Dicalcium silicate-induced mitochondrial dysfunction and autophagy-mediated macrophagic inflammation promotes osteogenic differentiation of BMSCs

Abstract Dicalcium silicate (Ca2SiO4, C2S) has osteogenic potential but induces macrophagic inflammation. Mitochondrial function plays a vital role in macrophage polarization and macrophagic inflammation. The mitochondrial function of C2S-treated macrophages is still unclear. This study hypothesized: (i) the C2S modulates mitochondrial function and autophagy in macrophages to regulate macrophagic inflammation, and (ii) C2S-induced macrophagic inflammation regulates osteogenesis. We used RAW264.7 cells as a model of macrophage. The C2S (75–150 μg/ml) extract was used to analyze the macrophagic mitochondrial function and macrophage-mediated effect on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs). The results showed that C2S extract (150 μg/ml) induced TNF-α, IL-1β and IL-6 production in macrophages. C2S extract (150 μg/ml) enhanced reactive oxygen species level and intracellular calcium level but reduced mitochondrial membrane potential and ATP production. TEM images showed reduced mitochondrial abundance and altered the mitochondrial morphology in C2S (150 μg/ml)-treated macrophages. Protein level expression of PINK1, Parkin, Beclin1 and LC3 was upregulated but TOMM20 was downregulated. mRNA sequencing and KEGG analysis showed that C2S-induced differentially expressed mRNAs in macrophages were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy. The conditioned medium from C2S-treated macrophage robustly promoted osteogenic differentiation in BMSCs. In conclusion, our results indicate mitochondrial dysfunction and autophagy as the possible mechanism of C2S-induced macrophagic inflammation. The promotion of osteogenic differentiation of BMSCs by the C2S-induced macrophagic inflammation suggests the potential application of C2S in developing immunomodulatory bone grafts.

Mounting evidence indicates that mitochondrial functions are involved in macrophage functions and immune responses [17][18][19].Mitochondrial dysfunction had been reported to prevent repolarization of polarized macrophages [16].However, the mitochondrial function in C 2 S-induced macrophagic inflammation is still unclear.Therefore, it is important to investigate the mitochondrial function of C 2 S-treated macrophages and the role of macrophagic inflammation on bone regeneration.
During the initial stage of bone defect healing, acute inflammation is essential to trigger bone regeneration [20][21][22].A low level of macrophagic inflammation promotes osteogenesis via autophagy regulation [23].Lack of recruitment and activation of macrophages impairs bone defect healing in animal models indicating the significant role of macrophages in bone regeneration [24].However, prolonged macrophagic inflammation impedes bone repair [25,26].This might be related to chronic inflammation caused by the failure of switching of macrophages from M1 to M2 phenotype [16] and the failure in the spatiotemporal expression of proinflammatory cytokines [27][28][29][30][31].There is an opposing role of proinflammatory cytokines such as interleukin (IL)-1b, IL-6 and tumor necrosis factor-alpha (TNF-a) that could inhibit osteogenic differentiation and some findings reported that these cytokines could promote osteogenic differentiation [25][26][27].Amorphous calcium phosphate nanoparticles-induced macrophagic inflammation had been recently reported to weaken osteogenic differentiation bone marrow-derived mesenchymal stem cells (BMSCs) [32,33].Our previous research results showed that C 2 S induces inflammation in RAW264.7 cells and promotes the release of TNF-a, IL-1b and IL-6 through toll-like receptor-2 (TLR2)-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB) and c-Jun N-terminal kinase (JNK) pathways [2].Horeover, the effect of C 2 S-induced macrophagic inflammation on osteogenic differentiation of precursor cells is still unknown.
Our previous studies focused on investigating the osteogenic potential of C 2 S and the underlying mechanisms [7].In this study, we aimed to elucidate the role of mitochondrial function in C 2 Sinduced macrophagic inflammation as well as the role of C 2 S-induced macrophagic inflammation on the osteogenic differentiation of precursor cells.Our findings revealed that C 2 S regulates macrophagic inflammation via inducing mitochondrial dysfunction and autophagy.Interestingly, C 2 S-induced macrophagic inflammation robustly promoted osteogenic differentiation of precursor cells.

