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Introduction

Titanium prostheses exhibit widespread uses in the clinic, including joint replacements and dental implants. However, in the majority of clinical cases, patients may experience ongoing inflammation in the absence of infection, which may lead to peri-prosthetic osteolysis (PPO) and subsequent aseptic loosening (1–4). The results of numerous studies have indicated that titanium implants undergo corrosion via various mechanisms inside the human body, including mechanical wear, biological activity and electrochemical processes (5–7). The aforementioned corrosion may cause gradual deterioration of the titanium implant; thus, impacting the structural integrity. In addition, the results of previous studies have demonstrated that the major cause of aseptic loosening is the gradual accumulation of wear particles around the titanium implant (8–10). Following 10 years of follow up, this phenomenon resulted in an implant failure rate of 32–62% following orthopedic surgery (11,12). For dental implants, unexplained mucosal inflammation impacts 43% of implants, and ongoing bone loss occurs in 6–29% of cases (13,14).

Numerous studies have focused on the molecular and cellular interactions between humans and wear particles (15,16). Previous studies have defined aseptic loosening as an immune-mediated biological complication (2,17–19). However, the complex interplay among the innate or adaptive immune response, the skeletal system and wear particles remains to be fully understood. Previous studies have revealed that the released particles may induce a cascade of biological effects, including potent proinflammatory immune responses, suppression of osteoblast function and activation of osteoclasts, leading to peri-implant bone loss (20–25). Thus, research has focused on the specific molecular mechanisms and potential targeted drugs for use in the clinic. Previous studies have revealed numerous inhibitors that exert protective effects on titanium particle-induced bone loss, and multiple associated downstream signaling pathways, such as the MAPK, NF-κB, GSK-3β/β-catenin and TGF-β pathways (10,22,26–31). Notably, multiple pathways are simultaneously activated by titanium particles. Thus, previous studies that focused on a single target or downstream pathway were unsuccessful in uncovering the specific mechanisms, and therapies that target single pathways to attenuate particle-induced osteogenic inhibition or inflammatory responses are limited (30,32,33).

N6-methyladenosine (m6A), a dynamic methylation at the N6 site of adenosine, is the most common post-transcriptional RNA modification. As an epigenetic regulator, it regulates numerous biological processes through mediating RNA metabolism, including degradation and translation (34–36). The methyltransferase-like (Mettl)3/Mettl14 catalytic heterodimer serves a role in the methylation of m6A. The process of demethylation is implemented by demethylases, including ALKB homolog 5 and fat mass and obesity-associated protein. This specific modification is recognized and bound by m6A reader proteins, mainly from YT521-B homology (YTH)-domain family proteins (37–41). Previous studies have revealed the association between m6A modifications and bone metabolism. RNA methylation serves a significant role in regulating bone homeostasis. Notably, Mettl3 and/or Mettl14 deficiency may disrupt the proliferation and differentiation of stem cells, such as bone marrow mesenchymal stem cells, causing bone disorders, such as osteoporosis (42–46). In addition, previous studies have demonstrated that post-transcriptional modification via changes in expression of methyltransferases, demethylases or reader proteins concurrently regulated multiple downstream pathways, achieving regulation in diverse cellular reactions (46,47).

As m6A modifications are essential in bone homeostasis (48), the present study aimed to explore the role of m6A modification in titanium particle-induced osteolysis. In addition, the present study aimed to provide novel insights into potential therapeutic targets for aseptic loosening. It was hypothesized that titanium particles may utilize RNA methylation as a form of upstream regulation to induce multiple subsequent pathways; thus, causing osteogenic inhibition and inflammatory responses.

Materials and methods

Titanium particle preparation

The titanium particles used in the present study were obtained from Alfa Aesar; Thermo Fisher Scientific, Inc. and prepared as previously described (10,22,29). Briefly, the particles were incubated at 180°C for 12 h, and subsequently immersed in 75% ethanol at room temperature for 48 h to eliminate endotoxins. Endotoxin levels in particles were detected using a Limulus assay kit (Xiamen Bioendo Technology Co., Ltd.) to ensure that endotoxin levels were <0.02 EU/ml.

Cell culture and treatment

The cells used in the present study were cultured at 37°C with 5% CO2. The murine osteoblast cell line, MC3T3-E1 (iCell Bioscience, Inc.) was cultured in α-minimum essential medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). For osteogenic differentiation, cells were cultured in the aforementioned growth medium with the addition of 50 µg/ml ascorbic acid (Sigma-Aldrich; Merck KGaA) and 10 mM β-glycerophosphate (Sigma-Aldrich; Merck KGaA). The prepared titanium particles were used at a concentration of 100 µg/ml. For titanium particle treatment groups, particles were cocultured with osteogenic induction medium to figure out the effect of titanium particles on osteoblasts in an osteogenic environment (10,22).

