaining metabolic homeostasis in eukaryotic cell types (Kudo et al. 1995). The anti-inflammatory effect of metformin is associated with AMPK phosphorylation. In addition, AMPK promoted the polarization of macrophages to an anti-inflammatory functional phenotype (Sag et al. 2008). Therefore, we hypothesized that metformin could limit the osteolysis induced by implant debris. Here, how metformin affects the in vitro production of inflammatory factors in primary mouse bone marrow macrophages (BMMs) in response to ultra-high molecular weight polyurethane (UHMWPE) particles was investigated. The ability of metformin to attenuate osteolysis induced in mouse calvaria by the particles was examined and found to be related to a reduction in osteoclast number and polarization of macrophages to an anti-inflammatory functional phenotype.
Preparation of UHMWPE-coated coverslips
The average diameter of the UHMWPE particles (provided by Mr. Ernst Krendlinger of Clariant, Gersthofen, Germany) was 1.74 ± 1.43 μm (range, 0.05–11.06 μm), with 90% particles smaller than 9 μm. (The particle diameter in membranes around total joint prostheses ranged from approximately 0 to 1 μm.) Notably, this particle diameter is within the range considered biologically active and differs from the diameter of particles isolated from the periprosthetic membranes. All particles were rinsed to remove the endotoxins using ethanol before desiccation in drying oven, and correspondingly, tests for endotoxins using the Limulus assay (Sigma-Aldrich, St. Louis, MO, USA) were negative. Before use in cell culture, the particles were disinfected under γ-irradiation in air (2.5 MR) and kept at 4 °C. The particles were exposed to UV light for 1 h immediately before use.
Glass coverslips were coated with UHMWPE particles. Previously characterized UHMWPE particles were placed in dimethyl sulfoxide (DMSO; 1 mg particles in 0.5 mL DMSO) in a sterile quartz pestle and mortar. Subsequently, the materials were suspended in 14.5 mL of collagen type-I monomer (0.01%) solution prepared from calf skin (C-8919; Sigma-Aldrich) and maintained at 4 °C. The final particle concentration was set at 107 particle/mL. The microscope coverslips with dimensions of 22 × 22 mm (Dingjie Biological Technology Co., Ltd., Shanghai, China) were disinfected for 2 h at 200 °C. Aliquots of collagen-suspended particles (105 particles/10 μL) were distributed onto the coverslip uniformly, before collagen polymerization was allowed to occur at room temperature. Coverslips not coated with UHMWPE were also created and used in control samples. The coverslips were placed into 6-well tissue culture plates before irradiation with ultraviolet light overnight in a sterile tissue culture hood. The plates were placed in PE packages and covered with aluminum foil. The storage temperature was 5 °C. Another 1 h exposure was performed immediately before usage of the coverslips (Nich et al. 2011).
Cell culture and IL-6, and TNF-α measurements
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco) and penicillin (100 U/mL) was used for primary mouse BMM culture, and the cells were obtained from mouse femurs as described previously (Shah et al. 2010). RPMI-1640 supplemented with streptomycin (100 mg/mL), 10% FBS (Gibco), penicillin (100 U/mL), 10% heat-inactivated fetal calf serum, and L-glutamine (2 mM) was used for RAW264.7 cell culture, and the cells were obtained from American Type Culture Collection (Rockville, MD, USA).
The cells were seeded at 1 × 106 cells/2 mL/well and exposed to UHMWPE particles on the coated coverslips for 24 h in 6-well tissue culture plates. To examine how the particles affected both cells, the cells were treated with metformin at a concentration of 0.05, 0.5, or 5 mM with or without various concentrations of compound C (Sigma-Aldrich), a chemical inhibitor of AMPK, as well as 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR, Sigma-Aldrich), which stimulates AMPK activity. The control samples were exposed to deionized water and incubated for 24 h. After each incubation period, the media samples were collected for cytokine measurement. Enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN, USA) were used to measure the concentrations of tumor necrosis factor (TNF)-α, IL-6, and IL-10. Cells were lysed and centrifuged at 1000 g at 4 °C for 5 min and then at 10,000 g at 4 °C for 10 min. After the supernatants were collected, ARG-1 in the supernatants was quantified using an ELISA kit (LifeSpan BioSciences) according to the manufacturer’s instructions.