Characterization of C 2 S and C 2 S extract preparation
C 2 S is a well-characterized biomaterial frequently used in dental capping and drug delivery [1,6].Our previous studies had performed characterization and analysis of the C 2 S micro-particles used in this experiment [2,7,10].C 2 S micro-particles were synthesized and characterized in the Shanghai Institute of Ceramics, Chinese Academy of Sciences as described previously [7].C 2 S particles with a purity of 99% were obtained.Different concentrations (75, 100, 125, or 150 lg/ml) of C 2 S particles were dispersed in high glucose Dulbecco's modified eagle medium (DMEM) and sonicated for 4 h to prepare the C 2 S extracts [7].The C 2 S extracts were filtered with a 0.22-lm filter and stored at 4 C.

Macrophage cell culture
The mouse macrophage cell line, RAW264.7 (National Collection of Authenticated Cell Cultures, TCM13, Shanghai, China) was used as a model of macrophage.The cells were maintained in high glucose DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin solution (Gibco, USA), under 37 C, supplied with 5% CO 2 and 95% air atmosphere.

Effect of C 2 S extract on macrophage vitality
Macrophages (3000 cells/well) were cultured in 96-well plates in presence of 0, 75, 100, 125 or 150 lg/ml C 2 S extract groups and incubated the cells for 4, 24, 48 and 72 h.The cell vitality was evaluated using the cell counting kit-8 assay (CCK8, BestBio, China) as described previously [7].

Cytokine expression analysis by ELISA
After 24 h culture of macrophages with C 2 S extract (150 lg/ml), the conditioned medium was collected and centrifuged at 3000 rpm for 10 min.Levels of TNF-a, IL-1b and IL-6 were measured in the supernatant using mouse ELISA kit for TNF-a, IL-1b and IL-6 (Thermo, USA) following the manufacturer's instruction.

mRNA sequencing
The macrophages were treated with and without C 2 S (150 mg/ml) for 24 h were used for RNA sequencing.Total RNA was extracted by using the Trizol kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol.The RNA concentration was determined using Qubit, and the RNA amount and purity of each sample were assessed with a NanoDrop spectrophotometer (NanoDrop2000, Wilmington, DE, USA).RNA was isolated in 40 ml of DEPC water and stored at À80 C. RNA-seq libraries were prepared by using the Illumina TruseqTM RNA sample prep Kit and were sequenced using an Illumina NovaSeq PE150.Low-quality base and adapters were removed by using "fastp" [34].Clean reads were aligned to the mouse genome (mm10) reference genome using STAR [35].Transcript per million value and count number of reads mapped to each gene by using RNA-Seq by expectation-maximization [36].And the 'edgeR' R software package was used to calculate the differential expression analysis between the two groups [37].Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed using the 'clusterProfiler' R package [38].KEGG terms with corrected P < 0.05 were considered significantly enriched.

Reactive oxygen species generation
The C 2 S extract-induced oxidative stress response in the macrophage was analyzed using a reactive oxygen species (ROS) kit (BestBio, China) as described previously [39].RAW264.7 cells were cultured with C 2 S extract (150 lg/ml) for 4 h.ROS generation efficiency was visualized under a confocal microscope using a green laser (502 nm) (Leica, TCS SP8, Wetzlar, Germany).ROS quantitative analysis was performed in a fluorescence microplate reader with an excitation wavelength of 502 nm and an emission wavelength of 530 nm.Hydrogen peroxide (100 lM) was used as a ROS inducer positive control.

