Abstract Background We aimed to investigate the effect and underlying mechanism of titanium (Ti) implant on the polarization of macrophages and subsequent effects on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs) affected by macrophages. Methods Firstly, the regulatory effect of Ti implant on macrophage polarization was investigated. Levels of M1 polarization markers and M2 polarization markers in macrophages were evaluated by immunofluorescence staining method and qPCR analysis. The osteogenic differentiation capacity of PDLSCs cultured with supernatants of macrophages of each group was evaluated by the Alizarin Red S (ARS) staining method and qPCR. Angiogenesis related genes were also evaluated in HUVECs cultured in supernatants of macrophages. To explore whether RNA m^5C modification can modulate the effects of Ti implant on macrophage regulation of osteogenic differentiation and angiogenesis, we analyzed the main genes related to m^5C in macrophages using RNA m^5C dot blotting and qPCR methods. The interaction between NSUN2 and IRF4 was verified by m^5C-RIP, RIP, and double-luciferase gene report experiments. Results Macrophages were activated as M1 macrophages under the interference of LPS, and macrophages attached to Ti implants were more easily activated as M2 macrophages under the action of LPS. Macrophages activated by Ti implant enhanced osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. NSUN2 level was up-regulated in macrophages treated with LPS and was down-regulated by Ti implant. Over-expression of NSUN2 attenuated the effect of Ti implant on M1 polarization promotion of macrophages and enhanced the M2 polarization promotion of macrophages. Up-regulation of NSUN2 weakened the effects of Ti implant on promotion the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. The KEGG analysis suggested that IRF4 was enriched in several inflammatory signaling pathways. Moreover, NSUN2 methylates IRF4 to affect the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. Conclusions Taken together, macrophages of M1 type can be stimulated by Ti implants in vitro, promoting osteogenic differentiation of PDLSCs and angiogenesis in HUVECs through NSUN2-mediated methylation of IRF4. Keywords: Titanium, Implant, Macrophage polarization, Osteogenesis, Angiogenesis Introduction In recent years, implant restoration has become the first choice for patients with denture defects, and has been widely recognized and applied in clinical practice. Titanium (Ti) implant is an ideal material for dental implant because of its good histocompatibility with human body, not easy to cause rejection, and is conducive to the formation of bone union. Ti also has high strength and hardness and light weight, can withstand chewing pressure, maintain the stability of the implant, and reduce the patient’s foreign body sensation [[34]1]. Ti implants with micro-nano surface are still the most widely used materials in clinical practice because of their fantastic mechanical properties and biocompatibility [[35]2]. Implant surface morphology plays an important role in regulating protein adsorption and cell surface integrin, and changes intracellular signal transduction pathways [[36]3]. Moreover, Ti implants with sandblasted, large-grit and acid-etched treatment can promote tissue healing and increase bone union [[37]4, [38]5]. Meanwhile, peri-implant tissues are susceptible to the same host-modulated plaque-induced factors that initiate and sustain periodontitis [[39]6]. Therefore, it is of significance to investigate the underlying mechanism of Ti implant with micro-nano surface on the dental implant therapy. After implantation, the Ti implant can induce innate immune response, and macrophages play an important role in this process as the defense line of the innate immune system [[40]7, [41]8]. It has been concluded that macrophages play an important role in tissue repair and reconstruction by secreting chemokines and cytokines [[42]9, [43]10]. Macrophages were activated after migration to the site of injury, showing two polarization states related to function: M1 type (promote inflammatory response) and M2 type (anti-inflammatory and promote tissue repair) [[44]11]. Relevant studies have shown that Ti implants can affect the differentiation and function of macrophages [[45]8, [46]11]. Therefore, it is very important to explore the influence of Ti implants on the polarization of macrophages. After implantation, macrophages secrete various factors to attract mesenchymal stem cells and fibroblasts to migrate to the injured site and initiate the process of tissue healing and bone regeneration [[47]12, [48]13]. It can be seen that in addition to inflammatory response, the participation of bone forming cells such as mesenchymal stem cells and osteoblasts in osteogenesis is also a necessary step in the treatment of peri-implant inflammation [[49]14–[50]16]. 