Monocyte Chemoattractant Protein-1 from a Conditioned Medium of Bone Marrow-Derived Mesenchymal Stem Cells Promotes Bone Regeneration by Enhancing Macrophage Phenotype Switching

Monocyte Chemoattractant Protein-1 from a Conditioned Medium of Bone Marrow-Derived Mesenchymal Stem Cells Promotes Bone Regeneration by Enhancing Macrophage Phenotype Switching | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Monocyte Chemoattractant Protein-1 from a Conditioned Medium of Bone Marrow-Derived Mesenchymal Stem Cells Promotes Bone Regeneration by Enhancing Macrophage Phenotype Switching Kosuke Hashizume, Wataru Katagiri, Ryoko Takeuchi, Daisuke Suda, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8186435/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Maxillofacial Plastic and Reconstructive Surgery → Version 1 posted 14 You are reading this latest preprint version Abstract Background We have reported that the cytokines and chemokines contained in conditioned media of human mesenchymal stem cells (MSC-CM), which were derived from bone marrow, promote bone regeneration. We recently reported macrophage phenotype switching towards the anti-inflammatory M2 phenotype induced by MSC-CM and its potential to establish regenerative condition and assist subsequent bone regeneration. However, the specific factors in the MSC-CM responsible for this process remain unclear. Monocyte chemoattractant protein (MCP) -1, present in MSC-CM, promotes cell migration and activation of the monocyte-macrophage lineage; therefore, we hypothesized that MCP-1 is one of the key factors in MSC-CM-induced macrophage phenotype switching. The effect of MCP-1 on MSC-CM-induced macrophage phenotype switching and subsequent bone regeneration was investigeted in this study. Methods MCP-1 was depleted from MSC-CM (depMSC-CM) and used in subsequent experiments. Rat bone marrow macrophages were incubated in MSC-CM or depMSC-CM and expression of macrophage markers was examined in vitro . In addition, the effect of MSC-CM and depMSC-CM on bone regeneration and macrophage phenotype switching were evaluated using rat calvaria defect model in vivo . Results MSC-CM enhanced M2 macrophage marker expression in rat bone marrow macrophages compared to those treated with depMSC-CM in vitro . In addition, MSC-CM increased the number of M2 macrophage marker-positive cells in bone defects and enhanced subsequent bone regeneration in a rat calvaria bone defect model. Conclusions MCP-1 seemed to play an essential role in MSC-CM-induced macrophage phenotypic switching and subsequent bone regeneration. bone regeneration conditioned media conditioned Medium MCP-1 macrophage phenotype switching mesenchymal stem cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Autogenous or allogenic mesenchymal stem cell (MSC) implantation is one of the strategies in regenerative medicine in oral and maxillofacial regions. MSC implantation for bone regeneration is applied clinically based on the finding that MSCs from multiple tissues promote bone regeneration [1–3]. Although this approach is valuable in regenerative medicine, some issues such as tumorigenesis [4], poor survival rate of implanted cells [5, 6], and transmission of infectious diseases remain to be resolved. Furthermore, while bone regeneration therapy using stem cells in the oral and maxillofacial region is revolutionary, it is expensive, and alternative methods such as autogenous bone graft and artificial materials have developed, so it cannot be said to be widely used. However, among bone regenerative therapies using stem cells, bone regenerative therapy using the conditioned medium of human mesenchymal stem cells which were derived from bone marrow (MSC-CM) is expected to complement the drawbacks of stem cell transplantation, as it is low-cost and does not involve cell transplantation. Osugi et al. have shown not only that mixing MSC-CM with existing bone graft materials and transplanting it can promote early bone regeneration through enhanced osteoblast migration [7], but also that it can maintain osteoclast function in animal models of drug-induced osteonecrosis of the jaw [8]. MSC-CM also supports extracellular matrix reconstruction and promotes cartilage regeneration and mandibular condylar resorption [9]. MSC-CM contains numerous factors that promote bone regeneration. Insulin like growth factor-1 (IGF-1), vascular endothelial growth factor-A (VEGF), and transforming growth factor-β1 (TGF-β1) in MSC-CM promote cell migration, angiogenesis, and cell differentiation; they accelerate bone regeneration [7]. Furthermore, we considered that not only the effects of these cytokines, but also the anti-inflammatory effects of MSC-CM and the creation of a regenerative environment are important for early bone regeneration. Katagiri et al. have reported that MSC-CM elicit macrophage phenotype switching and contribute to the establishment of an anti-inflammatory milieu during the early phases of bone regeneration [10]. Macrophages are activated by diverse stimuli and categorized into two major subtypes according to their functions. Classically activated M1 macrophages produce pro-inflammatory cytokines, whereas alternatively activated M2 macrophages release anti-inflammatory cytokines and growth factors. Recent studies have revealed that MSC-CM affects macrophage activation and subsequently creates an anti-inflammatory milieu that promotes tissue regeneration [11–13]. Katagiri et al. recently reported that MSC-CM promotes phenotype switching towards M2 macrophages during the early phase of bone regeneration in a rat calvaria bone defect model [10]. Furthermore, a previous study showed that monocyte chemoattractant protein (MCP)-1, suggested to be one of the factors that promote macrophage phenotype switching, is present in MSC-CM by cytokine antibody array analysis [14]. However, the direct contribution of M2 macrophages to the MSC-CM-induced early osteogenesis remains unclear. MCP-1 is a cytokine that plays an important role in inflammatory responses, particularly activating the monocyte/macrophage lineage [15]. MCP-1 promotes inflammation by promoting cell migration and the production of inflammatory factors, supporting immune responses in the host. In this study, the role of MCP-1 in MSC-CM-induced bone regeneration was investigated. Methods Preparation of Conditioned Medium We used the commercially available human bone marrow-derived mesenchymal stem cells (hMSCs) (Lonza inc., Walkersville, MD, USA). hMSCs were cultured at 37℃ in 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco; Thermo Fisher Scientific, Waltham, Ma, USA) containing 10% fetal bovine serum (Biowest, Nuaillé, France). The cells were then subcultured to the third to fifth passage and used in the experiment. At about 80% confluence, hMSCs were then cultured in DMEM without serum [DMEM(-)]. After being incubated for 48 hours, the medium was filtered through a 0.22 µm filter sterilizer and stored at 4 or -80℃ until use. MCP-1 Depletion from MSC-CM MCP-1 was depleted from the MSC-CM using rabbit anti-human polyclonal antibodies against MCP-1 (ab9669; Abcam, Cambridge, UK). Briefly, Protein G magnetic beads (SureBeads Protein G; Bio-Rad, USA), pre-bound with 100 ng/mL anti-MCP-1 antibodies, were added to MSC-CM and mixed gently at 4℃ for 1 h. Antibody beads were magnetized, and the supernatant was collected. MCP-1 depletion was confirmed using MCP-1 enzyme-linked immunosorbent assay (ELISA) kit (DCP00; R&D, USA) and Quantikine immunoassay control group 1 (QC01-1; R&D) as a negative control, according to the manufacturer’s instructions. Depleted MSC-CMs were defined as depMSC-CM and used in subsequent experiments. Bone Marrow Macrophage Isolation and Activation Bone marrow cells were isolated from the femurs of 8-week-old male Wistar rats (Japan SLC, Shizuoka, Japan) and plated on 60-mm cell culture dishes or cover slips. They were differentiated into bone marrow macrophages (BMMs) in DMEM supplemented with 20 ng/mL macrophage colony stimulating factor (Peprotech, NJ, USA) at 37℃ in 5% CO 2 for 7 days and used in subsequent experiments. Immunocytochemical Analysis The cells were fixed in 4% Paraformaldehyde Phosphate Buffer Solution (PFA), Fetal bovine serum (FBS) (Sigma-Aldrich), permeabilized in 0.1% Triton X-100 (Sigma-Aldrich), blocked in 5% goat serum, and incubated with primary antibodies against CD11b (1:1000; Ab1211, Abcam), inducible nitric oxide synthase (iNOS) (1:100; Ab15323, Abcam), and CD206 (1:10000; ab64693, Abcam). Next, secondary antibodies, AF647-conjugated anti rabbit (1:1000; Ab150079, Abcam), AF488-conjugated anti mouse (1:1000; Ab150113, Abcam) were used and cells were counterstained with DAPI (D9542, Sigma Aldrich). Samples were observed using a fluorescence microscope (Axioplan 2; Carl Zeiss AG, Oberkochen, Germany). The positive cell numbers within three randomly selected areas in each well were averaged to determine the positive cell rate for each well. n = 3 (3 wells) for each group were used to evaluate the data. Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) hMSCs and BMMs were cultured with MSC-CMs, depMSC-CM, or DMEM(-) for 48 h, and total RNA was extracted using the RNeasy Mini kit (Qiagen N. V., Venlo, Netherlands). Reverse-transcribed into cDNA and qRT-PCR were performed using PrimeScript RT Master Mix and TB Green Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) with a Thermal Cycler Dice Real-Time System III (TaKaRa Bio). Table 1 showes the sequences of primers of markers for M1 macrophages ( iNOS and CD80 ), markers for M2 macrophages [ CD206 and Arginase-1 ( Arg-1 )], and osteogenesis-related genes [osteopontin ( OPN ), type I collagen ( COLⅠ ), alkaline phosphate ( ALP ), and osteocalcin ( OCN )]. The obtained results were normalized to Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) and the 2 -ΔΔCt method was used to calculate relative expression levels. Table 1 Primer sequences used for qRT-PCR. Gene Sequence Accession no. OPN F 5’-ACACATATGATGGCCGAGGTGA-3’ NM_000582.2 R 5’-GTGTGAGGTGATGTCCTCGTCTGTA-3' COL Ⅰ F 5’-CCCGGGTTTCAGAGACAACTTC-3’ NM_000088.3 R 5’-TCCACATGCTTTATTCCAGCAATC-3’ ALP F 5’-GCCATTGGCACCTGCCTTAC-3’ NM_000478.5 R 5’-AGCTCCAGGGCATATTTCAGTGTC-3’ OCN F 5’-CATGAGAGCCCTCACACTCCT-3’ NM_199173.5 R 5’-CACCTTTGCTGGACTCTGCAC-3’ iNOS F 5'-GCTGCCAAGCTGAAATTGAATG-3' NM_000625.4 R 5'-TCTGTGCCGGCAGCTTTAAC-3' CD80 F 5'-CACCTCCATTTGCAATTGACC-3' NM_005191.4 R 5'-TCCTGCAAAGCAACTGAAGTGA-3' CD206 F 5'-ATGCCCGGAGTCAGATCACAC-3' NM_002438.4 R 5'-TTCTGCAGCACTTTCAATGGAAAC-3' Arg-1 F 5'-CTGGCAAGGTGGCAGAAGTC-3' NM_000045.3 R 5'-ATGGCCAGAGATGCTTCCAA-3' GAPDH F 5’-AGGCTAGCTGGCCCGATTTC-3’ NM_001256799.2 R 5’-TGGCAACAATATCCACTTTACCAGA-3’ Rat Calvarial Bone Defect Model All animal experiments were performed in strict accordance with the protocols reviewed by the Animal Care and Use Committee of Niigata University (No. SA00456). 10-week-old male Wistar rats were anesthetized with an intraperitoneal injection of a mixture of medetomidine, midazolam, and butorphanol. After shaving the parietal region, a transverse incision was made posterior to the eyes, and a longitudinal incision was made at the left ear base. The periosteum was raised to expose the calvarial bones. Two calvarial bone defects, 5 mm in diameter, were created using a trephine bur (Dentech, Tokyo, Japan) and rinsed with PBS to remove bone debris. MSC-CM (30 µl), depMSC-CM (30 µL), or DMEM(-) (30 µL) were implanted to the bone defect using atelocollagen sponges (Terudermis1, Olympus Terumo Bio-materials Corp., Tokyo, Japan) as a scaffold. Finally, the periosteum and skin were sutured using a 4 − 0 nylon thread. The rats were sacrificed at 72 h, 1 week, and 2 weeks after implantation, and the specimens were harvested. The rats were randomly divided into four groups. The experimental groups were as follows: MSC-CM group, depMSC-CM group, DMEM(-) group, or defect group (defect only) (n = 6 in each group). Microcomputed Tomography (micro-CT) Analysis Specimens from all groups were harvested at 72 h, 1 week, and 2 weeks after surgery and analyzed using a micro-CT system (CosmoScan Gx, Rigaku Co., Tokyo, Japan). The specimens were observed by micro-CT, and three-dimensional (3D) images were reconstructed using Analyze software (version 12.0; AnalyzeDirect Inc., KS, USA). The newly formed bone area was evaluated as a percentage of surgically created bone defects. Histological Analysis Specimens from all groups were harvested at 72 h, 1 week, or 2 weeks after implantation. The samples were fixed in 10% neutral formalin and decalcified with 10% EDTA (pH 7.4) for four weeks. Samples were dehydrated with graded ethanol, embedded in paraffin, and cut into 3 µm thickness in the coronal plane using a microtome (REM-710, YAMATO KOHKI Industrial Co., Ltd., Saitama, Japan). The sections were rehydrated, stained with hematoxylin and eosin (H&E), and analyzed under a light microscope (FX630, OLYMPUS Co., Tokyo, Japan). Immunohistochemical Analysis Immunohistochemical analysis for iNOS (1:100; Ab15323, Abcam) to evaluate M1 macrophages and for CD206 (1:10000; ab64693, Abcam) to detect M2 macrophages were performed. The sections were dewaxed, rehydrated, and antigen retrieval was performed with citrate buffer (pH 6.0) for 10 minutes at 121˚C. The sections were then incubated with 0.3% H 2 O 2 in methanol for 30 min to block endogenous peroxidase activity. After washing with PBS, the sections were blocked for non-specific binding using 10% goat serum for 1 h at room temperature, and then incubated with the primary antibody overnight at 4˚C. Subsequently, the sections were reacted with EnVision Plus (Dako, CA, USA) for 1 h and developed with 3,3’-diaminobenzidine (DAB) solution. Finally, the sections were counterstained with hematoxylin after DAB staining. At 72 hours, 1 week, and 2 weeks after transplantation, six specimens from each experimental group were investigated. Three areas with a diameter of 200 µm were randomly set at the edge of each bone defect, and the number of positive cells contained within the area was averaged to determine the number of positive cells for each specimen. Data was evaluated using n = 6 for each group. Statistical Analysis All data are expressed as mean ± standard deviation (SD). Differences between groups were compared using Tukey’s honestly significant difference test. Differences were considered statistically significant at p < 0.05. Results MCP-1 Concentration in MSC-CM and depMSC-CM The concentration of MCP-1 in MSC-CM, depMSC-CM, and DMEM(-) were 414.9 ± 138.2 pg/mL, 11.8 ± 1.3 pg/mL, and 5.2 ± 2.1 pg/mL, respectively. MSC-CMs contained significantly higher levels of MCP-1 than depMSC-CM or DMEM(-). MSC-CM Regulated Macrophage Phenotype-Related Gene Expression in BMMs Immunocytochemical analysis revealed that the percentage of iNOS and CD11b + M1 cells decreased, while that of CD11b and CD206 positive M2 cells increased in MSC-CM-treated BMMs compared to that in depMSC-CM-treated BMMs (Fig. 1 a-d). Consistent with these results, qRT-PCR revealed that the expression levels of the pro-inflammatory M1 macrophage markers ( iNOS and CD80 ) were significantly downregulated, and that the M2 macrophage markers (CD206 and Arg-1) were significantly upregulated in BMMs cultured with MSC-CM compared to those cultured with depMSC-CM ( p < 0.01) (Fig. 2 ). In contrast, the expression levels of anti-inflammatory M2 markers ( CD206 and Arg-1 ) were significantly upregulated in BMMs cultured with MSC-CM compared with those cultured with depMSC-CM. MSC-CMs Enhanced Osteogenesis-Related Gene Expression in hMSC The expression levels of ALP and OPN were upregulated significantly in hMSC cultured with MSC-CM compared to those cultured with DMEM(-) and depMSC-CM ( p < 0.01 and p < 0.05, respectively). The expression of COL I was significantly higher in hMSCs cultured with MSC-CM compared to those cultured with DMEM(-) ( p < 0.01). However, there was no significant difference between those cultured with MSC-CM and depMSC-CM. MSC-CM enhanced OCN expression in hMSCs; however, the difference between the DMEM(-) and depMSC-CM groups was not statistically significant (Fig. 3 ). MSC-CM Enhanced Bone Regeneration Compared to depMSC-CM Areas with the newly regenerated bone within the bone defect were evaluated using micro-CT scanning and three-dimensional images were reconstructed at 72 h, 1 week, and 2 weeks after implantation (Fig. 4 a). Areas with the newly regenerated bone was calculated as a percentage of the graft area. Seventy-two hours after implantation, bone regeneration was not evident in any group. After 1 week, areas with the newly regenerated bone in the MSC-CM group (10.8 ± 3.9%) were significantly higher compared to those in the depMSC-CM (2.6 ± 1.2%), DMEM(-) (3.3 ± 2.8%), and defect (2.9 ± 1.5%) groups (Fig. 4 b). After 2 weeks, areas with the newly regenerated bone in the depMSC-CM (19.9 ± 1.7%), DMEM(-) (16.4 ± 2.5%), and defect (5.3 ± 1.0%) groups were confirmed, whereas the areas in the MSC-CM group (30.3 ± 5.8%) were higher compared to those in other groups significantly (Fig. 4 c). Histological analysis also showed that MSC-CMs increased bone regeneration compared with the depMSC-CM, DMEM(-), and defect groups. Seventy-two hours after implantation, migrated inflammatory cells were found around the defect margins in the MSC-CM and depMSC-CM groups; however, bone regeneration was not obvious in any of the groups (Fig. 5 a). After 1 and 2 weeks, MSC-CM increased the newly regenerated bone compared to the other groups, and trabecular bone formation along with bone defect margins was observed in the MSC-CM group (Fig. 6 a, 7 a). MSC-CM Induced Macrophage Phenotype Switching at an Early Phase of Bone Regeneration Cells stained with the pro-inflammatory M1 macrophage marker iNOS and anti-inflammatory macrophage marker CD206 were observed around the margin of bone defect at 72 h, 1 week, and 2 weeks after implantation to investigate macrophage phenotype switching at an early phase of bone regeneration. After 72 hours, the number of iNOS-positive cells in the MSC-CM group (5.6 ± 1.9) was significantly lower than that in the depMSC-CM (17.4 ± 4.1), DMEM(-) (13.4 ± 2.4), and defect (20.0 ± 4.9) groups. In contrast, the number of CD206-positive cells in the MSC-CM group (14.8 ± 2.3) was significantly higher compared to that in the depMSC-CM (4.6 ± 1.6), DMEM(-) (4.2 ± 1.4), and defect (2.7 ± 1.3) groups at 72 hours after implantation (Fig. 5 a, b). After 1 week, the number of iNOS-positive cells in the MSC-CM group (1.4 ± 0.9) was significantly lower compared to that in the depMSC-CM (13.6 ± 2.9), DMEM(-) (10.2 ± 2.5), and defect (12.8 ± 5.6) groups. However, significant difference in the number of CD206 positive cells between each group was not obvious (Fig. 6 a, b). After 2 weeks, the number of iNOS-positive cells was significantly lower in the MSC-CM group (1.3 ± 0.6) compared to that in the depMSC-CM (6.3 ± 1.3), DMEM(-) (4.9 ± 0.9), and defect (4.3 ± 1.6) groups. In contrast, no significant difference in the number of CD206 positive cells was found between each group (Fig. 7 a, b). From 72 h to 2 weeks, the M2/M1 ratio was higher in the MSC-CM group than that in the other groups (Fig. 8 ) Discussion Since the bone-regenerative effect of MSC-CM was first reported, numerous studies have attempted to elucidate the underlying mechanism of its therapeutic