Mitochondrial membrane potential analysis
Mitochondrial membrane potential (MtMP) was analyzed using a JC-1 kit (BestBio, China) according to the manufacturer's instructions.RAW264.7 cells were cultured with C 2 S extract (150 lg/ml) for 4 h.A confocal fluorescence microscope with the laser (488 nm) was used to visualize the fluorescence of the JC-1 monomers and aggregates.Flow cytometry analysis was used to quantify the MtMP of the macrophages.Carbonyl cyanide m-chlorophenylhydrazone (CCCP,

Intracellular calcium ion concentration analysis
The cytoplasmic free calcium ion concentration in macrophages upon treatment with C 2 S was analyzed by the intracellular calcium staining kit (Bestbio, China).RAW264.7 cells were cultured with C 2 S extract (150 lg/ml) for 4 h.A confocal fluorescence microscope with the laser (488 nm) was used to visualize the intracellular calcium.Flow cytometry (526 nm) analysis was used to quantify the concentration of free calcium ions in the cytoplasm.

Adenosine triphosphate production
ATP production in macrophages was analyzed using a bioluminescence ATP assay kit (Beyotime, China).Briefly, after culture with C 2 S extract (150 lg/ml) for 24 h, the RAW264.7 cells were lysed using the lysate solution provided in the kit, centrifuged at 12 000 g (4 C for 5 min), and the supernatant was collected.The intracellular ATP was analyzed according to the manufacturer's instructions.

Autophagy analysis
Autophagy in macrophages was detected by enumerating the autophagosome formation using MDC (Monodansyl cadaverine) kit (Bestbio, China) and MitoTracker (Thermo, USA) following the manufacturer's instruction by confocal microscopy and flow cytometric analysis.Briefly, the C 2 S extract (150 lg/ml) treated macrophages were cultured for 4 h and then stained with 50 lM of MDC and costained with 500 nM of MitoTracker and incubated in the dark for 20 min, After incubation, the cells were washed with 1Â PBS and the autophagosome formation was visualized under confocal microscopy with a blue laser (335 nm) and the mitochondria were visualized with a red laser (579 nm).The autophagosome formation was quantitatively analyzed by using flow cytometry (518 nm).

Transmission electron microscope analysis
TEM analysis was performed to observe the formation of autophagosomes and the morphology of mitochondria.Macrophages were cultured with or without C 2 S extract (150 lg/ml).After 4 h culture, cells were fixed with precooled glutaraldehyde and stored at 4 C overnight.The images were taken by using TEM (Tecnai Spirit, FEI, Hillsboro, USA) with maximum resolution and magnification as described previously [40].

Western blot analysis
The cells were lysed with radioimmunoprecipitation lysis buffer (Beyotime, China) containing protease inhibitors (PMSF, Beyotime, China) on ice for 20 min.Cell lysates were used for western blot assay of Beclin1, LC3B and TOMM20.For PINK1 and Parkin, western blot analysis mitochondrial protein was extracted using a cell mitochondrial isolation kit (Beyotime, China).The protein concentrations in cell lysate and mitochondrial lysate were determined with a BCA protein assay kit (Bestbio, China).Lysate containing 20 lg protein was separated using 10-15% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) using wet blotting techniques.The PVDF membranes were blocked with 3% skim milk powder for 1 h and washed thrice with 1Â PBST and then incubated overnight at 4 C with primary antibodies.Protein from macrophages was detected using primary antibodies against PINK1 (Abcam, Cambridge, UK), Parkin (Abcam, Cambridge, UK), Beclin1 (Abcam, Cambridge, UK) and LC3B (Abcam, Cambridge, UK) in 1:2000 dilution.Protein from BMSCs was detected using primary antibody against RUNX2 (Abcam, Cambridge, UK), OPN (Bioss, USA) and GAPDH (Abcam, Cambridge, UK).After three washes with PBST, the PVDF membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA, USA, 1:3000 dilution) for 2 h and the bands were visualized using suitable chemiluminescent substrate and the band images were captured by using Gel doc system equipped with a luminometer Gelview 6000 Pro (BLT, Guangzhou, Guangdong, China).GAPDH (Cell Signaling Technology) was used as a housekeeping protein for quantitative analysis of Beclin1, LC3B and TOMM20.b-tubulin (Cell Signaling Technology) was used as a housekeeping protein for quantitative analysis of PINK1 and Parkin.The Image J software (http://rsb.info.nih.gov/ij/) was used to quantify the band densities.