The RAW264.7 mouse macrophage cell line was purchased from American Type Culture Collection and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS and 100 U/ml penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The supernatants of osteoblast cells with different treatments, including titanium particle treatment and the knockdown or overexpression of the Mettl3 gene, were harvested and then centrifuged at 500 × g at room temperature for 5 min to remove cell debris or titanium particles. Subsequently, the supernatant was mixed with culture medium at a ratio of 1:1 (conditioned medium) and then RAW264.7 cells were cultured in conditioned medium for the detection of osteoclastic differentiation. Cells cultured in growth medium were used as the negative control (NC), while cells treated with 50 ng/ml recombinant murine receptor activator of NF-κB ligand (RANKL; PeproTech, Inc.) were used as the positive control. The samples were collected at 2 and 3 days for reverse transcription-quantitative PCR (RT-qPCR) and western blotting, respectively.

For nucleotide binding oligomerization domain (NOD)1 pathway inhibition experiments, osteoblast cells were pretreated with 30 µM ML130 (MedChemExpress) and 10 µM WEHI-345 (Abmole Bioscience Inc.) at 37°C for 2 h (49,50).

Transmission electron microscopy (TEM)

Following co-culture with titanium particles for 24 h, osteoblast cells were collected and fixed with 2.5% glutaraldehyde in PBS at 4°C for 1 h. No stain was used as the stain would highlight various intracellular structures. Cells were fixed with 1% osmium tetroxide in H2O for 2 h, followed by sequential dehydration with a graded ethanol series. Samples were embedded in epoxy resin at 60°C for 12 h, cut into 70–90-nm ultrathin sections and subsequently observed using a JEM-1200EX electron microscope (JEOL, Ltd.).

RT-qPCR

Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using total RNA and HiScript III RT SuperMix (Vazyme Biotech Co., Ltd.) at 37°C for 15 h and then 85°C for 5 sec. ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) and a Light Cycler 480 (Roche Diagnostics GmbH) were employed to amplify cDNA. The initial denaturation condition was 95°C for 10 sec. The thermocycling (40 cycles) conditions were as follows: 95°C for 10 sec and 60°C for 30 sec. mRNA levels were quantified using the 2−ΔΔCq method and normalized to the internal reference gene GAPDH (51). The specific primers are listed in Table SI.

Western blotting

Following rinsing three times with PBS, cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology) supplemented with protease and phosphatase inhibitors (CoWin Biosciences). Cell homogenates were obtained and centrifuged at 14,000 × g for 20 min at 4°C. The cell supernatant was obtained and a BCA protein assay kit (CoWin Biosciences) was used to determine the concentration of total proteins. Samples were subsequently incubated at 95°C for 10 min in SDS-PAGE sample loading buffer (Beyotime Institute of Biotechnology) for denaturation. 20 µg total protein samples were separated via 4–12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (MilliporeSigma). Membranes were blocked in 5% skimmed milk powder supplemented with 1X TBS-1‰ Tween-20 (TBS-T) at room temperature for 1 h, and subsequently incubated with primary antibodies at 4°C for 12 h. All primary antibodies are listed in Table SII. Following rinsing with TBS-T, the membranes were incubated with the HRP goat anti-rabbit IgG secondary antibody (Beijing Emarbio Science & Technology Co., Ltd.) at room temperature for 1 h. Protein bands were visualized using ECL luminophore (MilliporeSigma) and an enhanced chemiluminescence detection system (Bio-Rad Laboratories, Inc.). Protein expression was semi-quantified using ImageJ software v1.53e (National Institutes of Health). For the MAPK pathway detection, vinculin (124 kDa) was used as internal reference as it has been widely used in other studies (52,53), to ensure there is no overlap with the molecular weight of the experimental proteins (40–54 kDa). In all other cases, β-actin (42 kDa) was used as the internal reference.

Alizarin red staining

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and incubated with alizarin red solution (Wuhan Servicebio Technology Co., Ltd.) at room temperature for 10 min. Cells were incubated in PBS to remove excess stain. Matrix calcium depositions were imaged using a light microscope (Zeiss AG) and subsequently dissolved in 10% cetylpyridinium chloride (Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. The absorbance was measured at 562 nm using Gene5 CHS 3.11 software (BioTek; Agilent Technologies, Inc.) on a microplate reader (BioTek; Agilent Technologies, Inc.).

ELISA

Cell culture supernatants were collected from each group and centrifuged at 500 × g for 5 min at 4°C. ELISA kits (Beijing winter song Boye Biotechnology Co. Ltd.) were used to measure the secreted cytokine expression of RANKL (Mouse RANKL ELISA Kit; cat. no. DG30574M), osteoprotegerin (OPG; Mouse OPG ELISA Kit; cat. no. DG30295M), IL-6 (Mouse IL-6 ELISA Kit; cat. no. DG30754M), TNF-α (Mouse TNF-α ELISA Kit; cat. no. DG30048M) and IL-1β (Mouse IL-1β ELISA Kit; cat. no. DG94767Q) according to the manufacturer's protocol.

RNA stability

Cells were treated with 5 µg/ml actinomycin D (ACMEC biochemical) at 37°C to inhibit mRNA transcription. Total RNA was extracted at 0, 3 and 6 h. RT-qPCR was carried out as aforementioned to examine mRNA expression and calculate the degradation rate of target genes, which was presented as the relative expression of mRNA at each time point relative to that at the 0-h timepoint (54).