effects. To date, several cytokines in MSC-CM and their effects on bone regeneration have been revealed. Previous studies have reported that VEGF, IGF-1, and TGF-β, which were contained in MSC-CMs, promote bone regeneration through cell proliferation, cell recruitment, and angiogenesis and VEGF as a key factor in tissue regeneration that enhances capillary formation, thus supporting cell migration and blood supply to damaged tissues [7, 16]. Recently, the immune regulatory effects of MSC-CMs through macrophage phenotype switching and subsequent anti-inflammatory effects have been reported by several studies [11,12]. We planned to identify the macrophage phenotype-switching factor in MSC-CM and its effect on bone regeneration in this study. Macrophages are activated by two major pathways (classical and alternative) and perform a wide range of functions [13]. Classically activated macrophages (also known as pro-inflammatory M1 macrophages) are induced by microbial products, T cell-derived signals, and foreign substances. They actively ingest and produce cytokines that stimulate inflammation, thereby playing an essential role in host defense against chronic inflammatory diseases. Alternatively, M2 macrophages are activated by interleukin (IL)-4 and IL-13 produced by T and mast cells. These macrophages produce TGF-β and other growth factors to terminate inflammation and enter the tissue regenerative phase [11,17,18]. Therefore, macrophage phenotype switching is the principal process for resolving inflammation and initiating tissue regeneration. MCP-1 is a chemokine that promotes chemotaxis of immune cells and plays a crucial role in inflammation and pathological circumstances [19]. Recently, the ability of MCP-1 to convert the macrophage phenotype towards the M2 phenotype has been reported. Hernan et al. reported that MCP-1 promotes macrophage phenotype switching towards the M2 type through inhibition of apoptosis and caspase 8 cleavage [20]. In this study, we confirmed that the MSC-CM contained MCP-1 at a concentration of 414.9 ± 138.2 pg/mL. Therefore, we hypothesized that MCP-1 plays a central role in MSC-CM-induced macrophage phenotypic switching. MSC-CM-induced immunoregulatory effects and subsequent tissue regeneration are closely associated with macrophage phenotype switching. Gao et al. reported that MSC-CMs reduced the expression level of tumor necrosis factor (TNF)-α but enhanced the expression levels of IL-10, arginase-1, and CD206 in mouse macrophage RAW264.7 cells. In addition, MSC-CM activated signal transducer and activator of transcription (STAT) 3 but inhibited nuclear factor (NF)-κB pathways in RAW264.7 cells [21]. Our previous report revealed that MSC-CM enhanced macrophage phenotype switching within 72 h after MSC-CM implantation and promoted bone regeneration in rat calvaria bone defects [12]. In this study, using BMMs in vitro , we revealed that the expression levels of M2 macrophage markers ( CD206 and Arg-1 ) were significantly upregulated, whereas the expression levels of M1 macrophage markers ( iNOS and CD80 ) were significantly downregulated in the MSC-CM group compared to those in the depMSC-CM group (Fig. 2 ) Therefore, MCP-1 in MSC-CM is likely to induce macrophage phenotype switching towards the M2 type. In addition, MSC-CM increased the number of M2 macrophages around the bone edge 72 h after MSC-CM implantation compared to the depMSC-CM group in vivo . Interestingly, the number of M1 macrophage in the MSC-CM group was significantly smaller compared to that in the depMSC-CM group. However, after 1 and 2 weeks, there was no significant difference between the number of M2 macrophages. These results suggest that MSC-CM promote macrophage phenotype switching towards the M2 type within 72 h after implantation; however, this effect is limited to the early phase of bone regeneration. The interaction between MSCs and M2 macrophages is a key component in bone regeneration. Gong et al. previously reported that M2 macrophages accelerate MSCs differentiation into osteoblasts by releasing pro-regenerative cytokines (such as TGF-β, VEGF, and IGF-1), whereas M1 macrophages suppress osteoblast cell differentiation by producing pro-inflammatory cytokines (including IL-6, IL-12, and TNF-α) [22]. In our study, MSC-CM enhanced osteogenesis-related gene expression in MSCs. However, the expression levels of OPN and ALP were significantly downregulated in the depMSC-CM group compared to those in the MSC-CM group in vitro (Fig. 3 ). These results indicate that MCP-1 in MSC-CM contributes to MSC osteogenesis. In a rat calvaria bone defect model, MSC-CM increased the number of M2 macrophages around the bone edge 72 h after implantation and maintained a high M2/M1 ratio for 2 weeks, resulting in enhanced bone regeneration. In contrast, depMSC-CM did not increase the M2/M1 ratio and subsequent bone regeneration was not obvious (Figs. 4 – 8 ). Overall, these results indicate that MSC-CM promotes bone regeneration not only via a direct effect on MSCs themselves but also via MCP-1-induced macrophage phenotype switching towards the M2 type within 72 h after implantation. MSC-CM contains numerous cytokines, some of which have been addressed in our previous studies to elucidate the underlying mechanism of bone regeneration induced by MSC-CM. We demonstrate the positive effects of MCP-1 on macrophage phenotypic switching and subsequent bone regeneration. However, the precise mechanism underlying MCP-1-induced macrophage phenotypic switching and the mechanism by which M2 macrophages promote bone regeneration remain elucidated. Clarifying these mechanisms will contribute to establishing the clinical use of MSC-CM not only for bone regeneration, but also in other inflammatory diseases and bone-resorptive diseases in the oral and maxillofacial regions. Abbreviations MSC Mesenchymal stem cell MSC-CM the conditioned medium of human mesenchymal stem cells derived from bone marrow IGF-1 Insulin like growth factor-1 VEGF Vascular endothelial growth factor-A TGF-β1 Transforming growth factor-β1 MCP-1 Monocyte chemoattractant protein-1 DMEM Dulbecco’s modified Eagle’s medium ELISA Enzyme-linked immunosorbent assay BMM Bone marrow macrophage PFA Paraformaldehyde Phosphate Buffer Solution FBS Fetal bovine serum iNOS inducible nitric oxide synthase RT-PCR Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction Arg-1 Arginase-1 OPN Osteopontin COL I Type I collagen ALP Alkaline phosphate OCN Osteocalcin GAPDH Glyceraldehyde-3-phosphate dehydrogenase Micro-CT Microcomputed Tomography Declarations Ethics approval and consent to participate All animal experiments were performed in strict accordance with the protocols reviewed by the Animal Care and Use Committee of Niigata University (No. SA00456). Consent for publication Not applicable. Funding This work was supported by Grants-in-Aid for Scientific Research (C) from the Ministry of Health, Labour, and Welfare of Japan (Nos. 20K10113 and 21K21060). Author Contribution K.H. and W.K. contributed equally to this work and co-first authors.W.K. performed the conception and design of this study. K.H. and W.K. collected and generated the data. K.H., W.K., R.T. and D.S. performed the data analysis and interpretation. K.H. and W.K. wrote the manuscript and it was revised by T.K.All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors would like to thank the members of the Division of Reconstructive Surgery for Oral and Maxillofacial Region, Faculty of Dentistry & Graduate School of Medical and Dental Sciences for their help and contributions to this study. 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Cite Share Download PDF Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Maxillofacial Plastic and Reconstructive Surgery → Version 1 posted Editorial decision: Revision requested 26 Dec, 2025 Reviews received at journal 25 Dec, 2025 Reviews received at journal 18 Dec, 2025 Reviewers agreed at journal 15 Dec, 2025 Reviewers agreed at journal 14 Dec, 2025 Reviews received at journal 13 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviews received at journal 06 Dec, 2025 Reviewers agreed at journal 05 Dec, 2025 Reviewers agreed at journal 03 Dec, 2025 Reviewers invited by journal 03 Dec, 2025 Editor assigned by journal 02 Dec, 2025 Submission checks completed at journal 02 Dec, 2025 First submitted to journal 23 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8186435","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":555236950,"identity":"cec5e1a1-6028-48ea-94ad-c72862ebe110","order_by":0,"name":"Kosuke Hashizume","email":"","orcid":"","institution":"Niigata University Graduate School of Medical and Dental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kosuke","middleName":"","lastName":"Hashizume","suffix":""},{"id":555236951,"identity":"40e936d8-d11c-431d-b035-a9d36b8cdd4d","order_by":1,"name":"Wataru 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1","display":"","copyAsset":false,"role":"figure","size":209107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of MSC-CM and depMSC-CM on BMM gene expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative images of BMMs immunocytologically stained for CD11b (red), iNOS (green), DAPI (blue) (a), CD11b (red), CD206 (green), and DAPI (blue, b) 48 h after incubation with MSC-CMs or depmSC-CMs. Scale bar: 50 μm. Ratios of iNOS and CD11b positive cells (c) and CD206 and CD11b positive cells (d). Gene expression in BMMs 48 h after incubation with MSC-CM and depMSC-CM (Fig.2). The results are expressed relative to the mRNA expression levels in DMEM(-)-treated cells (n = 3 per group). Data are represented as mean ± SD; *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/53eb4b7f793e478cda39f89e.jpeg"},{"id":97672795,"identity":"aff14e39-f818-4947-9411-9592dec0db8d","added_by":"auto","created_at":"2025-12-08 09:38:50","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of MSC-CM and depMSC-CM on BMMs gene expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression in BMMs 48 h after incubation with MSC-CM and depMSC-CM (Fig.2). The results are expressed relative to the mRNA expression levels in DMEM(-)-treated cells (n = 3 per group). Data are represented as mean ± SD; *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/6b45fc48e082617beb6e0004.jpeg"},{"id":97558497,"identity":"8feb1a3a-a479-4323-bb93-ebf765b11f67","added_by":"auto","created_at":"2025-12-05 19:36:01","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":76621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of MSC-CM and depMSC-CM on hMSC gene expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative expression levels of osteogenesis-related genes in MSC- and depMSC-CM. The results are expressed relative to the mRNA expression levels in DMEM(-)-treated cells (n = 3 per group). Data are represented as mean ± SD; *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/06a57927cda2d6c6622b3adb.jpeg"},{"id":97672877,"identity":"6a8de7b8-7a58-420a-bd40-f24943b6f790","added_by":"auto","created_at":"2025-12-08 09:38:59","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicro-CT analysis of bone formation after implantation of MSC-CM and depMSC-CM. \u003c/strong\u003eReconstructed images of rat calvaria bone defects at 72 h, 1 week, and 2 weeks after implantation of MSC-CM, depMSC-CM, or DMEM(-) (a). The defect group did not undergo implantation. Areas with the newly regenerated bone were measured, indicating that MSC-CM significantly promoted bone regeneration at 1 and 2 weeks after implantation compared to the other groups (b, c). Data are represented as mean ± SD; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/38255ff20ad3ebb2493dace3.jpeg"},{"id":97673355,"identity":"6bc7636e-098b-4c98-884b-68b59d66647d","added_by":"auto","created_at":"2025-12-08 09:39:57","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":70574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry for the newly regenerated bone 72 hours after implantation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E staining revealed the orientation of the specimens, indicating newly formed bone edges and surrounding tissue (a). Seventy-two hours after implantation, iNOS-positive cells were few in the MSC-CM group than in the other groups, whereas CD206 positive cells was the highest among all groups (b). Scale bar: 50 μm. Data are represented as mean ± SD; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/f65557e95f24dc80ab5933c1.jpeg"},{"id":97558509,"identity":"ec7fb726-6e1c-4572-a905-e459bcf10850","added_by":"auto","created_at":"2025-12-05 19:36:01","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":70173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry for the newly regenerated bone 1 week after implantation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 1 week, iNOS-positive cells were few in the MSC-CM group; however, there were no significant differences in the number of CD206 positive cells between the groups (a, b). Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/4cf9abb0a19540b17aa19e44.jpeg"},{"id":97671537,"identity":"de604083-8082-45b8-9d23-b678bb2f7a8f","added_by":"auto","created_at":"2025-12-08 09:32:42","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":76569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry for the newly regenerated bone two weeks after implantation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 2 weeks, the number of iNOS-positive cells was lower in the MSC-CM group; however, there were no significant differences in the number of CD206 positive cells between the groups (a, b). Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/0657c9c591ac49e02a44223c.jpeg"},{"id":97558500,"identity":"ccc11f47-866f-486e-b34e-40d5b970137d","added_by":"auto","created_at":"2025-12-05 19:36:01","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":58557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ratio of CD206-positive (M2) and iNOS-positive (M1) cells after implantation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt Seventy-two hours, one week and two weeks after implantation, MSC-CM improved the M2/M1 ratio around the newly formed bone edge, while it remained low in the other three groups.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/b14bfa9acea720bc1878c0b9.jpeg"},{"id":103766250,"identity":"2ec181bc-fad8-4f6e-b5b4-f24296939b5a","added_by":"auto","created_at":"2026-03-02 16:13:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1858675,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8186435/v1/2cff51b4-94dd-4f1f-8087-cea65ea3b3fc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Monocyte Chemoattractant Protein-1 from a Conditioned Medium of Bone Marrow-Derived Mesenchymal Stem Cells Promotes Bone Regeneration by Enhancing Macrophage Phenotype Switching","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAutogenous or allogenic mesenchymal stem cell (MSC) implantation is one of the strategies in regenerative medicine in oral and maxillofacial regions. MSC implantation for bone regeneration is applied clinically based on the finding that MSCs from multiple tissues promote bone regeneration [1\u0026ndash;3]. Although this approach is valuable in regenerative medicine, some issues such as tumorigenesis [4], poor survival rate of implanted cells [5, 6], and transmission of infectious diseases remain to be resolved. Furthermore, while bone regeneration therapy using stem cells in the oral and maxillofacial region is revolutionary, it is expensive, and alternative methods such as autogenous bone graft and artificial materials have developed, so it cannot be said to be widely used.\u003c/p\u003e\u003cp\u003eHowever, among bone regenerative therapies using stem cells, bone regenerative therapy using the conditioned medium of human mesenchymal stem cells which were derived from bone marrow (MSC-CM) is expected to complement the drawbacks of stem cell transplantation, as it is low-cost and does not involve cell transplantation.\u003c/p\u003e\u003cp\u003eOsugi et al. have shown not only that mixing MSC-CM with existing bone graft materials and transplanting it can promote early bone regeneration through enhanced osteoblast migration [7], but also that it can maintain osteoclast function in animal models of drug-induced osteonecrosis of the jaw [8]. MSC-CM also supports extracellular matrix reconstruction and promotes cartilage regeneration and mandibular condylar resorption [9].\u003c/p\u003e\u003cp\u003eMSC-CM contains numerous factors that promote bone regeneration. Insulin like growth factor-1 (IGF-1), vascular endothelial growth factor-A (VEGF), and transforming growth factor-β1 (TGF-β1) in MSC-CM promote cell migration, angiogenesis, and cell differentiation; they accelerate bone regeneration [7]. Furthermore, we considered that not only the effects of these cytokines, but also the anti-inflammatory effects of MSC-CM and the creation of a regenerative environment are important for early bone regeneration.\u003c/p\u003e\u003cp\u003eKatagiri et al. have reported that MSC-CM elicit macrophage phenotype switching and contribute to the establishment of an anti-inflammatory milieu during the early phases of bone regeneration [10].\u003c/p\u003e\u003cp\u003eMacrophages are activated by diverse stimuli and categorized into two major subtypes according to their functions. Classically activated M1 macrophages produce pro-inflammatory cytokines, whereas alternatively activated M2 macrophages release anti-inflammatory cytokines and growth factors. Recent studies have revealed that MSC-CM affects macrophage activation and subsequently creates an anti-inflammatory milieu that promotes tissue regeneration [11\u0026ndash;13]. Katagiri et al. recently reported that MSC-CM promotes phenotype switching towards M2 macrophages during the early phase of bone regeneration in a rat calvaria bone defect model [10]. Furthermore, a previous study showed that monocyte chemoattractant protein (MCP)-1, suggested to be one of the factors that promote macrophage phenotype switching, is present in MSC-CM by cytokine antibody array analysis [14]. However, the direct contribution of M2 macrophages to the MSC-CM-induced early osteogenesis remains unclear.\u003c/p\u003e\u003cp\u003eMCP-1 is a cytokine that plays an important role in inflammatory responses, particularly activating the monocyte/macrophage lineage [15]. MCP-1 promotes inflammation by promoting cell migration and the production of inflammatory factors, supporting immune responses in the host.\u003c/p\u003e\u003cp\u003eIn this study, the role of MCP-1 in MSC-CM-induced bone regeneration was investigated.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of Conditioned Medium\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe used the commercially available human bone marrow-derived mesenchymal stem cells (hMSCs) (Lonza inc., Walkersville, MD, USA). hMSCs were cultured at 37℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco; Thermo Fisher Scientific, Waltham, Ma, USA) containing 10% fetal bovine serum (Biowest, Nuaill\u0026eacute;, France). The cells were then subcultured to the third to fifth passage and used in the experiment.\u003c/p\u003e\u003cp\u003eAt about 80% confluence, hMSCs were then cultured in DMEM without serum [DMEM(-)]. After being incubated for 48 hours, the medium was filtered through a 0.22 \u0026micro;m filter sterilizer and stored at 4 or -80℃ until use.