Inhibition of autophagy in C 2 S-treated macrophages
An autophagy inhibitor 3-methyladenine (3MA, 0.1 mM) was added in macrophage culture to block autophagy.After 1 h, C 2 S (150 lg/ml) was added to the culture.After culturing for 24 h, the conditioned medium was collected and the concentrations of TNF-a, IL-1b and IL-6 were detected by ELISA.

Osteogenic differentiation of bone marrowderived mesenchymal stem cells
After 24 h of macrophage culture with or without C 2 S, the cultures were washed with PBS to eliminate C 2 S extract from the culture.The fresh culture medium was added to the culture and the conditioned medium (CM) was collected after 12 h of incubation.This CM contains C 2 S-free secretomes produced by the C 2 S-treated macrophages.Use of this macrophage-CM during BMSCs culture rules out the possible direct effect of C 2 S on osteogenic differentiation of BMSCs.Mouse BMSCs were cultured with CM and high glucose DMEM (in 1:1 ratio) in the presence of an osteogenic medium (OM) (10 nM dexamethasone, 50 lg/ml ascorbic acid, 10 mM b-sodium glycerophosphate).A total of three groups were allocated in this group: control group (OM), CM group (macrophage-CM and OM, 1:1 ratio) and C 2 S-CM group (C 2 S-treated macrophage-CM and OM, 1:1 ratio).All these three groups were maintained for osteogenic differentiation and the media were changed every 3-4 days time interval.

Alkaline phosphatase staining and activity
ALP staining and ALP activity were performed in osteogenic induced BMSCs with or without C 2 S at day 10.ALP staining was performed with the BCIP/NBT alkaline phosphatase color kit (Nanjing Jiancheng Biology Engineering Institute, China) according to the manufacturer's instructions.ALP activity was performed using an ALP detection kit (Bestbio, China) as described previously [7].Total protein concentration in the cell lysate was analyzed by the BCA protein assay kit.The ALP activity was normalized to the total protein content.

Alizarin red staining
Alizarin red (Sigma, USA) staining was performed on day 14 to visualize the mineralized matrix in BMSCs culture.For semiquantitative analysis of 200 ll of 10% CPC (Sigma, USA) solution was added to each well (48-well culture plate) to dissolve the mineralized nodules.The supernatant was transferred to a 96-well plate, optical density was measured at 562 nm, quantitative analysis was performed as described previously [7].

RT-qPCR analysis
The total RNA was extracted from BMSCs culture on day 7 using RNAEX reagent (Accurate Biotechnology, Hunan, China).Extracted 500 ng total RNA from each sample was reverse transcribed to cDNA using Evo M-MLV RT Premix for qRT-PCR (Accurate Biotechnology, Hunan, China).The reverse-transcribed cDNA was subjected to RT-qPCR (SYBR Green Premix Pro Taq HS qPCR Kit, Accurate Biotechnology, Hunan, China) using the following cycling conditions: 95 C for 10 min (initial denaturation), 40 cycles of 95 C for 15 s and 60 C for 60 s and then 60 C for 5 min for the terminal extension.The temperature was increased by 1 degree every 20 s to obtain the melting curve.The relative quantitative DD Ct method was used to determine the fold change of expression.The expressions of osteogenic gene RUNX2, ALP, OCN and BMP2 were analyzed by RT-qPCR.GAPDH was used as an internal control to normalize the expression of specific genes.The primer sequences used for RT-qPCR are listed in Table 1.