Animals and surgical procedure
Ninety 12-week-old C57BL/J6 male mice were utilized to establish the calvarial model of particle-induced osteolysis. All experiments involving animals were approved by the Institutional Review Board of Xijing Hospital (permit No. 20161003). All methods were performed in accordance with National Institute of Health Guide for the Care and Use of Laboratory Animals. The mice were divided into four groups randomly (n = 22/group). Five mice in each group were used to collect calvaria for tissue culture, five for RNA extraction and qRT-PCR, five for Western blot analysis, and the other seven for histologic evaluation after micro-computed tomography scanning. For surgery, mice were placed under general anesthesia via inhalation of isoflurane. An area (0.5 × 0.5 cm2) of the periosteum was exposed via a midline sagittal incision of 10 mm on the calvaria. The incision in the sham control was sutured without further procedure. In the experimental groups in which UHMWPE particles were implanted, a sterile sharp surgical spoon was used to uniformly distribute 20 μg of particles over the intact periosteum. A simple, interrupted 4-0 Ethicon suture was utilized to close the incision. The mice were subsequently placed into cages and provided food and water ad libitum. All animals were given metformin (80 mg/kg, once a day for 2 weeks in a row), alendronate (ALN, Sigma-Aldrich, as positive control, 50 μg/kg, once a day for 2 weeks as previously described as anti-osteoporosis use), or normal saline as negative control via intragastric administration immediately after surgery. The animals were sacrificed 14 days after the operation via an overdose of intraperitoneal sodium pentobarbital.
The detailed descriptions of micro-CT analyses, histologic evaluation of osteolysis, quantitative real-time polymerase chain reaction (qRT-PCR), calvaria culture, and western blot analysis are provided in Additional file 1: Supporting Information S3.
All experiments were performed at least three times. All data are presented as mean ± standard deviation (SD). The SPSS software package (SPSS 11.0, Chicago, IL, USA) was used for statistical analyses. One-way analysis of variance (ANOVA) was performed to compare the different groups. Post-hoc testing of discrepancies among groups was performed via Duncan’s test when the ANOVA results were significant. All P values < 0.05 were considered indicative of significance.
Effects of metformin on TNF-α, IL-6 and IL-10 production and macrophage transition in primary mouse BMMs
Role of AMPK activation in the effect of metformin on the exposed primary mouse BMMs
Metformin inhibition of inflammatory cytokine by lipopolysaccharide (LPS)-treated macrophages depended on the AMPK phosphorylation (Musi et al. 2002; Tsoyi et al. 2011). Whether AMPK activation played a role in the effect of metformin on cytokine release by primary mouse BMMs exposed to UHMWPE particles was examined. Immunoblot analysis demonstrated negative regulation of phosphorylated AMPK with particle exposure, whereas the expression of total AMPK was not significantly altered upon exposure to the particles (Fig. 1i). The inhibition of AMPK phosphorylation in primary mouse BMMs with particles was recovered by metformin also in a dose-dependent manner. This effect was similar to that observed in relation to cytokine release. However, AMPK phosphorylation was not affected by ALN (Fig. 1j). Metformin thus stimulated the activation of AMPK in macrophages exposed to UHMWPE particles.
We also investigated in vitro cytokine production by RAW246.7 cells exposed to UHMWPE particles and treated with metformin at different concentrations with or without 5-aminoimidazole-4-carboxamide ribonucleoside to activate or inhibit AMPK (see Additional file 1: Supporting Information S2 and Figure S2). Similar results were obtained, which confirmed the influence of metformin.