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMCP-1 Depletion from MSC-CM\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMCP-1 was depleted from the MSC-CM using rabbit anti-human polyclonal antibodies against MCP-1 (ab9669; Abcam, Cambridge, UK). Briefly, Protein G magnetic beads (SureBeads Protein G; Bio-Rad, USA), pre-bound with 100 ng/mL anti-MCP-1 antibodies, were added to MSC-CM and mixed gently at 4℃ for 1 h. Antibody beads were magnetized, and the supernatant was collected. MCP-1 depletion was confirmed using MCP-1 enzyme-linked immunosorbent assay (ELISA) kit (DCP00; R\u0026amp;D, USA) and Quantikine immunoassay control group 1 (QC01-1; R\u0026amp;D) as a negative control, according to the manufacturer\u0026rsquo;s instructions. Depleted MSC-CMs were defined as depMSC-CM and used in subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eBone Marrow Macrophage Isolation and Activation\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBone marrow cells were isolated from the femurs of 8-week-old male Wistar rats (Japan SLC, Shizuoka, Japan) and plated on 60-mm cell culture dishes or cover slips. They were differentiated into bone marrow macrophages (BMMs) in DMEM supplemented with 20 ng/mL macrophage colony stimulating factor (Peprotech, NJ, USA) at 37℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e for 7 days and used in subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eImmunocytochemical Analysis\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe cells were fixed in 4% Paraformaldehyde Phosphate Buffer Solution (PFA), Fetal bovine serum (FBS) (Sigma-Aldrich), permeabilized in 0.1% Triton X-100 (Sigma-Aldrich), blocked in 5% goat serum, and incubated with primary antibodies against CD11b (1:1000; Ab1211, Abcam), inducible nitric oxide synthase (iNOS) (1:100; Ab15323, Abcam), and CD206 (1:10000; ab64693, Abcam). Next, secondary antibodies, AF647-conjugated anti rabbit (1:1000; Ab150079, Abcam), AF488-conjugated anti mouse (1:1000; Ab150113, Abcam) were used and cells were counterstained with DAPI (D9542, Sigma Aldrich). Samples were observed using a fluorescence microscope (Axioplan 2; Carl Zeiss AG, Oberkochen, Germany). The positive cell numbers within three randomly selected areas in each well were averaged to determine the positive cell rate for each well. n\u0026thinsp;=\u0026thinsp;3 (3 wells) for each group were used to evaluate the data.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eReal-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ehMSCs and BMMs were cultured with MSC-CMs, depMSC-CM, or DMEM(-) for 48 h, and total RNA was extracted using the RNeasy Mini kit (Qiagen N. V., Venlo, Netherlands). Reverse-transcribed into cDNA and qRT-PCR were performed using PrimeScript RT Master Mix and TB Green Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) with a Thermal Cycler Dice Real-Time System III (TaKaRa Bio). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showes the sequences of primers of markers for M1 macrophages (\u003cem\u003eiNOS\u003c/em\u003e and \u003cem\u003eCD80\u003c/em\u003e), markers for M2 macrophages [\u003cem\u003eCD206\u003c/em\u003e and Arginase-1 (\u003cem\u003eArg-1\u003c/em\u003e)], and osteogenesis-related genes [osteopontin (\u003cem\u003eOPN\u003c/em\u003e), type I collagen (\u003cem\u003eCOLⅠ\u003c/em\u003e), alkaline phosphate (\u003cem\u003eALP\u003c/em\u003e), and osteocalcin (\u003cem\u003eOCN\u003c/em\u003e)]. The obtained results were normalized to Glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) and the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method was used to calculate relative expression levels.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer sequences used for qRT-PCR.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAccession no.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eOPN\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-ACACATATGATGGCCGAGGTGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_000582.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-GTGTGAGGTGATGTCCTCGTCTGTA-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCOL Ⅰ\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-CCCGGGTTTCAGAGACAACTTC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_000088.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-TCCACATGCTTTATTCCAGCAATC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eALP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-GCCATTGGCACCTGCCTTAC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_000478.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-AGCTCCAGGGCATATTTCAGTGTC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eOCN\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-CATGAGAGCCCTCACACTCCT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_199173.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-CACCTTTGCTGGACTCTGCAC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eiNOS\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-GCTGCCAAGCTGAAATTGAATG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eNM_000625.4\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-TCTGTGCCGGCAGCTTTAAC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCD80\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-CACCTCCATTTGCAATTGACC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_005191.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-TCCTGCAAAGCAACTGAAGTGA-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCD206\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ATGCCCGGAGTCAGATCACAC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_002438.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-TTCTGCAGCACTTTCAATGGAAAC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eArg-1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-CTGGCAAGGTGGCAGAAGTC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_000045.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ATGGCCAGAGATGCTTCCAA-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-AGGCTAGCTGGCCCGATTTC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_001256799.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-TGGCAACAATATCCACTTTACCAGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eRat Calvarial Bone Defect Model\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e All animal experiments were performed in strict accordance with the protocols reviewed by the Animal Care and Use Committee of Niigata University (No. SA00456).\u003c/p\u003e\u003cp\u003e10-week-old male Wistar rats were anesthetized with an intraperitoneal injection of a mixture of medetomidine, midazolam, and butorphanol. After shaving the parietal region, a transverse incision was made posterior to the eyes, and a longitudinal incision was made at the left ear base. The periosteum was raised to expose the calvarial bones. Two calvarial bone defects, 5 mm in diameter, were created using a trephine bur (Dentech, Tokyo, Japan) and rinsed with PBS to remove bone debris. MSC-CM (30 \u0026micro;l), depMSC-CM (30 \u0026micro;L), or DMEM(-) (30 \u0026micro;L) were implanted to the bone defect using atelocollagen sponges (Terudermis1, Olympus Terumo Bio-materials Corp., Tokyo, Japan) as a scaffold. Finally, the periosteum and skin were sutured using a 4\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon thread. The rats were sacrificed at 72 h, 1 week, and 2 weeks after implantation, and the specimens were harvested.\u003c/p\u003e\u003cp\u003eThe rats were randomly divided into four groups. The experimental groups were as follows: MSC-CM group, depMSC-CM group, DMEM(-) group, or defect group (defect only) (n\u0026thinsp;=\u0026thinsp;6 in each group).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMicrocomputed Tomography (micro-CT) Analysis\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSpecimens from all groups were harvested at 72 h, 1 week, and 2 weeks after surgery and analyzed using a micro-CT system (CosmoScan Gx, Rigaku Co., Tokyo, Japan). The specimens were observed by micro-CT, and three-dimensional (3D) images were reconstructed using Analyze software (version 12.0; AnalyzeDirect Inc., KS, USA). The newly formed bone area was evaluated as a percentage of surgically created bone defects.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eHistological Analysis\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSpecimens from all groups were harvested at 72 h, 1 week, or 2 weeks after implantation. The samples were fixed in 10% neutral formalin and decalcified with 10% EDTA (pH 7.4) for four weeks. Samples were dehydrated with graded ethanol, embedded in paraffin, and cut into 3 \u0026micro;m thickness in the coronal plane using a microtome (REM-710, YAMATO KOHKI Industrial Co., Ltd., Saitama, Japan). The sections were rehydrated, stained with hematoxylin and eosin (H\u0026amp;E), and analyzed under a light microscope (FX630, OLYMPUS Co., Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemical Analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eImmunohistochemical analysis for iNOS (1:100; Ab15323, Abcam) to evaluate M1 macrophages and for CD206 (1:10000; ab64693, Abcam) to detect M2 macrophages were performed. The sections were dewaxed, rehydrated, and antigen retrieval was performed with citrate buffer (pH 6.0) for 10 minutes at 121˚C. The sections were then incubated with 0.