Cell transfection

Specific lentiviral vectors carrying short hairpin RNA (shRNA/sh) or control shRNA (GeneChem, Inc.) were used for Mettl3 knockdown. shRNA sequences are shown in Table SIII. Lentiviral vectors were used for Mettl3 overexpression, and blank vectors were prepared by Guangzhou IGE Biotechnology, Ltd. shRNA sequences were synthesized and Mettl3 overexpression products were amplified. The lentivirus transfer plasmid pLVX (Thermo Fisher Scientific, Inc.)-shRNA was used for recombinant plasmid construction and lentivirus production. And for Mettl3 overexpression, pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector (Thermo Fisher Scientific, Inc.) was employed and the Mettl3 coding region was inserted into the MCS sites of the vector. At 18 h before transfection, HEK 293T cells (Procell Life Science &Technology Co., Ltd.) were inoculated in six-well cell culture plates at a density of 4×105 cells per well. Before transfection, 2 µg of the plasmid was diluted in 100 µl Opti-MEM medium (Gibco) without serum (tube A), and 6 µl polyethylenimine reagent was diluted in another 100 µl serum-free Opti-MEM (tube B). We then added the dilution in tube B into tube A and mixed it gently. The mixture was incubated at room temperature for 12 min and then added to the cell culture medium. A 3rd generation system including four-plasmid lentivirus, pLP/VSVG, pLP1, pLP2, and empty/recombinant pLVX vector (Thermo Fisher Scientific, Inc.), was applied at a molar ratio of 1:1:1:2. Cells were incubated with transfection reagent-plasmid mixture at 37°C for 6 h before replacement. At 24 h after replacement, cell culture medium containing lentiviral particles was collected. The above protocols were completed by company and we carried out the subsequent transfection and selection. In brief, lentiviruses were cultured with MC3T3-E1 cells at a MOI of 50 at 37°C for 12 h. Green fluorescence protein labeling was used to observe transfection using a light microscope (Zeiss AG). Cell lines with stable expression were selected using 6 µg/ml puromycin for 3 days and maintained using 2 µg/ml puromycin (Biosharp Life Sciences). For YTH domain family 2 (Ythdf2) knockdown, 50 nM small interfering RNA (si)Ythdf2 or NC (Guangzhou IGE Biotechnology, Ltd.), named siCtrl, was employed to transfect cells with the addition of Lipofectamine® 2000 Transfection reagent (Biosharp Life Sciences) at 37°C for 24 h (Table SIII). The transfected cells were employed for subsequent experimentations at 24 h after transfection. The transfection efficiency was determined using RT-qPCR and western blot analysis and at 24 and 48 h after the transfection, respectively.

RNA dot blot for total m6A content analysis

Total RNA was extracted as aforementioned and incubated at 95°C for 3 min to disrupt secondary structures. Denatured total RNA was loaded onto nitrocellulose membranes and subsequently crosslinked to the membrane with UV light at 254 nm for 30 min. Unbound RNA was removed by washing with TBS-T. Western blot analysis was carried out as aforementioned using the anti-m6A antibody (Table SII).

Methylated RNA immunoprecipitation (MeRIP)-qPCR

The EpiQuik CUT&RUN m6A RNA Enrichment kit (EpigenTek Group, Inc.) was used for the MeRIP assay as previously described (55,56). Briefly, total RNA was collected and randomly fragmented. RNA fragments were immunoprecipitated with magnetic beads pre-coated with anti-immunoglobulin (IgG), anti-m6A or anti-Ythdf2 antibodies. Targeted RNA fragments were released, purified and eluted, followed by RT-qPCR as aforementioned. Relative enrichment was normalized to the input sample.

Statistical analysis

All data were representative of three independent experiments and presented as the mean ± standard deviation. Data were plotted and analyzed using GraphPad Prism v9.0 (Dotmatics). Comparisons between two groups were analyzed using an unpaired Student's t-test. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test and two-way ANOVA followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Titanium particles inhibit osteogenic differentiation and mediate osteoblast-osteoclast communication

Titanium particles engulfed into osteoblastic cells were observed using TEM (Fig. 1A). In the present study, RT-qPCR and western blot analysis were used to show early trends, and alizarin red staining was used to study later trends. The results of the present study demonstrated that titanium particle treatment significantly inhibited the expression of osteogenesis-associated markers and the formation of calcium nodules compared with those in the osteogenic induction group (Fig. 1B and C). Furthermore, titanium particles induced the mRNA expression of proinflammatory cytokines (Fig. 1D), and these results were verified via ELISA. To further investigate the regulatory role of titanium particles in osteoblast-osteoclast communication, which is critical for bone homeostasis (57), the supernatant of titanium-treated osteoblasts was harvested and co-cultured with preosteoclasts. As shown in Fig. 1E, mRNA and protein expression levels of markers associated with osteoclast differentiation of RAW264.7 cells were increased in the titanium particle treatment group (CM) compared with the NC group. Thus, the expression levels of additional soluble factors released from osteoblasts that mediate osteoblast-osteoclast communication were investigated. Notably, RANKL expression was significantly increased, leading to a higher ratio of RANKL/OPG (Fig. 1F).