5-methylcytosine (m^5C) modification of RNA is a common chemical modification of RNA in eukaryotes. It is found in a variety of RNA types, including mRNA, tRNA, and rRNA. m^5C modification is related to mRNA stability, splicing, cytoplasmic shuttling, and DNA damage repair [[51]17]. In addition, the abnormality of m^5C modification has been associated with the occurrence and development of various diseases [[52]18]. Two m^5C immune subtypes were reported in macrophages in prostate cancer [[53]19]. m^5C methylation can inhibit the transcription of macrophage-related chemokines, thereby inhibiting the recruitment of M2 macrophages in bladder cancer [[54]20]. Whether m^5C methylation regulates macrophages during Ti implantation is unknown. Accelerating the biological process of osseointegration at the implant-bone interface, as well as enhancing the quality of osseointegration, are crucial objectives within the realm of dental implant materials and surface modification [[55]21]. Therefore, we aimed to evaluate the effects of Ti on regulating the immune microenvironment and the polarization of macrophages. Furthermore, the osteogenesis of periodontal ligament stem cells (PDLSCs) and angiogenesis process of human umbilical vein endothelial cells (HUVECs) regulated by the conditioned medium of macrophages attached with Ti implant were also studied. Materials and methods Cell collection and culture Human monocytic cell line (THP-1) obtained from the Cell Bank of the Chinese Academy of Sciences were cultured in RPMI 1640 medium (HyClone) containing 10% fetal bovine serum (FBS; Bovogen) in a 37℃ constant temperature incubator with 5% CO[2] concentration. The THP-1 monocytes were differentiated to macrophages with 10 ng/ml phorbol-12-myristate-13-acetate (PMA) for 24 h [[56]22]. Macrophages were divided into three groups: control group macrophages at a density of 5 × 10^4 cells/cm^2 were plated onto tissue culture polystyrene (TCPS) in 24-well plates; LPS group macrophages at a density of 5 × 10^4 cells/cm^2 were plated onto TCPS in 24-well plates, and treated with 100 ng/mL Porphyromonas gingivalis lipopolysaccharide (LPS; InvivoGen) for 24 h [[57]23]; LPS + implant group macrophages at a density of 5 × 10^4 cells/cm^2 were plated onto rough-hydro grade 2 unalloyed Ti (2010.601-STM; 15 mm-diameter; Ti + polyoxymethylene, Institut Straumann AG) surfaces in 24-well plates, and treated with 100 ng/mL LPS for 24 h. PDLSCs were purchased from Bohu BiologicalTechnology Co.,Ltd. (Shanghai, China), and were incubated at 37℃ in DMEM containing 10% FBS. The PDLSCs in the 3–5 passage were inoculated in a 12-well plate at 5 × 10^5 cells/well and cultured in a medium containing β-glycerophosphate sodium (5 mmol/L), vitamin C (50 µg/mL), dexamethasone (100 mmol/L), and FBS (10%) (all purchased from Sigma-Aldrich) [[58]24]. HUVECs were purchased from the ATCC. HUVEC cells in the 3–5 passage were selected and achieved a fusion rate of 80% within 24 h after inoculation. Then the cells were starved, the complete culture medium was replaced with DMEM containing 0.2% FBS, and cultured for 24 h. Matrigel was diluted to 8–12 mg/mL and 50 µl-80 µL was added to each well in the 96-well plate. The plate is then placed in a cell incubator and incubated at 37℃ for 30 min to allow Matrigel to form a gel base. HUVECs were suspended in a medium containing 10% FBS at a concentration of 3 × 10^5 cells/ml, and cell suspension was added to each well. 6 h later, the expression of genes associated with angiogenesis were detected by quantitative reverse transcription PCR (RT-qPCR). Cell transfection The coding sequences of NSUN2 or IRF4 were inserted into the lentiviral vector pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro (HanHeng Biotechnology) to over-express NSUN2 or IRF4 in macrophages. The empty lentiviral vector pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro vector was used as the negative control. The packaging plasmids (4:3:1) were co-transfected into HEK293T cells for lentivirus packaging. After 48 h, lentivirus particles from the medium were collected and filtered. Subsequently, macrophages were infected with lentivirus according to the instructions using Lipofectamine 3000 (Thermo Fisher Scientific). 