Statistical analysis
The data was analyzed using SPSS 20.0 software (SPSS, Inc., Chicago, Illinois).All data are expressed as mean 6 SD.All experiments were performed at least three times.Differences between groups were analyzed using analysis of variance followed by Bonferroni post hoc comparison and Dunnett's test, when appropriate.When P values < 0.05, it is considered statistically significant.

Availability of data and materials
All raw sequencing data can be accessed from the Sequence Read Archive (project accession GSE171979).

Characteristics of C 2 S particles
Based on the results from the scanning electron microscopy, the C 2 S particles appear as spherical particles aggregated in blocks (Supplementary Fig. S1).The laser particle size analysis showed that the main size of the particles is about 9.4160.65 lm.The energy dispersive spectrometry showed that the surface of the particles is mainly calcium, silicon, oxygen and carbon.X-ray diffraction analysis showed that the particle collection of illustration matched calcium silicate-matching Ca 2 SiO 4 .Inductively coupled plasma optical emission spectrometer showed that C 2 S particles dissolved in DMEM release calcium, silicon and phosphorus ions.

Effect of C 2 S on macrophage viability, inflammation induction and oxidative stress response
Different concentration C 2 S extracts did not affect macrophage viability, ruling out any cytotoxic effect of C 2 S on macrophages (Fig. 1A).Results from our previous studies showed that C 2 S triggers macrophagic inflammation [2,4,10].Based on the result of cell viability, we tested the inflammation induction potential of the highest concentration of C 2 S (150 lg/ml) extract tested in this study on inflammatory cytokine release by macrophages.C 2 S extract treatment for 24 h robustly enhanced TNF-a, IL-1b and IL-6 production by macrophages (Fig. 1B).The C 2 S extract induced ROS production in macrophages by 1.3-fold compared with control (Fig. 2A and B).

mRNA sequencing data analysis
Results of mRNA sequencing showed that the C 2 S-treated macrophages showed differential mRNA expression patterns compared with the control group.A total of 1475 mRNAs were differentially upregulated and 924 mRNAs were differentially downregulated in C 2 S-treated macrophages (Supplementary Figs S2 and S3).The R package 'ClusterProfiler' was used to perform KEGG pathway enrichment analysis of these differentially expressed mRNAs.We found that differentially expressed mRNAs are mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy, for example MAPK, PI3K-Akt, TNF and Rap1 signaling pathway (Fig. 3A).Among the mitochondrial function and autophagy-related mRNAs, PINK1, Parkin, Beclin1 and LC3 were differentially upregulated (Fig. 3B-F).Based on these results, we choose to analyze mitochondrial function and autophagy as a possible mechanism of C 2 S-induced macrophagic inflammation.

Effect of C 2 S on macrophage mitochondrial function
MtMP reflects electron transport and oxidative phosphorylation process and is an indicator of mitochondrial activity.MtMP was analyzed using JC-1 dye.This dye exhibits MtMP-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from red to green when MtMP declines due to membrane damage (Fig. 4A).The C 2 S extract-treated macrophages showed higher intensity of JC-1 staining (green fluorescence) compared with the control group (Fig. 4A).The quantitative analysis of JC-1 stained cells showed 2.55-fold higher numbers of JC-1 stained macrophages in the C 2 S-treated group compared with the control group (Fig. 4B).This result indicates that the C 2 S impairs the mitochondrial activity of macrophages.
The disrupted mitochondrial function increases intracellular calcium ion concentration.We analyzed the intracellular calcium ion concentration in C 2 S extract-treated macrophages using the Fluo-3 probe.C 2 S extract-treated macrophages showed higher intensity of green color Fluo-3 compared with the control group (Fig. 5A).Quantitative analysis of Fluo-3 staining intensity in macrophages by flow cytometry analysis revealed a 2.33-fold overload of calcium ion in the C 2 S extract-treated group compared with the control group (Fig. 5B).The ATP production decreases during mitochondrial dysfunction [41].The ATP level was drastically reduced in C 2 S extract-treated macrophages compared with the control group (Fig. 5C).The results of calcium ion concentration and ATP release assays indicate the disrupted mitochondrial function in C 2 S extract-treated macrophages.