Figure 1. Titanium particles inhibit osteogenic differentiation and mediate osteoblast-osteoclast communication. (A) Transmission electron microscopy of osteoblasts co-cultured (A-a) without and (A-b) with titanium particles (red arrows; scale bar, 5 µm). (B) Titanium particle treatment inhibited the formation of mineralized nodules at 14 days. (B-a) Blank, (B-b) osteogenic induction medium and (B-c) titanium particle treatment. Scale bar, 500 µm. (C) Titanium particle treatment inhibited the expression of osteogenesis-associated markers. The samples were collected at 2 and 3 days for RT-qPCR and western blotting, respectively. (D) Titanium particle treatment promoted the expression of inflammatory cytokines. The samples were collected at 2 and 3 days for RT-qPCR and ELISA, respectively. (E) Supernatants of titanium-treated osteoblasts promoted the expression of osteoclast differentiation-associated markers in preosteoclasts. (F) Titanium treatment promoted the expression of RANKL, leading to the higher ratio of RANKL/OPG. The samples were collected at 2 and 3 days for RT-qPCR and ELISA, respectively. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Acp5, acid phosphatase 5, tartrate resistant; Col1a1, collagen type I α1 chain; Ctsk, cathepsin K; CM, conditioned medium; Dcstamp, dendrocyte expressed seven transmembrane protein; M-CSF, macrophage colony-stimulating factor; NC, negative control; Nfatc1, nuclear factor of activated T cells 1; ns, not significant; OD, optical density; OM, osteogenic induction medium; OPG, osteoprotegerin; PC, positive control; RANKL, receptor activator of NF-κB ligand; RT-qPCR, reverse transcription-quantitative PCR; Runx2, RUNX family transcription factor 2; Ti, titanium particle.

Role of m6A modification in titanium particle treatment

To explore the role of m6A modification in titanium particle treatment of osteoblasts, m6A levels were evaluated. The results of the RNA dot blot analysis demonstrated that m6A levels were reduced in the titanium treatment group compared with the osteogenic induction group (Fig. 2A). Subsequently, expression levels of common methyltransferases and demethylases were further determined via RT-qPCR and western blotting (Fig. 2B and C). The expression levels of Mettl3, a key methyltransferase, were significantly decreased during titanium treatment, and these results were consistent with the measurement of m6A levels.

Figure 2. Role of m6A modification in titanium particle treatment. (A) Titanium particle treatment reduced total m6A levels compared with the osteogenic induction group after 2 days. (B) Titanium particle treatment significantly reduced the mRNA expression of the methylation-associated enzyme Mettl3. Samples were collected at 2 days for reverse transcription-quantitative PCR. (C) Titanium particle treatment significantly reduced the protein expression levels of Mettl3. Samples were collected at 3 days for western blotting. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001. Alkbh5, alkB homolog 5, RNA demethylase; Fto, FTO α-ketoglutarate dependent dioxygenase; m6A, N6-methyladenosine; Mettl3, methyltransferase-like 3; ns, not significant; OM, osteogenic induction medium; Ti, titanium particle.

Mettl3 knockdown induces osteogenic inhibition and proinflammatory responses

To explore the association between titanium particle-induced bioactivities and the reduced expression levels of Mettl3, Mettl3 knockdown was carried out in MC3T3-E1 cells, referred to as shMettl3 cells (Fig. 3A). The results of the present study demonstrated that Mettl3 mRNA and protein expression was significantly reduced following transfection compared with the NC or blank groups (Fig. 3B). The results of the RNA dot blot analysis revealed that m6A levels were lowest in the shMettl3 group compared with the control and titanium particle treatment groups (Fig. 3C). Notably, mRNA and protein expression levels of osteogenic markers were significantly reduced in the shMettl3 group (Fig. 3D). In addition, the alizarin red staining assay demonstrated consistent results, indicating that Mettl3 knockdown induced osteogenic inhibition comparable with that induced by titanium particles (Fig. 3D). Furthermore, Mettl3 knockdown increased the expression of proinflammatory cytokines (Fig. 3E). Mettl3 knockdown increased the RANKL expression and the ratio of RANKL/OPG compared with the control group (shCtrl osteoblasts without titanium particle) (Fig. 3F). Additionally, the supernatant of Mettl3-knockdown osteoblasts was harvested and co-cultured with RAW264.7 cells. mRNA and protein expression levels of markers associated with osteoclastogenesis were increased in RAW264.7 cells cultured with supernatant of Mettl3-knockdown osteoblasts compared with the control group (shCtrl osteoblasts without titanium particle) (Fig. 3G).