48 h later, infected cells were selected with puromycin to establish stable knockdown or overexpression cells. The mRNA levels of NSUN2 or IRF4 were detected by RT-qPCR to confirm the over-expression effect. Transfected macrophages were treated with LPS and/or attached to Ti implants. Immunofluorescence staining Firstly, macrophages were cultured in 24-well plates. macrophages were fixed with 4% paraformaldehyde to block the non-specific binding site when grew to about 80%. The cells were treated with 0.1% Triton X-100 to increase membrane permeability. The non-specific binding site was blocked with 5% bovine serum albumin (BSA) and incubated at room temperature for 30 min. Subsequently, primary antibodies including anti-iNOS (ab178945, 1:500, Abcam) and anti-CD206 (ab64693, 1 µg/mL, Abcam) were added to the sample and incubated at 4 °C overnight. After washed with PBS for three times with 5 min each time, the fluorescence labeled secondary antibody (1:1,000) was added to the sample and incubated at room temperature for 1–2 h. Cells were washed with PBS for three times with 5 min each time, and DAPI was added for 5 min staining. Finally, the cover glass was sealed with a sealing medium and sealed with nail polish to prevent the sample from drying out and moving under the microscope. The sample is placed in darkness and cells were observed with a fluorescence microscope. Conditioned medium collection and treatment Macrophages at a density of 5 × 10^4 cells/cm^2 were plated onto Tiimplants in 24-well plates, and treated with 100 ng/mL LPS for 24 h. Subsequently, the supernatant was collected, and was mixed with low-glucose DMEM (containing 10% FBS) in a ratio of 1:1 to form the conditioned medium for PDLSCs and HUVECs. PDLSCs are grouped as follows: PDLSCs cultured in supernatant of macrophages, PDLSCs cultured in supernatant of macrophages treated with LPS, and PDLSCs cultured in supernatant of LPS-treated macrophages attached to Ti. Mineralization by differentiated PDLSCs was detected after 21 d [[59]25]. HUVECs are grouped as follows: HUVECs cultured in supernatant of macrophages, HUVECs cultured in supernatant of macrophages treated with LPS, and HUVECs cultured in supernatant of LPS-treated macrophages attached to Ti. RT-qPCR The mRNA levels of M1 polarization markers (IL6, TNFA, and IL1B) and M2 polarization markers (TGFB1, IL10 and Arg1) from macrophages were detected by RT-qPCR. At the same time, levels of angiogenesis-related markers (VEGF, ACTA2, and COL1A1), osteogenesis-related markers (BGLAP, SPP1, and BMP2), and m^5C related genes (NOP2, NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, and TRDMT1) were also measured. Total RNA was extracted by Trizon method, and the absorbance of RNA at 260 nm and 280 nm was determined by ultraviolet spectrophotometry, and the RNA content was calculated. RNA was obtained for reverse transcription reaction by Promega MMLV reverse transcriptase (9PIM170). RT-qPCR was performed on 0.5 µL RT products by SYBR Premix Ex TaqTM (TaKaRa). The reaction conditions were as follows: PCR program was set at 94℃ 4 min, 94℃ 30 s, 56℃ 60 s, 72℃ 40 s for 40 cycle. GAPDH acted as the endogenous control, and the 2^−ΔΔCT method was for transcript expression level measurement. Primer sequences of this study are listed in Table [60]1. Table 1. Primer sequences Primer HGNC ID Sequence (5’ -> 3’) IL6 (interleukin-6) 6018 Forward: CTGCAAGAGACTTCCATCCAG Reverse: AGTGGTATAGACAGGTCTGTTGG TNFA (tumor necrosis factor-alpha) 11,892 Forward: CGGGCAGGTCTACTTTGGAG Reverse: ACCCTGAGCCATAATCCCCT IL1B (interleukin-1 beta) 5992 Forward: ATGATGGCTTATTACAGTGGCAA Reverse: GTCGGAGATTCGTAGCTGGA TGFB1 (transforming growth factor-beta) 11,766 Forward: GGCCAGATCCTGTCCAAGC Reverse: GTGGGTTTCCACCATTAGCAC IL10 (interleukin-10) 5962 Forward: GACTTTAAGGGTTACCTGGGTTG Reverse: TCACATGCGCCTTGATGTCTG ARG1 (arginase-1) 663 Forward: TGTCCCTAATGACAGCTCCTT Reverse: GCATCCACCCAAATGACACAT VEGF (vascular endothelial growth factor) 12,680 Forward: AGGGCAGAATCATCACGAAGT Reverse: AGGGTCTCGATTGGATGGCA ACTA2 (actin alpha 2, smooth muscle) 130 Forward: AAAAGACAGCTACGTGGGTGA Reverse: GCCATGTTCTATCGGGTACTTC COL1A1 (collagen type I alpha 1 chain) 2197 Forward: GAGGGCCAAGACGAAGACATC Reverse: CAGATCACGTCATCGCACAAC BGLAP (bone gamma-carboxyglutamate protein) 1043 Forward: CACTCCTCGCCCTATTGGC Reverse: CCCTCCTGCTTGGACACAAAG SPP1 (secreted phosphoprotein 1) 11,255 Forward: CTCCATTGACTCGAACGACTC Reverse: CAGGTCTGCGAAACTTCTTAGAT BMP2 (bone morphogenetic protein-2) 1069 Forward: ACCCGCTGTCTTCTAGCGT Reverse: TTTCAGGCCGAACATGCTGAG NOP2 (nucleolar protein 2, also known as NUM1) 7867 Forward: CGAAAGGCCCGAAAACAGAAG Reverse: TGGATAACTCTCCAGGCAATGT NSUN2 (NOL1/NOP2/Sun domain family member 2) 25,994 Forward: AGGTGGCTATCCCGAGATCG Reverse: GACTCCATGAATTGGTCCCATT NSUN3 (NOL1/NOP2/Sun domain family member 3) 26,208 Forward: CAATATGCCATCCTCTTCAACCG Reverse: AGGACTGTGTGATAGCCCCTC NSUN4 (NOL1/NOP2/Sun domain family member 4) 31,802 Forward: TGGGATAGTGTGAGTGCTAAGC Reverse: AAGCATCGAAGATTTGGGCTG NSUN5 (NOL1/NOP2/Sun domain family member 5) 16,385 Forward: ACCTGAAGCAGTTGTACGCTC Reverse: CCCCTTCCCCAGCAATAATTC NSUN6 (NOL1/NOP2/Sun domain family member 6) 23,529 Forward: AAGACAACAGGGTGAAGTGATTG Reverse: TCCATCAAATTCTTTGGCTCCTT NSUN7 (NOL1/NOP2/Sun domain family member 7) 25,857 Forward: TCTCAAGGTGGTCTACCGAAA Reverse: TTCATTGCGTGTGTTAGCTGT TRDMT1 (tRNA aspartic acid methyltransferase 1) 2977 Forward: GCGCTGCGAGAAAGTCATATC Reverse: CCCTGTAGGCCAATTCTTGTG IRF4 (interferon regulatory