Metformin attenuated particle-induced mouse calvarial osteolysis without changing the blood glucose level
Metformin inhibited particle-induced osteoclastogenesis in association with a reduction in the expression of osteoclastic genes
Metformin inhibited UHMWPE particle-induced osteoclastogenenic cytokine production in vivo
Inhibitory effect of metformin on mouse calvarial osteolysis is dependent on promotion of macrophage polarization to M2 phenotype by AMPK activation
Osteolysis around orthopedic implants can develop via the formation of an inflamed periprosthetic membrane containing high levels of inflammatory cytokines, macrophages, and implant wear debris. Implant-derived wear particles can induce a unique immune response based on the particle size and concentration. The chemotactic cytokines secreted in response to UHMWPE particles can activate macrophages located in the tissues surrounding joint replacements (Frokjaer et al. 1995), creating an environment conducive to osteoclastogenesis and osteolysis. Bone marrow-derived macrophages may play dual roles in the osteolysis associated with TJA. One role is as the major cell type responsible for cytokine production in the host defense against UHMWPE particles, and the other is as the precursor cells for osteoclasts responsible for subsequent bone resorption. An additional study reported that the immune response to UHMWPE wear particles is characterized by cells of monocytic or osteoclastogenic lineage (Ren et al. 2011).
Macrophages produce varied arrays of mediators with pro- and anti-inflammatory capabilities in response to different tissue environments or stimuli (Malyshev and Malyshev 2015; Martinez et al. 2008). Macrophages expressing a pro-inflammatory versus an anti-inflammatory functional profile are distinguished using the designations M1 and M2 (Davies and Taylor 2015) . IL-10 is a regulating factor in macrophage polarization that inhibits the UHMWPE particle-induced increase in phospho-STAT1 and phospho-nuclear factor (NF)-κB p65 production and promotes phospho-STAT3 expression (Jiang et al. 2016). Therefore, manipulation of macrophage functional phenotypes can represent a potential therapeutic target for some diseases (Singh et al. 2014).
The AMPK pathway has emerged recently as a critical sensing mechanism in regulating cellular energy homeostasis and maintaining normal bone physiology (Patel et al. 2015; Wang et al. 2016). The bone mass in mice is decreased with deletion of the α or β subunit of AMPK (Shah et al. 2010; Quinn et al. 2010). Activation of AMPK stimulates bone formation in vitro (Kanazawa et al. 2008; Wang et al. 2016) and negatively regulates RANKL expression in the differentiation of osteoclasts (Kang et al. 2013). In addition, AMPK can promote macrophage polarization to an anti-inflammatory functional phenotype (Sag et al. 2008). Many studies have reported that AMPK phosphorylation was reduced in cells under the condition of inflammation or exposed to cytokines such as TNF-α (Vasamsetti et al. 2015). Activation of macrophages by UHMWPE particles induced inflammation and secretion of TNF-α, which reduced AMPK phosphorylation. Metformin has been discovered as a potent AMPK agonist that can suppress inflammatory cytokine production and osteoclast activation. Therefore, we hypothesized that it may be an effective drug against osteolysis induced by particles representing implant wear debris.
The effects of metformin on UHMWPE particle-induced production of inflammatory cytokines by primary mouse BMMs in vitro were examined. Incubation with the particles increased IL-6 and TNF-α production. Metformin not only inhibited the elevated production of IL-6 and TNF-α induced by UHMWPE particles, but promoted the release of the anti-inflammatory cytokine IL-10 and shifted polarization to M2 type in a dose-dependent manner. More importantly, the suppressed production of inflammatory cytokines was associated with phosphorylation of AMPK. Given the effects of metformin on both primary mouse BMMs and RAW264.7 cells as well as the fact that AMPK plays a significant role in the metabolism of both cell types, the results indicate that the effect of metformin on the production of macrophage-derived cytokines is dependent on AMPK activation.
Metformin attenuated the particle-induced mouse calvarial osteolysis, in association with a reduction in the severity of inflammation and osteoclast numbers. Consistent with in vitro results, activation of AMPK was involved in the protective effect of metformin against UHMWPE particle-induced osteolysis, via the promotion of macrophage polarization to an anti-inflammatory phenotype.