Figure 3. Mettl3 knockdown induces osteogenic inhibition and proinflammatory responses. (A) Construction of Mettl3 knockdown cells (scale bar, 100 µm). (B) The verification of transfection efficiency using reverse transcription-quantitative PCR and western blotting. (C) Mettl3 knockdown and titanium particle treatment were associated with reduced m6A content in total RNA compared with the blank group (osteoblasts without transfection). (D) Mettl3 knockdown inhibited the expression of osteogenic markers and the formation of mineralized nodules (scale bar, 500 µm). (E) Mettl3 knockdown promoted the expression of inflammatory cytokines. (F) Mettl3 knockdown led to a higher ratio of RANKL/OPG compared with that of the control group (shCtrl cells without Ti). (G) Supernatant of Mettl3 knockdown osteoblasts promoted the expression of osteoclast differentiation-associated markers in preosteoclasts. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Acp5, acid phosphatase 5, tartrate resistant; Col1a1, collagen type I α1 chain; Ctrl, control; Ctsk, cathepsin K; Dcstamp, dendrocyte expressed seven transmembrane protein; m6A, N6-methyladenosine; Mettl3, methyltransferase-like 3; Nfatc1, nuclear factor of activated T cells 1; ns, not significant; OD, optical density; OPG, osteoprotegerin; RANKL, receptor activator of NF-κB ligand; sh, short hairpin RNA; Runx2, RUNX family transcription factor 2; Ti, titanium particle.

Mettl3 overexpression attenuates titanium particle-induced inhibition of osteogenesis and proinflammatory activities

To further verify whether titanium particles mediated the aforementioned activities through reduced Mettl3 expression, a Mettl3-overexpression osteoblast cell line was constructed, and a blank vector was used as the NC. RT-qPCR and western blotting were performed to determine the transfection efficiency following overexpression (Fig. 4A). TEM analysis demonstrated that Mettl3 overexpression did not impact the engulfment capability of osteoblasts (Fig. 4B). Notably, m6A levels in the Mettl3 overexpression group were significantly increased compared with those in the blank group (Fig. 4C). The results of the present study also demonstrated that Mettl3 overexpression attenuated titanium-induced inhibition of osteogenesis (Fig. 4D). In addition, titanium-induced proinflammatory activities were also attenuated, as well as the ratio of RANKL/OPG (Fig. 4E and F). The aforementioned results in Fig. 1E indicated the regulatory role of titanium particles in osteoblast-osteoclast communication, while this indirect regulatory effect of titanium particles on osteoclastogenesis of RAW264.7 cells was also reversed by Mettl3 overexpression (Fig. 4G).

Figure 4. Mettl3 overexpression attenuates titanium particle-induced osteogenesis inhibition and proinflammatory responses. (A) Construction of Mettl3 overexpression cells and the verification of transfection efficiency using reverse transcription-quantitative PCR and western blotting (scale bar, 100 µm). (B) Transmission electron microscopy of Mettl3 overexpression cells co-cultured (B-a) without or (B-b) with titanium particles (red arrows; scale bar, 5 µm). (C) Mettl3 overexpression increased the m6A content in total RNA compared with that of the blank group (osteoblasts without transfection). (D) Mettl3 overexpression attenuated titanium particle-induced osteogenesis inhibition (scale bar, 500 µm). (E) Mettl3 overexpression attenuated titanium particle-induced proinflammatory responses. (F) Mettl3 overexpression attenuated the titanium particle-induced increase in the RANKL/OPG ratio. (G) Mettl3 overexpression attenuated osteoclast differentiation promotion induced by the supernatant of Ti-treated osteoblasts. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Acp5, acid phosphatase 5, tartrate resistant; Col1a1, collagen type I α1 chain; Ctsk, cathepsin K; Dcstamp, dendrocyte expressed seven transmembrane protein; Lv, lentivirus; m6A, N6-methyladenosine; Mettl3, methyltransferase-like 3; Nfatc1, nuclear factor of activated T cells 1; ns, not significant; OD, optical density; OPG, osteoprotegerin; RANKL, receptor activator of NF-kB ligand; Runx2, RUNX family transcription factor 2; Ti, titanium particle.

Titanium particle treatment targets Smad7 and SMAD specific E3 ubiquitin protein ligase 1 (Smurf1) via Mettl3, leading to bone morphogenetic protein (BMP) signaling inhibition

To explore the downstream signaling molecules affected by Mettl3, the canonical BMP-Smad signaling pathway was evaluated. The results of RT-qPCR analysis demonstrated that titanium particle treatment increased Smad7 and Smurf1 expression in osteoblasts, while Mettl3 overexpression attenuated the upregulated expression of Smad7 and Smurf1 induced by titanium particle treatment (Fig. 5A). The results of western blotting further demonstrated that titanium particle treatment inhibited the phosphorylation levels of Smad1/5/9 and induced the increased expression of Smad7 and Smurf1, and these reactions induced by titanium particle treatment were also reversed by Mettl3 overexpression (Fig. 5B). The results of the present study also demonstrated that both titanium particle treatment and Mettl3 knockdown increased the mRNA stability of these two genes, leading to increased expression levels (Fig. 5C). Furthermore, MeRIP-qPCR analysis revealed that the m6A enrichment of Smad7 and Smurf1 transcripts was significantly reduced in the titanium particle treatment and Mettl3 knockdown groups compared with the control group (Fig. 5D).