factor 4) 6119 Forward: GCTGATCGACCAGATCGACAG Reverse: CGGTTGTAGTCCTGCTTGC GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase) 4141 Forward: GGACACTGAGCAAGAGAGGC Reverse: TTATGGGGGTCTGGGATGGA [61]Open in a new tab Human gene names follow Gene/Protein Nomenclature Guidelines (HGNC, [62]http://www.genenames.org) Osteogenic differentiation and alizarin Red S staining Alizarin Red S (ARS) staining is usually used to detect and observe calcium salt deposits to assess the degree of osteogenic differentiation of cells after 21 days [[63]25]. The staining steps are as follows: osteogenic differentiated PDLSCs were fixed with 4% paraformaldehyde (pre-cooled at 4℃) at room temperature for 30 min. After washing twice with PBS or double steaming water to remove the remaining fixative, the configured ARS dye solution (1%, pH 4.2; Sigma-Aldrich, A5533) was added to the sample cells and incubated at room temperature for 1 h. Then the ARS dye was discarded and cells were gently washed with double steaming water several times to remove the background stain. The stained sample can be viewed and photographed under an optical microscope (Olympus, IX73, Japan), and the calcium deposit area appears to be bright red. The ARS was dissolved with 10% acetopyridine (w/v) and the staining intensity was quantified by measuring optical density values at 562 nm to assess the degree of calcification. RNA m5C dot blotting Total RNA was extracted by TRIZOL reagent (Invitrogen). Then a Bio-Dot apparatus (Bio‐Rad) was used to transfer the mRNA which has been denatured in advance onto a nitrocellulose membrane (Amersham). Then the separated RNA were transferred onto a positively charged nylon membrane followed by cross-linking with Ultraviolet light. Then the membranes were blocked, incubated m^5C antibody (ab214727, 1.0 µg/mL, Abcam), followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody. Finally, we used the enhanced chemiluminescence (Bio‐Rad) to visualize the membrane, and the m^5C level of each group was quantitatively determined by Image J software. Bioinformatic analysis The [64]GSE173078 dataset was downloaded from the GEO database. This expression profiling is related to mRNA profiles from 12 periodontitis, 12 gingivitis, and 12 healthy patients. Differentially expressed mRNA was analyzed using a paired t-test using the limma R package, and mRNA with adjusted p-value < 0.05 and |logFC > 1| were considered significant mRNA. To investigate the potential biological functions, KEGG pathway enrichment analysis was performed by the R package clusterProfiler. The top 10 pathways were selected based on the adjusted p-value ranking. RNAm^5Cfinder ([65]http://www.rnanut.net/rnam5cfinder/) was used to predict the potential m^5C modification site of IRF4. RNA immunoprecipitation (RIP) The RIP assay was performed using a Magna RIP^® RIP Kit (17–700, Millipore) according to the manufacturer’s instructions. Briefly, macrophages were harvested and washed twice with cold PBS, and the cell pellet was incubated with RIP lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, Protease Inhibitor, RNase Inhibitor) on ice for 30 min. One tenth portion of the cell lysate was used as input. The rest of the cell lysate was incubated with either Rabbit IgG (10285-1-AP, Proteintech)-coated beads or anti-NSUN2 (ab259941, 1/30; Abcam)-coated beads for 4 h at room temperature. Afterward, the beads-antibody-protein-RNA complex was washed five times with ice-cold washing buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, RNase inhibitor). Then, immunoprecipitated sample was digested with proteinase K and the RNA was precipitated with glycogen (Thermo Scientific, AM9516). Total RNA was extracted by TRIzol reagent followed by RT-qPCR. m5C-RIP Purified mRNA was fragmented RNA Fragmentation Reagents (Invitrogen, AM8740). Specific 2.5 µg m^5C antibodies (ab214727, Abcam) were incubated with 400 ng RNA fragments (100-nucleotide-long) in immunoprecipitation buffer and incubated by rotating at 25℃ for 1 h to bind the antibody to the m^5C modification site. The mixture was then immunoprecipitated by incubation with protein A/G magnetic beads at 4℃ for 5 h. The magnetic bead (Thermo Fisher Scientific, 10002D) was washed several times with IP buffer, and the bound RNA fragments were eluted from the beads by proteinase K digestion at 554℃ for 60 min. Finally, the RNA containing m^5C modification was isolated from the eluate by phenol-chloroform extraction and ethanol plus glycogen. Finally, qRT-PCR analysis was performed to evaluate the m^5C modification level of IRF4. Double luciferase reporter gene experiment The wild-type (WT) and mutant (Mut) fragments of IRF4 were constructed and inserted into the the pGM-CMV-Luc vector (Yeasen Biotech, Shanghai, China). Cells were planted in a 24-well plate with 100,000 cells per well and attached to the wall for 36 h. 900 ng