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M2 Macrophagy-derived exosomal miRNA-5106 induces bone mesenchymal stem cells towards osteoblastic fate by targeting salt-inducible kinase 2 and 3

Journal of Nanobiotechnology volume 18, Article number: 66 (2020) Cite this article

A Correction to this article was published on 27 March 2021

This article has been updated

Abstract

Background

Osteoblast differentiation is a vital process for fracture healing, and exosomes are nanosized membrane vesicles that can deliver therapeutic drugs easily and safely. Macrophages participate in the regulation of various biological processes in vivo, and macrophage-derived exosomes (MD-Exos) have recently been a topic of increasing research interest. However, few study has explored the link between MD-Exos and osteoblast differentiation. Herein, we sought to identify miRNAs differentially expressed between M1 and M2 macrophage-derived exosomes, and to evaluate their roles in the context of osteoblast differentiation.

Results

We found that microRNA-5106 (miR-5106) was significantly overexpressed in M2 macrophage-derived exosomes (M2D-Exos), while its expression was decreased in M1 macrophage-derived exosomes (M1D-Exos), and we found that this exosomal miRNA can induce bone mesenchymal stem cell (BMSC) osteogenic differentiation via directly targeting the Salt-inducible kinase 2 and 3 (SIK2 and SIK3) genes. In addition, the local injection of both a miR-5106 agonist or M2D-Exos to fracture sites was sufficient to accelerate healing in vivo.

Conclusions

Our study demonstrates that miR-5106 is highly enriched in M2D-Exos, and that it can be transferred to BMSCs wherein it targets SIK2 and SIK3 genes to promote osteoblast differentiation.

Background

Fracture is very common in clinic, which can bring heavy burden to fracture patients. The rapid healing of fracture can help patients to get early recovery, relieve the financial burden, and improve the quality of life. Bone mesenchymal stem cells (BMSCs) are capable of differentiating into osteoblasts, making them crucial in the process of fracture healing [1, 2]. Previous studies have found that many factors contribute to the differentiation of BMSCs, including microRNA-188, modulates age-related switching between osteoblast and adipocyte differentiation [3]. Similarly, myocardin-related transcription factor A (MRTFA) has been found to be a positive regulator of BMSCs [4]. The identification of further factors regulating BMSCs differentiation may therefore allow for a better understanding of osteoblast differentiation. Macrophages are vital for all stages of fracture healing [5]. They are among the earliest cells to migrate to fracture sites, stimulated by the tissue injury and the associated acute inflammatory phase [6]. Macrophages are also necessary for osteoblast differentiation in vitro, and have been shown to be important during both early and late stages of fracture healing [7, 8]. Particularly important for the healing process is macrophage phenotypic switching from the more inflammatory M1 subtype to the anti-inflammatory M2 subtype, with non-union often being associated with prolonged pro-inflammatory macrophage responses [9, 10]. However, the mechanisms governing the beneficial role of M2 macrophages in the context of osteoblasts differentiation remain elusive.

Nanomaterials, which could exert certain regulatory function in vivo, had obtained accumulated evidence of their roles in osteogenic differentiation [11,12,13]. Exosomes represent a promising type of nanomaterials from which miRNAs can be isolated [14]. MiRNAs in turn are able to bind with target mRNAs to regulate their rates of expression or degradation without being interfered by various of internal and external factors [15]. Indeed, miRNAs have been reported to be secreted extracellularly in exosomes and to exert a regulatory role in a wide array of biological processes [16, 17]. One recent study, for example, found that myocardial miRNAs encapsulated in circulating exosomes coordinate systemic responses to cardiac injuries, potentially offering novel therapeutic avenues for treating myocardial infarction [18].

Herein, we sought to investigate the effect of M2 macrophage-derived exosomal miRNAs on BMSCs differentiation. We demonstrate that miR-5106 is highly enriched in M2D-Exos, and that it can be transferred to BMSCs wherein it targets the SIK2 and SIK3 genes to induce osteoblastic differentiation in vitro and in vivo.

Results

M1 and M2 macrophages secrete exosomal miRNAs

We first assessed the expression of phenotypic markers associated with bone marrow-derived macrophages (BMDMs) by flow cytometry (Additional file 1: Figure S1A). To monitor M1 polarization, we assessed the expression of phenotypic markers associated with M1 macrophages, including F4/80 and CD11c. As expected, the rate of F4/80 and CD11c dual-positivity in BMDMs was significantly increased after 24 h of treatment with LPS and IFN-γ (Additional file 1: Figure S1B). Similarly, the rate of F4/80 and CD206 dual-positivity in BMDMs was significantly increased after 24 h of treatment with IL-4 (Additional file 1: Figure S1C). These results confirmed the success of the cellular M1 and M2 polarization model we used.