Gene
Forward primer (5 0 -3 0 ) Reverse primer (5 0 -3 0 ) Mitochondrial dysfunction induces autophagy and macrophagic inflammation [17].MDC/MitoTracker staining showed more numbers of autophagosomes around the mitochondria and a reduced number of healthy mitochondria in the C 2 S-treated macrophages compared with the control group (Fig. 6A).Flow cytometry analysis revealed the 1.26-fold higher intensity of blue  MDC staining in C 2 S-treated macrophages compared with the control group (Fig. 6B).TEM images showed abundant mitochondria with normal morphology in the control group (Fig. 7).In contrast, swollen mitochondria surrounded by autophagosomes were visualized in the C 2 S-treated macrophage (Fig. 7).

Partial inhibition of autophagy reversed the C 2 S-induced inflammatory response
To further analyze the role of autophagy on C 2 S-induced macrophagic inflammation, autophagy was partially inhibited in macrophage culture using 0.1 mM 3MA.Inhibition of autophagy in macrophages almost reversed the C 2 S-induced overexpression of inflammatory markers TNF-a, IL-1b and IL-6 (Fig. 9).

C 2 S-treated macrophages promote osteogenic differentiation of BMSCs
Osteogenesis is the key phenomenon of bone defect healing.Since C 2 S induces macrophagic inflammation via mitochondrial dysfunction and autophagy, we further tested the effect of C 2 Sinduced macrophagic inflammation on the osteogenic differentiation of precursor cells.C 2 S-treated-macrophage-CM induced higher ALP production in BMSCs compared with macrophage-CM or control group (Fig. 10A).ALP activity analysis revealed that C 2 S-treated macrophage-CM enhanced ALP activity in BMSCs by 1.4-, and 1.14-fold compared with macrophage-CM and control group, respectively (Fig. 10B).Alizarin red staining images showed higher intensity of mineralized matrix in C 2 S-treated macrophage-CM group compared with control or macrophage-CM group (Fig. 10A).Quantitative analysis of alizarin red staining showed 1.59-, and 1.26-fold higher mineralized matrix in C 2 S-treated macrophage-CM group compared with macrophage-CM and control group, respectively (Fig. 10C).