pGM-CMV-Luc and 40 ng Renilla luciferase plasmid were transfected per well (20:1) 12 h later. After 24 h, the fluorescence values of the lysed cells were determined according to the kit procedure. mRNA stability assay Macrophages were inoculated in 12-well plates overnight and then treated with 5 µg/mL actinomycin D (MedChemExpress) at 0, 1, 4, 8 and 12 h. Total RNA was then isolated and the results were analyzed by RT-qPCR. Statistical analysis The experimental data were analyzed by GraphPad Prism software version 8.3, and the data operation between the two groups was represented by mean ± SD and compared with t test. One-way ANOVA was used to compare the mean of multiple groups. Tukey′s post hoc test was used to compare pairwise comparisons between groups. There were three iterations of each experiment in this study. The p value less than 0.05 means the difference is statistically significant. Results Macrophages activated by Ti implant enhance osteogenic differentiation of PDLSCs and angiogenesis of HUVECs Firstly, the regulatory effect of Ti implant on macrophage polarization was investigated. The levels of M1 polarization markers and M2 polarization markers in macrophgages of different groups were detected. In LPS-stimulated macrophages, immunofluorescence staining with specific iNOS antibody showed that iNOS protein was mainly distributed in the cytoplasm, showing a strong fluorescence signal. This result indicated that LPS stimulation can effectively induce the expression of iNOS in macrophages, and the expression of iNOS is related to the activation of M1 polarization in macrophages. In contrast to that, LPS stimulation decreased the fluorescence signal of CD206 protein distributed in the cytoplasm of macrophages, indicating that activation of M1 polarization of macrophages was suppressed by LPS treatment. However, the fluorescence intensity of iNOS in LPS-treated macrophages attached to Ti implant was lower than that in LPS-treated macrophages while CD206 fluorescence intensity was higher (Fig. [66]1A and B). qPCR analysis demonstrated that levels of M1 polarization related genes (IL6, TNFA, and IL1B) were increased while levels of M2 polarization related genes (TGFB1, IL10, and ARG1) were decreased in LPS-treated macrophages. Ti implant attachement significantly weakened the effects of LPS treatment on regulating these genes levels (Fig. [67]1C and D, p < 0.01). Taken together, macrophages were activated into M1 macrophages under the interference of LPS, and M2 macrophages were more easily activated when the macrophages attached to the Ti implant were treated with LPS. Subsequently, the effects of Ti implant on macrophage regulation of osteogenic differentiation and angiogenesis was studied. As shown in Fig. [68]1E, angiogenesis related genes including VEGF, ACTA2, and COL1A1 were evaluated in three groups of HUVECs cultured in different conditioned medium for 6 h. qPCR analysis suggested that supernatant of macrophages treated with LPS suppressed the angiogenic capacity of HUVECs while HUVECs cultured in supernatant of LPS-treated macrophages attached to Ti implant showed higher angiogenic potential by enhancing the mRNA levels of VEGF, ACTA2, and COL1A1 (Fig. [69]1E, p < 0.01). At the same time, LPS-treated macrophage supernatants reduced calcium accumulation of PDLSCs after osteogenic differentiation induction for 21 days according to the ARS staining, and Ti implant attachment increased the mineralization of PDLSCs after osteogenic differentiation induction (Fig. [70]1F, p < 0.01). Osteogenesis-related genes including BGLAP, SPP1, and BMP2 in PDLSCs were decreased under the LPS interference, but increased by the Ti implant (Fig. [71]1G, p < 0.01). Fig. 1. [72]Fig. 1 [73]Open in a new tab Macrophages activated by Ti implant enhance osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. (A-B) The representative immunofluorescence images of untreated macrophages (control), macrophages treated with LPS (100 ng/mL) (LPS), and LPS-treated macrophages which plated on Ti implant (LPS + Implant). M1 macrophage marker iNOS and M2 macrophage marker CD206 were monitored using corresponding antibodies. n = 3. (C-D) qPCR analysis of M1 polarization related genes (IL6, TNFA, and IL1B) and M2 polarization related genes (TGFB1, IL10, and ARG1) in macrophages of each group. n = 3, **p < 0.01. (E) qPCR analysis of angiogenesis related genes including VEGF, ACTA2, and COL1A1 were evaluated in of HUVECs cultured in different conditioned medium of macrophages. n = 3, **p < 0.01. (F) The representative ARS staining images and quantitative analysis of PDLSCs cultured with different conditioned medium of macrophages. n = 3, **p < 0.01. (G) qPCR analysis of osteogenesis-related genes including BGLAP, SPP1, and BMP2 in PDLSCs cultured in different conditioned medium of macrophages. n = 3, **p < 0.01 Over-expression of m5C RNA