Figure 5. Titanium particle treatment targets Smad7 and Smurf1 via Mettl3, leading to BMP signaling inhibition. (A) Effects of titanium particle treatment and Mettl3 knockdown/overexpression on the mRNA expression of key signaling molecules of BMP signaling. (B) Effects of titanium particle treatment and Mettl3 knockdown/overexpression on BMP signaling activation. (C) Titanium particle treatment enhanced the mRNA stability of Smad7 and Smurf1 transcripts at the selected timepoints, following the addition of actinomycin D. (D) Titanium particle treatment significantly decreased the m6A modification of these two transcripts. The control group in this figure were osteoblasts without transfection. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. BMP, bone morphogenetic protein; Ctrl, control; Lv, lentivirus; m6A, N6-methyladenosine; Mettl3, methyltransferase-like 3; ns, not significant; p-, phosphorylated; sh, short hairpin RNA; Smurf1, SMAD specific E3 ubiquitin protein ligase 1; t-, total; Ti, titanium particle.

Titanium particles induce NOD-like receptors (NLRs) to exert proinflammatory activities

The results of western blotting revealed that titanium particle treatment induced the activation of the MAPK and NF-κB pathways, while Mettl3 overexpression attenuated this activation. For p38, it seemed that the titanium particle induced its activation while the Mettl3 overexpression failed to attenuated this activation. (Fig. 6A). The expression changes of key components of the NLR signaling pathway were also evaluated. The results of the present study demonstrated that titanium particle treatment increased the expression and the mRNA half-life of NOD1 and receptor interacting serine/threonine kinase 2 (RIPK2) in osteoblasts. However, the mRNA stability and expression of NOD2 were not enhanced (Fig. 6B and C). MeRIP-qPCR analysis demonstrated that NOD1 and RIPK2 transcripts functioned as the target of Mettl3, as titanium particle treatment and Mettl3 knockdown both significantly reduced the m6A enrichment of these two transcripts compared with the control group (Fig. 6D).

Figure 6. Titanium particle treatment induces NOD-like receptors to exert proinflammatory responses. (A) Titanium particle treatment induced the activation of the MAPK and NF-κB signaling pathways. (B) Titanium particle treatment induced the activation of the NOD-like receptor pathway, while Mettl3 overexpression attenuated these effects. (C) Titanium particle treatment and Mettl3 knockdown enhanced the mRNA stabilities of NOD1 and RIPK2 following the addition of actinomycin D. (D) Titanium particle treatment and Mettl3 knockdown decreased the m6A modification of NOD1 and RIPK2. The control group in this figure were osteoblasts without transfection. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Ctrl, control; Lv, lentivirus; m6A, N6-methyladenosine; Mettl3, methyltransferase-like 3; NOD, nucleotide binding oligomerization domain; ns, not significant; p-, phosphorylated; RIPK2, receptor interacting serine/threonine kinase 2; sh, short hairpin RNA; Ti, titanium particle.

To further verify the role of the NLR1 signaling pathway in the titanium particle-induced inflammatory response, cells were pretreated with ML130 (NOD1 inhibitor) and WEHI-345 (RIPK2 inhibitor) to block the NOD1 pathway since titanium particles can act on both NOD1 and RIPK2 mRNA based on the aforementioned results. In Fig. 7A, without the addition of inhibitors, titanium particle induced the activation of the MAPK and NF-κB signaling pathways, and the activation of these two pathways was significantly inhibited following treatment with both inhibitors from the comparison between titanium particle treatment without inhibitors group and titanium particle treatment with inhibitors group (Fig. 7A). In addition, inhibition of the NOD1 pathway attenuated the titanium particle-induced increased expression of IL-6, TNF-α, IL-1β and RANKL (Fig. 7B). Collectively, these data suggested that titanium particles may target the NOD1 signaling pathway to regulate subsequent inflammatory responses.

Figure 7. Inhibition of the NOD-like receptor signaling pathway inhibits titanium particle-induced proinflammatory responses. (A) Inhibition of the NOD1 signaling pathway inhibited MAPK and NF-κB signaling activation induced by titanium particle treatment. (B) Inhibition of the NOD1 signaling pathway inhibited proinflammatory cytokine expression induced by titanium particle treatment. The control group in this figure were osteoblasts without inhibitors. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001. Ctrl, control; Mettl3, methyltransferase-like 3; NOD, nucleotide binding oligomerization domain; p-, phosphorylated; sh, short hairpin RNA; Ti, titanium particle.

Ythdf2 participates in the Mettl3-mediated osteogenic inhibition and proinflammatory activities in titanium particle treatment

m6A readers recognize m6A modifications and then mediate regulatory effects, including RNA translation, decay and splicing (41). The aforementioned results (Figs. 5D and 6D) revealed that downregulated Mettl3 levels led to the enhancement of mRNA stabilities of Smad7, Smurf1, NOD1 and RIPK2, and thus, it was hypothesized that it was Ythdf2 that exerted the effects. Firstly, siYthdf2 was used to knockdown its expression (Fig. 8A). The mRNA and protein expression levels of Smad7, Smurf1, NOD1 and RIPK2 were increased in the Ythdf2 knockdown group compared with the control group (Fig. 8B). The mRNA stabilities of these four mRNAs were also enhanced in Ythdf2 knockdown cells at 3 and 6 h, while the stability of NOD at 6 h didn't show statistical difference (Fig. 8C), suggesting that Ythdf2 could affect the expression of these four mRNAs by regulating their stabilities. In Fig. 8D, we measured the relative expressions of these four mRNAs precipitated by Ythdf2, which were shown in anti-Ythdf2 column. Notably, titanium particle treatment reduced the expression levels of these mRNAs compared with control group, indicating that the binding between Ythdf2 and mRNAs was significantly decreased following titanium particle treatment (Fig. 8D).