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol for 30 min to block endogenous peroxidase activity. After washing with PBS, the sections were blocked for non-specific binding using 10% goat serum for 1 h at room temperature, and then incubated with the primary antibody overnight at 4˚C. Subsequently, the sections were reacted with EnVision Plus (Dako, CA, USA) for 1 h and developed with 3,3\u0026rsquo;-diaminobenzidine (DAB) solution. Finally, the sections were counterstained with hematoxylin after DAB staining. At 72 hours, 1 week, and 2 weeks after transplantation, six specimens from each experimental group were investigated. Three areas with a diameter of 200 \u0026micro;m were randomly set at the edge of each bone defect, and the number of positive cells contained within the area was averaged to determine the number of positive cells for each specimen. Data was evaluated using n\u0026thinsp;=\u0026thinsp;6 for each group.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between groups were compared using Tukey\u0026rsquo;s honestly significant difference test. Differences were considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMCP-1 Concentration in MSC-CM and depMSC-CM\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe concentration of MCP-1 in MSC-CM, depMSC-CM, and DMEM(-) were 414.9\u0026thinsp;\u0026plusmn;\u0026thinsp;138.2 pg/mL, 11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 pg/mL, and 5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 pg/mL, respectively. MSC-CMs contained significantly higher levels of MCP-1 than depMSC-CM or DMEM(-).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMSC-CM Regulated Macrophage Phenotype-Related Gene Expression in BMMs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImmunocytochemical analysis revealed that the percentage of iNOS and CD11b\u0026thinsp;+\u0026thinsp;M1 cells decreased, while that of CD11b and CD206 positive M2 cells increased in MSC-CM-treated BMMs compared to that in depMSC-CM-treated BMMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-d).\u003c/p\u003e\u003cp\u003eConsistent with these results, qRT-PCR revealed that the expression levels of the pro-inflammatory M1 macrophage markers (\u003cem\u003eiNOS\u003c/em\u003e and \u003cem\u003eCD80\u003c/em\u003e) were significantly downregulated, and that the M2 macrophage markers (CD206 and Arg-1) were significantly upregulated in BMMs cultured with MSC-CM compared to those cultured with depMSC-CM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, the expression levels of anti-inflammatory M2 markers (\u003cem\u003eCD206\u003c/em\u003e and \u003cem\u003eArg-1\u003c/em\u003e) were significantly upregulated in BMMs cultured with MSC-CM compared with those cultured with depMSC-CM.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMSC-CMs Enhanced Osteogenesis-Related Gene Expression in hMSC\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression levels of \u003cem\u003eALP\u003c/em\u003e and \u003cem\u003eOPN\u003c/em\u003e were upregulated significantly in hMSC cultured with MSC-CM compared to those cultured with DMEM(-) and depMSC-CM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively). The expression of \u003cem\u003eCOL I\u003c/em\u003e was significantly higher in hMSCs cultured with MSC-CM compared to those cultured with DMEM(-) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, there was no significant difference between those cultured with MSC-CM and depMSC-CM. MSC-CM enhanced \u003cem\u003eOCN\u003c/em\u003e expression in hMSCs; however, the difference between the DMEM(-) and depMSC-CM groups was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMSC-CM Enhanced Bone Regeneration Compared to depMSC-CM\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAreas with the newly regenerated bone within the bone defect were evaluated using micro-CT scanning and three-dimensional images were reconstructed at 72 h, 1 week, and 2 weeks after implantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Areas with the newly regenerated bone was calculated as a percentage of the graft area. Seventy-two hours after implantation, bone regeneration was not evident in any group. After 1 week, areas with the newly regenerated bone in the MSC-CM group (10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9%) were significantly higher compared to those in the depMSC-CM (2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%), DMEM(-) (3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8%), and defect (2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). After 2 weeks, areas with the newly regenerated bone in the depMSC-CM (19.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7%), DMEM(-) (16.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%), and defect (5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0%) groups were confirmed, whereas the areas in the MSC-CM group (30.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8%) were higher compared to those in other groups significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eHistological analysis also showed that MSC-CMs increased bone regeneration compared with the depMSC-CM, DMEM(-), and defect groups. Seventy-two hours after implantation, migrated inflammatory cells were found around the defect margins in the MSC-CM and depMSC-CM groups; however, bone regeneration was not obvious in any of the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). After 1 and 2 weeks, MSC-CM increased the newly regenerated bone compared to the other groups, and trabecular bone formation along with bone defect margins was observed in the MSC-CM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMSC-CM Induced Macrophage Phenotype Switching at an Early Phase of Bone Regeneration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells stained with the pro-inflammatory M1 macrophage marker iNOS and anti-inflammatory macrophage marker CD206 were observed around the margin of bone defect at 72 h, 1 week, and 2 weeks after implantation to investigate macrophage phenotype switching at an early phase of bone regeneration. After 72 hours, the number of iNOS-positive cells in the MSC-CM group (5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9) was significantly lower than that in the depMSC-CM (17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1), DMEM(-) (13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4), and defect (20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9) groups. In contrast, the number of CD206-positive cells in the MSC-CM group (14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3) was significantly higher compared to that in the depMSC-CM (4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6), DMEM(-) (4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4), and defect (2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3) groups at 72 hours after implantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). After 1 week, the number of iNOS-positive cells in the MSC-CM group (1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9) was significantly lower compared to that in the depMSC-CM (13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9), DMEM(-) (10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5), and defect (12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6) groups. However, significant difference in the number of CD206 positive cells between each group was not obvious (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). After 2 weeks, the number of iNOS-positive cells was significantly lower in the MSC-CM group (1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6) compared to that in the depMSC-CM (6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3), DMEM(-) (4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9), and defect (4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6) groups. In contrast, no significant difference in the number of CD206 positive cells was found between each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). From 72 h to 2 weeks, the M2/M1 ratio was higher in the MSC-CM group than that in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSince the bone-regenerative effect of MSC-CM was first reported, numerous studies have attempted to elucidate the underlying mechanism of its therapeutic effects. To date, several cytokines in MSC-CM and their effects on bone regeneration have been revealed. Previous studies have reported that VEGF, IGF-1, and TGF-β, which were contained in MSC-CMs, promote bone regeneration through cell proliferation, cell recruitment, and angiogenesis and VEGF as a key factor in tissue regeneration that enhances capillary formation, thus supporting cell migration and blood supply to damaged tissues [7, 16]. Recently, the immune regulatory effects of MSC-CMs through macrophage phenotype switching and subsequent anti-inflammatory effects have been reported by several studies [11,12]. We planned to identify the macrophage phenotype-switching factor in MSC-CM and its effect on bone regeneration in this study.\u003c/p\u003e\u003cp\u003eMacrophages are activated by two major pathways (classical and alternative) and perform a wide range of functions [13]. Classically activated macrophages (also known as pro-inflammatory M1 macrophages) are induced by microbial products, T cell-derived signals, and foreign substances. They actively ingest and produce cytokines that stimulate inflammation, thereby playing an essential role in host defense against chronic inflammatory diseases. Alternatively, M2 macrophages are activated by interleukin (IL)-4 and IL-13 produced by T and mast cells. These macrophages produce TGF-β and other growth factors to terminate inflammation and enter the tissue regenerative phase [11,17,18]. Therefore, macrophage phenotype switching is the principal process for resolving inflammation and initiating tissue regeneration.