methyltransferase NSUN2 attenuates the effects of Ti implant on macrophages To explore whether RNA m^5C modification can modulate the effects of Ti implant on macrophage regulation of osteogenic differentiation and angiogenesis, we analyzed the main genes related to m^5C in macrophages of each groups. The RNA m^5C dot blotting data suggested that m^5C levels in macrophages of LPS group were higher than that in control group while Ti implant attachement down-regulated the total m^5C levels in macrophages (Fig. [74]2A, p < 0.01). Furthermore, m^5C related genes including NOP2, NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, and TRDMT1 were detected in three groups of macrophages, and the qPCR analysis then demonstrated that NOP2, NSUN2, NSUN3, NSUN5, NSUN6, and NSUN7 were significantly highly expressed in macrophages of LPS group compared with the control group. After attached to Ti implant, NSUN2 level was further significantly down-regulated in macrophages while levels of NOP2, NSUN3, NSUN5, NSUN6, and NSUN7 were not significantly changed (Fig. [75]2B, p < 0.05). Subsequently, the regulatory role of NSUN2 was investigated. After transfection of macrophages, the expression of NUSN2 increased by more than 10 times compared with the control vector group according to the qPCR analysis (Fig. [76]3A, p < 0.01). Overexpression of NSUN2 weakened the promoting effect of Ti implant on M1 polarization of macrophages and enhanced the promoting effect of M2 polarization of macrophages, which showed an increase in iNOS protein level and a decrease in CD206 protein level, as well as an increase in the gene levels of IL6, TNFA and IL1B, and a decrease in the gene levels of TGFB1, IL10 and ARG1 (Fig. [77]3B - E, p < 0.01). Up-regulation of NSUN2 also weakened the effects of Ti implant on macrophages promoting the osteogenic differentiation of PDLSCs, which was manifested by decreased levels of VEGF, ACTA2 and COL1A1 genes and decreased levels of mineralization (Fig. [78]3F and G, p < 0.01). At the same time, the down-regulation of BGLAP, SPP1 and BMP2 genes also suggested that the up-regulation of NSUN2 impaired the ability of Ti implants to promote macrophage induction of HUVECs angiogenesis. (Fig. [79]3H, p < 0.01). Fig. 2. [80]Fig. 2 [81]Open in a new tab Over-expression of m^5C RNA methyltransferase NSUN2 attenuates the effects of Ti implant on macrophages. (A) The representative RNA m^5C dot blotting images and quantitative analysis of untreated macrophages (control), macrophages treated with LPS (100 ng/mL) (LPS), and LPS-treated macrophages which plated on Ti implant (LPS + Implant). n = 3, **p < 0.01. (B) The main genes related to m^5C in macrophages of each groups were analyzed by qPCR method. n = 3, *p < 0.05, **p < 0.01 Fig. 3. [82]Fig. 3 [83]Open in a new tab NSUN2 affects the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. (A) The relative mRNA expression of NSUN2 in macrophages transfected with NSUN2 over-expression vector. n = 3, **p < 0.01. (B-C) The representative immunofluorescence images of macrophages treated with LPS (100 ng/mL) and transfected with empty vector (LPS + vector), LPS-treated macrophages which plated on Ti implant and transfected with empty vector (LPS + Implant + vector), and LPS-treated macrophages which plated on Ti implant and transfected with NSUN2 over-expression vector (LPS + Implant + vector). M1 macrophage marker iNOS and M2 macrophage marker CD206 were monitored using corresponding antibodies. n = 3. (D-E) qPCR analysis of M1 polarization related genes (IL6, TNFA, and IL1B) and M2 polarization related genes (TGFB1, IL10, and ARG1) in macrophages of each group. n = 3, **p < 0.01. (F) qPCR analysis of angiogenesis related genes including VEGF, ACTA2, and COL1A1 were evaluated in of HUVECs cultured in different conditioned medium of macrophages. n = 3, **p < 0.01. (G) The representative ARS staining images and quantitative analysis of PDLSCs cultured with different conditioned medium of macrophages. n = 3, **p < 0.01. (H) qPCR analysis of osteogenesis-related genes including BGLAP, SPP1, and BMP2 in PDLSCs cultured in different conditioned medium of macrophages. n = 3, **p < 0.01 NSUN2 methylates IRF4 to affect the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs mRNAs in [84]GSE173078 dataset are related to periodontitis and gingivitis, and inflammatory factors associated with periodontal disease were screened. The KEGG analysis of differentially expressed genes from [85]GSE173078 dataset was performed, and the biological pathway suggested that IRF4 was enriched in several inflammatory signaling pathways (Fig. [86]4A). IRF4 has been reported to exert regulatory role in immune infiltration [[87]26], macrophage polarization [[88]27, [89]28], and cell cycle [[90]29]. The suppression effect of IRF4 on osteogenic differentiation was also been empathized according to various studies [[91]30, [92]31]. Therefore, we speculated that IRF4 may be m^5C modified mediated by NSUN2 in macrophages. The m^5C-RIP assay was implemented to assess the m^5C modification status of IRF4 mRNA. The results exhibited that the level of IRF4 can be enriched by m^5C antibody (Fig. [93]4B, p < 0.01). RIP followed by qPCR found that compared with IgG antibody, NSUN2 antibody significantly enriched IRF4 (Fig. [94]4C, p < 0.01). Subsequently, the potential m^5C modification sites of IRF4 predicted by RNAm^5Cfinder suggested that IRF4 may be m^5C modified at three sites (Fig. [95]4D). After mutation at each of the three sites, the results of the double-luciferase gene report experiment suggested that there was no significant change in relative luciferase activity before and after NSUN2 over-expression at mutation sites 1 and 3, but after mutation at site 2, up-regulation of NSUN2 could significantly reduce relative luciferase activity (Fig. [96]4E, p < 0.01). Moreover, over-expression of NSUN2 promoted the degradation of IRF4 mRNA (Fig. [97]4F, p < 0.01). After transfection of macrophages, the expression of IRF4 increased by more than 10 times compared with the control vector group according to the qPCR analysis (Fig. [98]5A, p < 0.01), the macrophage polarization related proteins and genes were evaluated. Both of the immunofluorescence staining and qPCR analysis suggested that NSUN2 promoted the M1 macrophage polarization while IRF4 weakened the effects of NSUN2 by promoting M2 macrophage polarization (Fig. [99]5B - E, p < 0.01). Meanwhile, IRF4 also reversed the effects of NSUN2 on the regulatory function of macrophages on inhibition of osteogenic differentiation of PDLSCs and angiogenesis of HUVECs (Fig. [100]5F - H, p < 0.01). Fig. 4. [101]Fig. 4 [102]Open in a new tab NSUN2 methylates IRF4 to affect its mRNA stability. (A) The KEGG analysis of differentially expressed genes from [103]GSE173078 dataset was performed, and the biological pathway suggested that IRF4 was enriched in several inflammatory signaling. (B) The interaction between IRF4 and m^5C antibody was verified by m^5C-RIP method. n = 3, **p < 0.01. (C) The interaction between IRF4 and NSUN2 antibody was verified by RIP method. n = 3, **p < 0.01. (D) The potential m^5C modification sites of IRF4 predicted by RNAm^5Cfinder. (E) Dual luciferase reporter assay is performed to evaluate the binding of IRF4 and NSUN2. n = 3, **p < 0.01. (F) qPCR analysis indicates that the levels of IRF4 expression in 293T cells treated with actinomycin D (2 µg/mL) at the indicated time points. n = 3, **p < 0.01 Fig. 5. [104]Fig. 5 [105]Open in a new tab NSUN2 methylates IRF4 to affect the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. (A) The relative mRNA expression of IRF4 in macrophages transfected with IRF4 over-expression vector. n = 3, **p < 0.01. (B-C) The representative immunofluorescence images of macrophages transfected with empty vector (vector), macrophages transfected with NSUN2 over-expression vector and empty vector (NSUN2 + vector), and macrophages transfected with NSUN2 over-expression and IRF4 vector (NSUN2 + IRF4). M1 macrophage marker iNOS and M2 macrophage marker CD206 were monitored using corresponding antibodies. n = 3. (D-E) qPCR analysis of M1 polarization related genes (IL6, TNFA, and IL1B) and M2 polarization related genes (TGFB1, IL10, and ARG1) in macrophages of each group. n = 3, **p < 0.01. (F) qPCR analysis of angiogenesis related genes including VEGF, ACTA2, and COL1A1 were evaluated in of HUVECs cultured in different conditioned medium of macrophages. n = 3, **p < 0.01. (G) The representative ARS staining images and quantitative analysis of PDLSCs cultured with different conditioned medium of macrophages. n = 3, **p < 0.01. (H) qPCR analysis of osteogenesis-related genes including BGLAP, SPP1, and BMP2 in PDLSCs cultured in different conditioned medium of macrophages. n = 3, **p < 0.01 Discussion Ti and its alloys are widely used in orthopedic implants due to their exceptional mechanical properties, chemical stability, and biocompatibility [[106]32]. The osteogenesis and angiogenesis capacity of peri-implant tissues are key indexes in the application of Ti implant [[107]33–[108]35]. Interestingly, a high proportion of M2 macrophages infiltrates the damaged tissue and inhibits inflammation around the implant, which is conducive to the formation of implant osseous union and angiogenesis [[109]36]. Therefore, macrophages play an important role in the process of Ti implant therapy. Our data suggested that Ti implant showed favorable effects on promoting the M2 polarization transformation. Meanwhile, the M2 type macrophages promoted the osteogenic differentiation of PDLSCs and angiogenesis of HUVECs in vitro. The role of macrophages in promoting bone formation by Ti implants is multifaceted. Macrophages are not only the first cells contacted by implants