Figure 8. Ythdf2 participates in the methyltransferase-like 3-mediated bioactivities in titanium particle treatment. (A) Knockdown of Ythdf2 and verification of transfection efficiency using reverse transcription-quantitative PCR and western blotting. (B) Ythdf2 knockdown promoted the expression of Smad7, Smurf1, NOD1 and RIPK2. (C) Ythdf2 knockdown enhanced the mRNA stabilities of Smad7, Smurf1, NOD1 and RIPK2 at 3 h. (D) Methylated RNA immunoprecipitation-quantitative PCR results demonstrated that titanium particle treatment decreased the relative expression levels of Smad7, Smurf1, NOD1 and RIPK2 precipitated by Ythdf2. Data are representative of three independent experiments and are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Ctrl, control; NOD, nucleotide binding oligomerization domain; RIPK2, receptor interacting serine/threonine kinase 2; si, small interfering RNA; Smurf1, SMAD specific E3 ubiquitin protein ligase 1; Ythdf2, YTH domain family 2; Ti, titanium particle.

Discussion

Bone homeostasis is a dynamic balance regulated by osteoblasts and osteoclasts. However, dysregulation of the balance between osteoblasts and osteoclasts may result in pathological bone loss, in which osteogenic inhibition and inflammatory responses serve a vital role (57,58). The activation of inflammatory responses promotes the secretion of several cytokines and the regulation of specific cell types, including osteoclasts, osteoblasts, macrophages and immune cells, thus leading to pathological osteolysis (57,59). The results of the present study revealed that titanium particles inhibited osteogenesis and proinflammatory responses, and these results are consistent with previous reports (10,22,60). Furthermore, the results of the present study demonstrated that titanium particles mediated the osteoblast-osteoclast communication, leading to enhanced osteoclast activity, and these underlying mechanisms were primarily explored. Notably, osteoblasts secrete several soluble factors, including macrophage colony-stimulating factor, RANKL, OPG and WNT5A, to act on osteoclasts (57). The elevated RANKL/OPG ratio may contribute to the enhanced osteoclast activity. However, additional mechanisms, such as secreted exosomes and membrane-bound mediators, were not further explored in the present study.

Previous studies have reported the critical role of m6A methylation in bone remodeling and inflammation (48,61). To explore the potential role of m6A methylation in titanium particle-induced bioactivities, total m6A levels were determined. Notably, the differentially expressed enzyme Mettl3 is the most critical methyltransferase and previous studies have indicated that Mettl3 exerts regulatory effects in several physiological and pathological processes (42–47). The results of the present study demonstrated that Mettl3 knockdown induced the inhibition of osteogenesis and proinflammatory responses, and these effects were comparable to those induced by titanium particles. In addition, Mettl3 overexpression reversed the bioactivities induced by titanium particles. Previous studies have demonstrated that Mettl3-mediated m6A modification regulated bone metabolism, including cell differentiation, and abnormal Mettl3 expression levels may induce bone metabolic diseases (44,46,48). Collectively, the results of the present study highlighted that Mettl3 may function as an upstream regulator in titanium particle-induced bioactivities.

Notably, the canonical Smad-dependent pathway serves a key role in osteogenesis and the activation and translocation of the Smad1/5/9 complex triggers the subsequent expression of osteogenesis-related genes (62,63). Inhibitory Smads negatively regulate Smad signaling by preventing Smad1/5/9 activation and degrading Smad1/5/9 with the assistance of the E3 ubiquitin ligase, Smurf1 (62,64). Thus, RT-qPCR and western blotting revealed the decreased levels of Smad1/5/9 phosphorylation, along with the increased expression of Smad7 and Smurf1, following treatment with titanium particles or Mettl3 knockdown. Notably, enhanced mRNA stability may account for the increased expression of Smad7 and Smurf1. To further confirm the regulation of Mettl3, MeRIP-qPCR was performed. The results of the present study indicated that the m6A enrichment of these two transcripts was significantly reduced following titanium particle treatment or Mettl3 knockdown. Thus, titanium particle treatment may suppress the mRNA decay of Smad7 and Smurf1 via Mettl3 downregulation, leading to inhibition of Smad-dependent signaling.