Discussion
C 2 S releases calcium and silica ions that affect the function of cells involved in bone regeneration [2,3,10,42].Results of previous studies from our research group and reports from literature had unraveled the anabolic effect of C 2 S on osteogenic differentiation of precursor cells and the underlying molecular mechanisms [4,7,43].Macrophages played a vital role in immunoregulation during bone defect healing [8,44,45].The development of macrophage-mediated immunomodulatory biomaterials is currently a hot research topic of bone tissue engineering.Results from our previous studies showed that C 2 S regulates macrophagic inflammation [2,4,10].In this study, we unraveled that the C 2 S induces macrophagic inflammation via mitochondrial dysfunction and autophagy.Moreover, the C 2 S-induced macrophagic inflammation robustly promoted osteogenic differentiation of BMSCs (Fig. 11).Our results showed that the C 2 S-induced macrophage-mediated immunomodulation positively regulates osteogenesis, indicating the potential application of C 2 S in developing immunomodulatory biomaterials for bone tissue engineering and implantology applications.
Reports from literature show that various immunomodulatory biomaterials have been designed to affect macrophage proliferation and activation [27,[46][47][48][49].In this study, C 2 S extracts (75, 100, 125, or 150 lg/ml) did not affect macrophage viability.This finding is in accordance with the findings of previous studies [2,10].Our previous study investigated the effect of C 2 S extract up to the dose of 100 lg/ml on macrophage cell viability, inflammatory markers production and ROS production [2].In the current study, we observed no cytotoxic effect of 150 lg/ml of C 2 S extract on macrophages.Therefore, we chose the highest concentration tested (150 lg/ml) to further analyze the effect of C 2 S on macrophagic inflammation.Moreover, among the concentration tested (75, 100, 125, 125 and 150 lg/ml), the 150 lg/ml of C 2 S extract showed the highest effect on autophagy and mitochondrial function markers expression in macrophages, indicating 150 lg/ml as a suitable concentration for the scope of this study (Fig. 8).We found that C 2 S treatment promoted the expression of proinflammatory cytokines TNF-a, IL-1b and IL-6 in macrophages.Our previous studies had also reported a similar effect of C 2 S on macrophages [2,4,10,42].Results from this study and reports from literature elucidate the macrophagic inflammationinducing potential of C 2 S [4,9,10,42].However, the mitochondrial function of C 2 S-treated macrophages is still not fully understood.In this study, we attempt to investigate the mitochondrial function and autophagy-mediated effect of C 2 S on the induction of macrophagic inflammation.
The ionic products released by silicon-based biomaterials affect mitochondrial membrane functions [50].Mitochondrial dysfunction induces macrophagic inflammation [16].We analyzed the effect of C 2 S on the mitochondrial function of macrophages.The C 2 S treatment robustly upregulated ROS production and intracellular calcium overload and reduced MtMP and ATP production in macrophages.TOMM20 plays a key role in mitochondrial protein import [51].C 2 S-treated macrophages showed downregulation of TOMM20 expression.C 2 S treatment also altered the mitochondrial morphology in macrophages.These results indicate that the C 2 S-induced mitochondrial dysfunction in macrophages.
C 2 S causes oxidative stress and mitochondrial damage in RAW264.7 cells, but it does not significantly inhibit the growth and proliferation of RAW264.7 cells.Autophagy prevents cell apoptosis and inflammation by forming autophagosomes in cells to remove the excessive accumulation of ROS in cells and removing damaged mitochondria [52].Based on the aforementioned facts, we speculated that autophagy may be a 'self-rescue' mechanism in cells that can resist the increase of ROS and the change of mitochondrial outer membrane potential.Autophagy plays important role in macrophage activation [53], via various signaling pathways, including, NF-jB, MAPK, TNFa, PI3K/Akt and mTOR [54,55].The damaged mitochondria upregulate PINK1/Parkin expression [56].Upregulated PINK1/Parkin is associated with macrophagic inflammation [57].C 2 S treatment robustly upregulated mitochondrial PINK1/Parkin expression in macrophages.Mitochondrial dysfunction induces autophagy in a tissue-specific manner [58].Moreover, autophagy is involved in macrophage function and inflammatory responses [55].In this study, C 2 S treatment-induced autophagy in macrophages as indicated by the higher numbers of autophagosomes in TEM images and MDC staining.The expression of autophagy-related proteins Beclin1 and LC3 II were robustly upregulated in C 2 S-treated macrophages.Enhanced LC3 II expression is associated with autophagy induction in various cells [59].KEGG analysis of mRNA sequencing of C 2 S-treated macrophages revealed that the differentially expressed mRNAs were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy, for example MAPK, TNF and PI3K-Akt signaling pathway.Findings from the literature and current study indicate the possible role of C 2 S-induced mitochondrial dysfunction and autophagy on the induction of macrophagic inflammation.
In the transient inflammatory response at the initial stage of bone repair and healing, the inflammatory macrophages (M1 phenotype) initially gathered at the repair site, which polarized into the M2 phenotype after about 3-4 days [27], and the whole process lasted about 7 days [60].Early-stage inflammation is essential for the effective healing of bone defects [11,13].Macrophage polarization induced by biomaterials and implants regulates osteoinductivity and osseointegration [61].The C 2 S-induced macrophagic inflammation robustly promoted the osteogenic differentiation of BMSCs.This result indicates that the C 2 Sinduced macrophagic inflammation acts as an early-stage inflammation required for triggering bone healing.However, chronic and intense inflammation impairs bone defect healing.Macrophages with M2 phenotype after the initial stage promotes bone formation [62], and participate in the formation of new bone by secreting bone formation-related molecules such as IL-10, TGF-b and BMP2 [63].In this study, we analyzed only the effect of 24 h C 2 S treatment on the induction of macrophagic inflammation.Our results indicated that the anabolic effect of C 2 Sinduced early-stage macrophagic inflammation on osteogenesis.The switching of macrophage polarization from M1 and M2 after the early stage of healing is closely related to effective bone Regenerative Biomaterials, 2022, Vol. 9, rbab075 | 11 regeneration [11,64].Therefore, the long-term effect of C 2 S on macrophage activation and phenotype switching should be further analyzed to better understand the macrophage-mediated role of C 2 S during the whole bone regeneration process.
In this study, we extensively analyzed the mitochondrial function and autophagy in C 2 S-treated macrophages.Our results revealed that C 2 S-induced macrophagic inflammation, mitochondrial dysfunction and autophagy.C 2 S-induced macrophagic inflammation robustly promoted osteogenic differentiation of BMSCs.The limitation of this study is that we did not investigate the exact molecular mechanism of C 2 S-induced mitochondrial dysfunction and autophagy.Furthermore, in vivo bone defect healing effect of C 2 S-induced macrophagic inflammation should be further tested.