after implantation, but also the main regulator of the integration of tissues and biomaterials in regulating the innate immune response [[110]37]. Macrophages play an important role in bone formation, bone remodeling and fracture healing, and can induce bone differentiation of mesenchymal stem cells [[111]34]. Different physicochemical and biological modifications on the surface of Ti implants can play a key role in implant bone binding by activating the M1 inflammatory polarization direction of macrophages or the M2 tissue healing direction [[112]4, [113]38]. Meanwhile, implant surface modification also has an important impact on the induction of bone formation by macrophages. The future development direction is to explain the healing mechanism of implant-host interaction from the perspective of immunology, and develop a new type of Ti implant, which can induce bone formation and obtain homeostasis of bone coupling through the immune regulation of macrophages, so as to achieve early and long-term stable bone union. In this study, we found that Ti implant showed favorable effects on promoting the M2 polarization transformation and subsequent osteogenesis and angiogenesis in vitro. Afterwards, the underlying mechanism was studied. The RNA modification of m^5C is mediated by the NSUN family (NSUN1-7) and DNMT homolog DNMT2. ALYREF and YBX1 RNA junction proteins called reader are responsible for identifying sites modified by m^5C. DNMT2 and NSUN2 are writers of methyltransferase involved in the production of m^5C modification. A methyltransferase called TET1 eraser can remove the m^5C modification. They worked together to keep the m^5C modification in dynamic balance [[114]39–[115]41]. There is growing evidence that m^5C methylation plays a role in gene expression and pathological processes in several human diseases by regulating mRNA stability splicing and protein translation. As for m^5C studies related to angiogenesis and osteogenesis, RNA-binding protein YBX1 in angiogenesis-dependent bone formation and provided a therapeutic approach for ameliorating osteoporosis [[116]42]. Moreover, m^5C modification of LINC00324 has been reported to promote angiogenesis in glioma [[117]43]. Therefore, we hypothesized that m^5C modification has a regulatory role in the regulation of macrophages on osteogenesis and angiogenesis in Ti implant. Our data suggested that total m^5C levels were elevated in LPS treated macrophages, and Ti implant decreased the m^5C levels. Further, we found that this m^5C modification is dominated by NSUN2. Interestingly, it has been found that NSUN2 mediated m^5C modification contributes to the angiogenesis in glioma [[118]43]. Our data was also in line with this study that over-expression of NSUN2 significantly weakened the effects of Ti implant on macrophages, and the osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. Subsequently, the bioinformatic analysis indicated that IRF4 is an aberrant expressed genes related to osteogenic differentiation, and is related to several inflammatory pathways. IRF4 has been reported to exert regulatory role in immune infiltration [[119]26], macrophage polarization [[120]27, [121]28], and cell cycle [[122]29]. The suppression effect of IRF4 on osteogenic differentiation was also been empathized according to various studies [[123]30, [124]31]. In this study, IRF4 can be enriched by m^5C antibody and NSUN2 antibody. Moreover, over-expression of NSUN2 promoted the degradation of IRF4 mRNA. The promotion of M2 macrophage polarization induced by IRF4 was also been verified in this study, which is inline with the previous studies [[125]27, [126]28]. Meanwhile, IRF4 also reversed the effects of NSUN2 on the regulatory function of macrophages on inhibition of osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. With further research on the mechanism of titanium implants, it is possible to develop new types of titanium implants targeting NUSN2 and IRF4 in the future. For instance, drug delivery systems targeting NUSN2 and IRF4 may be designed on the implant surface to achieve localized drug release. Additionally, these implants not only promote bone formation through the immunomodulation of macrophages, but also achieve early and long-term stable bone union by optimizing angiogenesis. This will provide a more efficient and stable implant selection for the clinic, and improve the treatment effect and quality of life of patients. There are some limitation in current work. Which cytokines are secreted after LPS treatment of macrophages and how these factors affect angiogenesis or osteogenic differentiation should be further studied. Conclusion This study suggests that Ti implant induces the M2 type macrophage, which subsequently promotes angiogenesis and bone formation. This effect is likely mediated through the NSUN2 mediated m^5C modification of IRF4. Acknowledgements