Previous studies have demonstrated that titanium particles may concurrently activate two classical proinflammatory pathways, including the MAPK and NF-κB pathways, and promote the expression of downstream proinflammatory cytokines (26,27,65). The results of the present study demonstrated that titanium particle treatment induced the activation of these two inflammation-associated pathways, while Mettl3 overexpression reversed the activation, suggesting that Mettl3 was involved in the activation of the MAPK and NF-κB signaling pathways. To further explore the target of titanium particles in these pathways, the mRNA stability of numerous molecules was determined. A previous study identified the activation of the NLR signaling pathway in Mettl3 knockdown macrophages via RNA sequencing analysis (66). Activation of key members of the NLR pathway, including NOD1, NOD2 and RIPK2, may trigger the subsequent activation of the MAPK and NF-κB signaling pathways (67–69). Thus, it was hypothesized that titanium particles may impact the NLR pathway, leading to the subsequent activation of inflammatory responses. Notably, titanium particle treatment increased the mRNA expression and stability of NOD1 and RIPK2. In addition, the m6A enrichment of these two transcripts was significantly reduced following titanium particle treatment. Inhibition of the NLR pathway inhibited the activation of MAPK and NF-κB pathways and the expression of proinflammatory cytokines, suggesting that titanium particles exert their effect via downregulation of Mettl3, which activates the NLR pathway and leads to the subsequent activation of inflammatory responses.

The m6A reader proteins are required to recognize and bind to the m6A modified transcript to regulate gene expression (70). Several m6A readers have been identified, including YTH domain-containing family proteins, heterogeneous nuclear ribonucleoproteins, insulin like growth factor 2 mRNA binding protein families and eukaryotic initiation factor (71). Ythdf2, a main member of the YTH domain-containing family proteins, is the most extensively studied m6A reader. Ythdf2 promotes targeted mRNA decay by recruiting the C-C motif chemokine receptor 4 negative on TATA-less deadenylase complex (72). Increasing evidence has indicated that Ythdf2 serves a critical role in pathological processes, including stress, viral infection and the inflammatory response (66,73–76). Considering that titanium particle treatment regulates RNA decay of specific mRNAs, it was hypothesized that Ythdf2may recognize the methylated modification of these mRNAs and promote the targeted mRNA decay. The results of the present study demonstrated that the knockdown of Ythdf2 led to significantly increased expression and enhanced stabilities of Smad7, Smurf1, NOD1 and RIPK2 transcripts at 3 h, and Smad7, Smurf1 and RIPK2 transcripts at 6 h. MeRIP-qPCR results (Figs. 5D and 6D) showed that titanium particle treatment could reduce the m6A sites of these four mRNAs, meanwhile, binding sites between Ythdf2 and targeted mRNAs was significantly decreased following titanium particle treatment (Fig. 8D). Taken together, it might be concluded that the reduced m6A sites of mRNAs led to the reduced recognition from Ythdf2, therefore reduced the mRNAs decay and increased the expression of these four molecules. Furthermore, it is possible that there are other reader proteins that participate in the titanium particle treatment. Previous studies have reported several m6A methylation-regulated bioactivities involving more than one reader protein (77–79), including YTH N6-methyladenosine RNA binding protein F1, which promotes mRNA translation, and YTH N6-methyladenosine RNA binding protein F3, which mediates the translation or degradation of targeted RNA, which could be potential readers that also participate in the titanium particle-induced bone loss (71). Further studies are needed to uncover the whole mechanism.

In addition, it is important to point out that the real-life concentration range of titanium particles within the body is wide (21). The in vivo concentration of particles is affected by various factors, including prosthesis type, implant time, sample location and detection method (80). It should be noted that the particle concentrations from different studies are difficult to be integrated as a specific range due to different measurement systems (81). Measured concentrations from several representative studies include 720–820 ppm (82), 66–1,734 µg/g tissue (83) and 103–5,759 µg/g tissue (84). It is possible to observe local high particle concentrations at specific timepoints for various reasons such as implant cracks (85). On the other hand, the cell density used in in vitro experiments can not simulate the real-life condition, and thus, it is difficult to directly employ the real-life concentration for in vitro experiments. The concentration used in the present study was drawn from previous studies (10,22,60); however, this specific concentration may not be physiologically relevant, which constitutes a potential limitation of the present study.

In conclusion, the results of the present study demonstrated that titanium particles reduced the expression of Mettl3, a key methyltransferase, and reduced the m6A modification of specific mRNAs. The reduced methylation of these transcripts decreased the specific sites recognized by reader protein Ythdf2, therefore decreased the mRNA degradation mediated by Ythdf2. And that might account for the increased expression levels of Smad7, Smurf1, NOD1 and RIPK2 transcripts, leading to the inhibition of osteogenesis and proinflammatory responses. These findings highlighted that Mettl3 may act as an upstream regulatory molecule in titanium particle-induced osteolysis. Based on the critical functions of Mettl3 in various types of cancer, Mettl3 has been considered a potential target in cancer treatment (86). Therefore, future studies might focus on the feasibility of employing Mettl3 as a therapeutic target of PPO, so that multiple pathways can be regulated simultaneously and to reach the targeted effect. The in vivo application and ideal drug concentration are also topics for further research. Thus, the present study may provide novel insights into potential therapeutic targets for the aseptic loosening of titanium prostheses.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by The Science and Technology Bureau of Nansha, Guangzhou (grant no. 2021ZD004).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XL, YY, XY and FD contributed to the study design. XL, YY, YH and XZ performed the experiments. EL and ZZ assisted in the data collection. ZZ and RX assisted in the data analysis. XL and YY wrote the manuscript and confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References