The anti-osteolysis effects of metformin and ALN as well-established leading drugs for treating osteoporosis (Black and Rosen 2016) show no significant difference, but their mechanisms are different. The classical pharmacological effects of ALNs are attributed to two key properties: the affinity for bone mineral and the ability to slow the activities of osteoclasts (Russell et al. 2008; Cantatore et al. 1999). Metformin, on the other hand, inhibited pro-inflammatory cytokine production, osteoclastogenesis, and osteolysis via the AMPK pathway. ALN cannot fundamentally inhibit osteolysis due to its limited capacity to inhibit the production of inflammatory cytokines and RANKL. For example, Zhang et al. (Zhang et al. 2005) reported that high local levels of TNF-α produced during periprosthetic inflammation prevented ALN-induced apoptosis among osteoclasts in vivo. Therefore, metformin should be more effective and useful than ALN in the treatment of periprosthetic osteolysis after TJA.
Oral administration of metformin hydrochloride (0.5–1.5 g) is routinely prescribed for the treatment of type II diabetes and has protective actions on the skeletal system (Monami et al. 2008; Hamann et al. 2012). According to our study, administration of 100 mg/kg metformin had a significant protective effect against the osteolysis induced by wear particles in the mouse calvarial osteolysis model. According to standard drug dose conversion between humans and animals, the dose of metformin used in this study is within the physiologically acceptable range. Since metformin can enhance insulin sensitivity and improve metabolic syndrome, it has been used not only for treating diabetes patients, but also for treating osteoporosis patients and individuals with metabolic syndromes barely affecting glucose levels. Many TJA patients are elderly and suffer from diabetes, which can cause osteoporosis (Voronov et al. 1998). Based on our results, we propose that long-term usage of metformin for treating type II diabetes might lessen the symptoms related to type II diabetes-related osteoporosis and offer some degree of protection against wear particle-derived periprosthetic osteolysis as well.
The mechanism by which UHMWPE particles reduced the AMPK phosphorylation was not explored in our study, and the long-term effects of metformin administration on bone metabolism remain to be explored in our future experiments. Despite of the limitations of the present study, our work still indicates that metformin may represent a promising therapeutic agent for preventing or correcting periprosthetic osteolysis and implant loosening.
Periprosthetic osteolysis and aseptic loosening are considered as proof of wear debris-induced disruption of local regulatory mechanisms related to excessive bone resorption associated with osteolysis and the damage at the bone-prosthesis interface. A crucial point in limiting and correcting the periprosthetic osteolysis and aseptic loosening is the inhibition of inflammatory factor production and osteoclast activation induced by activated macrophages. Metformin could limit the osteolysis induced by implant debris. The ability of metformin to attenuate osteolysis induced in mouse calvaria by the particles was related to a reduction in osteoclast number and polarization of macrophages to an anti-inflammatory functional phenotype.
This work was supported by the Project of Natural Science Foundation of Shaanxi Province (2014JQ4142) and the National Natural Science Foundation of China (81501936) and (31571215). The authors thank Xiaorui Cao and Qingsheng Zhu from the PLA Institute of Orthopaedics, Xijing Hospital, and the Fourth Military Medical University, China as well as Zifan Lu of the State Key Laboratory of Cancer Biology, Fourth Military Medical University, China, for their technical help and advice.
Availability of data and materials
Additional file 1 is available from the authors or on the journal website.
ZY, XC and QZ wrote the main manuscript text; XT, JZ and ZL contributed to the design the experiments. ZY and LY performed all the experiments. DZ, YL and CY prepared Figs. 1, 2, 3, 4, 5 and 6. ZY designed Fig. 7. All authors reviewed the manuscript. All authors read and approved the final manuscript.
The protocols for the animal experiments were approved by the Institutional Review Board of Xijing Hospital. All methods were performed in accordance with National Institute of Health Guide for the Care and Use of Laboratory Animals (No. 20161003).
The authors declare they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
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