Figure 1 .Figure 2 . 5 C 2 S
Figure 1.C 2 S extract did not affect macrophage viability but induced macrophagic inflammation.(A) Macrophage viability, and (B) TNF-a, IL-1b and IL-6 expression pattern in macrophage analyzed by ELISA.Data are presented as mean6SD, n ¼ 3. Significant effect of the treatment compared with the control group, ***P < 0.001

Figure 3 .
Figure 3. C 2 S extract treatment regulated the expression of mitochondrial function/autophagy-related genes and signaling pathways.(A) KEGG pathway enrichment analysis of differentially expressed genes.(B-E) The expression of PINK1, Parkin, Beclin1 and LC3 was analyzed by RNA-sequencing

Figure 4 .
Figure 4. C 2 S reduced macrophagic MtMP.(A) Representative fluorescence microscopy images showing JC-1-stained macrophages.(B-D) Representative flow cytometry analysis histogram images showing JC-1-stained cells in the Q4 region.(E) Flow cytometry quantitative analysis of the percentage of cells in Q4 region.Data are presented as mean6SD, n ¼ 3. Significant effect of the treatment: compared with the control group, ***P < 0.001, compared with the positive control group (CCCP), ### P < 0.001

Figure 5 .Figure 6 .
Figure 5. C 2 S enhanced intracellular calcium levels and reduced ATP production in macrophages.(A) Representative fluorescence microscopy images of Flu-3 stained, macrophages.(B) Quantitative analysis of Fluo-3-stained macrophages by flow cytometry analysis.(C) Quantitative measurements of ATP level in macrophages.Data are presented as mean6SD, n ¼ 3. Significant effect of the treatment: compared with the control group, **P < 0.01, ***P < 0.001, compared with the positive control group (CCCP), ### P < 0.001

Figure 7 .
Figure 7. C 2 S altered mitochondrial morphology changes and autophagosome formation in macrophages.The mitochondria and autophagosomes were observed under a transmission electron microscope.The blue arrows point to mitochondria and the red arrows point to autophagosomes

Figure 11 .
Figure 11.C 2 S-induces macrophagic inflammation via mitochondrial dysfunction and autophagy that promotes osteogenic differentiation of precursor cells