\u003c/p\u003e\u003cp\u003eMCP-1 is a chemokine that promotes chemotaxis of immune cells and plays a crucial role in inflammation and pathological circumstances [19]. Recently, the ability of MCP-1 to convert the macrophage phenotype towards the M2 phenotype has been reported. Hernan \u003cem\u003eet al.\u003c/em\u003e reported that MCP-1 promotes macrophage phenotype switching towards the M2 type through inhibition of apoptosis and caspase 8 cleavage [20]. In this study, we confirmed that the MSC-CM contained MCP-1 at a concentration of 414.9\u0026thinsp;\u0026plusmn;\u0026thinsp;138.2 pg/mL. Therefore, we hypothesized that MCP-1 plays a central role in MSC-CM-induced macrophage phenotypic switching.\u003c/p\u003e\u003cp\u003eMSC-CM-induced immunoregulatory effects and subsequent tissue regeneration are closely associated with macrophage phenotype switching. Gao \u003cem\u003eet al.\u003c/em\u003e reported that MSC-CMs reduced the expression level of tumor necrosis factor (TNF)-α but enhanced the expression levels of IL-10, arginase-1, and CD206 in mouse macrophage RAW264.7 cells. In addition, MSC-CM activated signal transducer and activator of transcription (STAT) 3 but inhibited nuclear factor (NF)-κB pathways in RAW264.7 cells [21]. Our previous report revealed that MSC-CM enhanced macrophage phenotype switching within 72 h after MSC-CM implantation and promoted bone regeneration in rat calvaria bone defects [12]. In this study, using BMMs \u003cem\u003ein vitro\u003c/em\u003e, we revealed that the expression levels of M2 macrophage markers (\u003cem\u003eCD206\u003c/em\u003e and \u003cem\u003eArg-1\u003c/em\u003e) were significantly upregulated, whereas the expression levels of M1 macrophage markers (\u003cem\u003eiNOS\u003c/em\u003e and \u003cem\u003eCD80\u003c/em\u003e) were significantly downregulated in the MSC-CM group compared to those in the depMSC-CM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) Therefore, MCP-1 in MSC-CM is likely to induce macrophage phenotype switching towards the M2 type. In addition, MSC-CM increased the number of M2 macrophages around the bone edge 72 h after MSC-CM implantation compared to the depMSC-CM group \u003cem\u003ein vivo\u003c/em\u003e. Interestingly, the number of M1 macrophage in the MSC-CM group was significantly smaller compared to that in the depMSC-CM group. However, after 1 and 2 weeks, there was no significant difference between the number of M2 macrophages. These results suggest that MSC-CM promote macrophage phenotype switching towards the M2 type within 72 h after implantation; however, this effect is limited to the early phase of bone regeneration.\u003c/p\u003e\u003cp\u003eThe interaction between MSCs and M2 macrophages is a key component in bone regeneration. Gong \u003cem\u003eet al.\u003c/em\u003e previously reported that M2 macrophages accelerate MSCs differentiation into osteoblasts by releasing pro-regenerative cytokines (such as TGF-β, VEGF, and IGF-1), whereas M1 macrophages suppress osteoblast cell differentiation by producing pro-inflammatory cytokines (including IL-6, IL-12, and TNF-α) [22]. In our study, MSC-CM enhanced osteogenesis-related gene expression in MSCs. However, the expression levels of \u003cem\u003eOPN\u003c/em\u003e and \u003cem\u003eALP\u003c/em\u003e were significantly downregulated in the depMSC-CM group compared to those in the MSC-CM group \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results indicate that MCP-1 in MSC-CM contributes to MSC osteogenesis. In a rat calvaria bone defect model, MSC-CM increased the number of M2 macrophages around the bone edge 72 h after implantation and maintained a high M2/M1 ratio for 2 weeks, resulting in enhanced bone regeneration. In contrast, depMSC-CM did not increase the M2/M1 ratio and subsequent bone regeneration was not obvious (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Overall, these results indicate that MSC-CM promotes bone regeneration not only via a direct effect on MSCs themselves but also via MCP-1-induced macrophage phenotype switching towards the M2 type within 72 h after implantation.\u003c/p\u003e\u003cp\u003eMSC-CM contains numerous cytokines, some of which have been addressed in our previous studies to elucidate the underlying mechanism of bone regeneration induced by MSC-CM. We demonstrate the positive effects of MCP-1 on macrophage phenotypic switching and subsequent bone regeneration. However, the precise mechanism underlying MCP-1-induced macrophage phenotypic switching and the mechanism by which M2 macrophages promote bone regeneration remain elucidated. Clarifying these mechanisms will contribute to establishing the clinical use of MSC-CM not only for bone regeneration, but also in other inflammatory diseases and bone-resorptive diseases in the oral and maxillofacial regions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMesenchymal stem cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMSC-CM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ethe conditioned medium of human mesenchymal stem cells derived from bone marrow\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIGF-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInsulin like growth factor-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVEGF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eVascular endothelial growth factor-A\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTGF-β1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransforming growth factor-β1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMCP-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMonocyte chemoattractant protein-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBMM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBone marrow macrophage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePFA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eParaformaldehyde Phosphate Buffer Solution\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFetal bovine serum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eiNOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einducible nitric oxide synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRT-PCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReal-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eArg-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eArginase-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOPN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOsteopontin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCOL I\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eType I collagen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAlkaline phosphate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOCN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOsteocalcin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMicro-CT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMicrocomputed Tomography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eAll animal experiments were performed in strict accordance with the protocols reviewed by the Animal Care and Use Committee of Niigata University (No. SA00456).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Grants-in-Aid for Scientific Research (C) from the Ministry of Health, Labour, and Welfare of Japan (Nos. 20K10113 and 21K21060).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eK.H. and W.K. contributed equally to this work and co-first authors.W.K. performed the conception and design of this study. K.H. and W.K. collected and generated the data. K.H., W.K., R.T. and D.S. performed the data analysis and interpretation. K.H. and W.K. wrote the manuscript and it was revised by T.K.All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank the members of the Division of Reconstructive Surgery for Oral and Maxillofacial Region, Faculty of Dentistry \u0026amp; Graduate School of Medical and Dental Sciences for their help and contributions to this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePelegrine AA, da Costa CES, Correa MEP, Marques JF (2010) Clinical and histomorphometric evaluation of extraction sockets treated with an autologous bone marrow graft. Clin. Oral Implants Res 21: 535\u0026ndash;542.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRickert D, Sauerbier S, Nagursky H, Menne D, Vissink A, Raghorbar GM (2011) Maxillary sinus floor elevation with bovine bone mineral combined with either autogenous bone or autogenous stem cells: a prospective randomized clinical trial. Clin Oral Implants Res 22: 251\u0026ndash;258.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaigler D, Pagni G, Park CH, Braun TM, Holman LA, Yi E, Tarle SA, Bartel RL, Giannobile WV (2013) Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial. Cell Transplant 22: 767\u0026ndash;777.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWislet-Gendebien S, Poulet C, Neirinckx V, Hennuy B, Swingland JT, Laudet E, Sommer L, Shakova O, Bours V, Rogister B (2012) \u003cem\u003eIn vivo\u003c/em\u003e tumorigenesis was observed after injection of \u003cem\u003ein vitro\u003c/em\u003e expanded neural crest stem cells isolated from adult bone marrow. PLOS ONE 7: e46425.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIde C, Nakai Y, Nakano N, Seo Tae-Beom, Yamada Y, Endo K, Noda T, Saito F, Suzuki Y, Fukushima M, Nakatani T (2010) Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat. Brain Res 1332: 32\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eToma C, Wagner WR, Bowry S, Schwartz A, Villanueva F (2009) Fate of culture-expanded mesenchymal stem cells in the microvasculature: \u003cem\u003ein vivo\u003c/em\u003e observations of cell kinetics. Circ Res 104: 398\u0026ndash;402.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOsugi M, Katagiri W, Yoshimi R, Inukai T, Hibi H, Ueda M (2012) Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects. Tissue Eng Part A 18: 1479\u0026ndash;1489.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatagiri W, Watanebe J, Toyama N, Osugi M, Sakaguchi K, Hibi H (2017) Clinical study of bone regeneration by conditioned medium from mesenchymal stem cells after maxillary sinus floor elevation. Implant Dentistry 26: 607\u0026ndash;612.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatagiri W, Endo S, Takeuchi R, Suda D, Saito N, Kobayashi T (2021) Conditioned medium from mesenchymal stem cells improves condylar resorption by mandibular distraction osteogenesis in a rat model. Heliyon 7: e06530.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatagiri W, Takeuchi R, Saito N, Suda D, Kobayashi T (2020) Migration and phenotype switching of macrophages at early-phase of bone-formation by secretomes from bone marrow derived mesenchymal stem cells using rat calvaria bone defect model. J. Dent. Sci 17: 421\u0026ndash;429.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMantovani A, Biswas SK, Galdiero MR, Sica A, Locati M (2013) Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol 229: 176\u0026ndash;185.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang MK, Raggatt LJ, Alexander KA, Kuliwaba JS, Fazzalari NL, Schroder K, Maylin ER, Ripoll VM, Hume DA, Pettit AR (2008) Osteal tissue macrophages are intercalated throughout human and mouse bone lining tissues and regulate osteoblast function \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. J Immunol 181: 1232\u0026ndash;1244.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang QZ, Su WR, Shi SH, Wilder-Smith P, Xiang AP, Wong A, Nguyen AL, Kwon CW, Le AD (2010) Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing. Stem Cells 28: 1856\u0026ndash;1868.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOgata K, Osugi M, Kawai T, Wakayama Y, Sakaguchi K, Nakamura S, Katagiri W (2018) Secretomes of mesenchymal stem cells induce early bone regeneration by accelerating migration of stem cells. J Oral Maxillofac Surg Med Pathol 30: 445\u0026ndash;451.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanjiv S, Anshita D, Ravichandiran V (2021) MCP-1: Function, regulation, and involvement in disease. Int. Immunopharmacol 101: 107598. doi:10.1016/j.intimp.2021.107598\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatagiri W, Kawai T, Osugi M, Sugimura-Wakayama Y, Sakaguchi K, Kojima T, Kobayashi T (2017) Angiogenesis in newly regenerated bone by secretomes of human mesenchymal stem cells. Maxillofac Plast Reconstr Surg 39: 8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMurray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11: 723\u0026ndash;737.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMurray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, Gordon S, Hamilton JA, Ivashkiv LB, Lawrence T, Locati M, Mantovani A, Martinez FO, Mege J, Mosser DM, Natoli G, Saeji JP, Schultze JL, Shirey KN, Sica A, Wynn TA (2014) Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 41: 14\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNarasaraju T, Ng HH, Phoon MC, Chow VTK (2010) MCP-1 antibody treatment enhances damage and impedes repair of the alveolar epithelium in influenza pneumonitis. Am J Respir Cell Mol Biol 42: 732\u0026ndash;743.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoca H, Varsos ZS. Sud S, Craig MJ, Ying C, Pienta KJ (2009) CCL2 and interleukin-6 promote survival of human CD11b\u0026thinsp;+\u0026thinsp;peripheral blood mononuclear cells and induce M2-type macrophage polarization. J Biol Chem 284: 34342\u0026ndash;34354.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao S, Mao F, Zhang B, Zhang L, Zhang X, Wang M, Yan Y, Yang T, Zhang J, Zhu W, Qian H, Xu W (2014) Mouse bone marrow-derived mesenchymal stem cells induce macrophage M2 polarization through the nuclear factor-κB and signal transducer and activator of transcription 3 pathways. Exp. Biol. Med 239: 366\u0026ndash;375.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGong L, Zhao Y, Zhang Y, Ruan Z (2016) The macrophage polarization regulates MSC osteoblast differentiation \u003cem\u003ein vitro\u003c/em\u003e. Ann Clin Lab Sci 46: 65\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"maxillofacial-plastic-and-reconstructive-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mprs","sideBox":"Learn more about [Maxillofacial Plastic and Reconstructive Surgery](http://jkamprs.springeropen.com/)","snPcode":"40902","submissionUrl":"https://submission.springernature.com/new-submission/40902/3","title":"Maxillofacial Plastic and Reconstructive Surgery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"bone regeneration, conditioned media, conditioned Medium, MCP-1, macrophage phenotype switching, mesenchymal stem cell","lastPublishedDoi":"10.21203/rs.3.rs-8186435/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8186435/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eWe have reported that the cytokines and chemokines contained in conditioned media of human mesenchymal stem cells (MSC-CM), which were derived from bone marrow, promote bone regeneration. We recently reported macrophage phenotype switching towards the anti-inflammatory M2 phenotype induced by MSC-CM and its potential to establish regenerative condition and assist subsequent bone regeneration. However, the specific factors in the MSC-CM responsible for this process remain unclear. Monocyte chemoattractant protein (MCP) -1, present in MSC-CM, promotes cell migration and activation of the monocyte-macrophage lineage; therefore, we hypothesized that MCP-1 is one of the key factors in MSC-CM-induced macrophage phenotype switching. The effect of MCP-1 on MSC-CM-induced macrophage phenotype switching and subsequent bone regeneration was investigeted in this study.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eMCP-1 was depleted from MSC-CM (depMSC-CM) and used in subsequent experiments. Rat bone marrow macrophages were incubated in MSC-CM or depMSC-CM and expression of macrophage markers was examined \u003cem\u003ein vitro\u003c/em\u003e. In addition, the effect of MSC-CM and depMSC-CM on bone regeneration and macrophage phenotype switching were evaluated using rat calvaria defect model \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eMSC-CM enhanced M2 macrophage marker expression in rat bone marrow macrophages compared to those treated with depMSC-CM \u003cem\u003ein vitro\u003c/em\u003e. In addition, MSC-CM increased the number of M2 macrophage marker-positive cells in bone defects and enhanced subsequent bone regeneration in a rat calvaria bone defect model.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eMCP-1 seemed to play an essential role in MSC-CM-induced macrophage phenotypic switching and subsequent bone regeneration.\u003c/p\u003e","manuscriptTitle":"Monocyte Chemoattractant Protein-1 from a Conditioned Medium of Bone Marrow-Derived Mesenchymal Stem Cells Promotes Bone Regeneration by Enhancing Macrophage Phenotype Switching","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 19:35:56","doi":"10.21203/rs.3.rs-8186435/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-26T06:37:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-26T02:14:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T16:32:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283136110094623368828937669880326341236","date":"2025-12-15T17:22:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91420398506185119107312349509333755065","date":"2025-12-14T22:38:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-13T06:05:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172234734455280946877872522453713546908","date":"2025-12-09T03:35:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-07T02:37:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156693800813007942528750369677482335761","date":"2025-12-06T03:46:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85621621376440625073612849965627062397","date":"2025-12-04T03:22:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-04T02:58:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-02T08:20:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-02T08:17:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Maxillofacial Plastic and Reconstructive Surgery","date":"2025-11-23T15:20:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"maxillofacial-plastic-and-reconstructive-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mprs","sideBox":"Learn more about [Maxillofacial Plastic and Reconstructive Surgery](http://jkamprs.springeropen.com/)","snPcode":"40902","submissionUrl":"https://submission.springernature.com/new-submission/40902/3","title":"Maxillofacial Plastic and Reconstructive Surgery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6fbc81c-d1a6-46b8-8a76-b5c0c085eff2","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:07:54+00:00","versionOfRecord":{"articleIdentity":"rs-8186435","link":"https://doi.org/10.1186/s40902-026-00503-1","journal":{"identity":"maxillofacial-plastic-and-reconstructive-surgery","isVorOnly":false,"title":"Maxillofacial Plastic and Reconstructive Surgery"},"publishedOn":"2026-02-24 15:59:34","publishedOnDateReadable":"February 24th, 2026"},"versionCreatedAt":"2025-12-05 19:35:56","video":"","vorDoi":"10.1186/s40902-026-00503-1","vorDoiUrl":"https://doi.org/10.1186/s40902-026-00503-1","workflowStages":[]},"version":"v1","identity":"rs-8186435","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8186435","identity":"rs-8186435","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}