We next assessed the ability of these macrophages to secrete miRNAs that can be internalized by other cells. To do this, we used a model system wherein miRNA-26a-5p (miR-26a-5p), which is an osteoblast-selective miRNA, 19 was labeled with Cy3 and transfected into these M1 and M2 macrophages in the upper chamber of a transwell chamber system. BMSCs were added to the lower chamber, and we assessed the delivery of Cy3-miR-26a-5p from the upper to the lower chamber in this assay system. As we observed increasing red fluorescence among BMSCs over time, which confirmed that miR-26a-5p was released from both types of macrophages in a format that was internalized by BMSCs (Fig. 1a). In contrast, when free Cy3 was used to treat M1 or M2 macrophages prior to their use in this assay system, minimal Cy3 was detectable in BMSC2 following a 12 h co-culture (Additional file 1: Figure S2). As such, these results show that M1 and M2 macrophages can secrete extracellular miRNAs that can be internalized by BMSCs.

Fig. 1
figure 1

Macrophages secrete exosomal miRNAs. a M1 and M2 macrophages transfected with a Cy3-labeled miR-26a-5p mimic were co-cultured with BMSCs in a transwell (membrane pore = 0.4 nm) plate; b Particle size of the vesicles secreted from M1 and M2 macrophages were measured by NanoSight analysis; c Represent picture of the ultrastructure of the two kind of exosomes observed by TEM. Scar bar = 200 nm; d The protein levels of CD 63 and CD 81 in the two kind exosomes; e The two kind of exosomes were marked with red flurescence dye PKH26 and co-cultured with BMSCs, red flurescence represents exosomes in BMSCs, scar bar = 50 µm. Data are mean ± SD of triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001

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To explore whether M1 and M2 macrophage secrete exosomes, we extracted exosomes via ultracentrifugation and analyzed the isolates via transmission electron microscopy (TEM), dynamic light scattering (DLS), and flow cytometry. DLS suggested particles with sizes ranging from 30 to 200 nm were present within samples (Fig. 1b). TEM revealed these particles to have cup- or sphere-shaped morphology (Fig. 1c). Flow cytometry analysis further give evidence that exosomal surface markers such as CD63 and CD81 were present on these particles (Fig. 1d). Together these results suggested that the isolated circulating nanoparticles are exosomes.

Subsequently, we tested whether these MD-Exos can be taken up by BMSCs. These MD-Exos were labeled with the fluorescent dye PKH26 and then added into the culture medium of BMSCs. After 12 h, the BMSCs exhibited efficient uptake of the MD-Exos, as indicated by the presence of red fluorescence staining in these cells (Fig. 1e). CCK-8 assay was performed to test the biocompatibility of M1D- and M2D- Exos (Additional file 1: Figure S3).Together these results suggested that macrophages can secrete miRNA-containing exosomes, which are then efficiently transported into recipient cells.

M1D-Exos and M2D-Exos regulate BMSCs differentiation

We next investigated how MD-Exos affect osteogenic differentiation in vitro. BMSCs were treated with phosphate buffered saline (PBS), 100 µg/ml M1D-Exos, or 100 µg/ml M2D-Exos respectively. Expression of the osteogenic genes collagen I, alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2) were then measured by western blotting and quantitative reverse transcriptase PCR (qRT-PCR) 48 h post transfection. As compared with the PBS group, significantly higher mRNA levels of these genes were detectable in the M2D-Exos group, while levels were lower in the M1D-Exos group (Fig. 2a–c). To explore the impact of M1D-Exos and M2D-Exos on extracellular matrix mineralization, cells were continuously cultured for 21 days and then assessed for alizarin red staining, revealing reduced mineral deposition in M1D-Exos-treated cells, and enhanced mineral deposition in M2D-Exos-treated cells (Fig. 2d–f). Similarly, M1D-Exos reduced ALP activity and staining, whereas M2D-Exos enhanced ALP activity and staining (Fig. 2e–g).

Fig. 2
figure 2

M2D-Exos induce osteoblast activity and matrix mineralization. a Osteogenic genes were upregulated in M2D-Exos-treated BMSCs measured by western blotting analysis; b The relative intensity of western blotting analysis; c Overexpression of the four osteognic genes can be detected in M2D-Exos groups measured by qRT-PCR analysis; d Alizarin red-mediated calcium staining in BMSCs following treated by PBS (control group), M1D-Exos, and M2D-Exos for 21 days. Scale bar = 10 mm; e ALP staining in BMSCs following treated by PBS (control group), M1D-Exos, and M2D-Exos for 14 days. Scale bar = 10 mm; f, g The statistical data of Alizarin red-mediated calcium staining and ALP staining. Data are mean ± SD of triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001

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M2D-Exos accelerates murine femoral fracture healing

To explore the impact of M1D-Exos and M2D-Exos on murine fracture healing, fracture mice model were locally injected a total of 100 µl samples with PBS, 100 µg/ml M1D-Exos, or 100 µg/ml M2D-Exos respectively in the fracture sites on days 0, 4, and 7 after surgery. X-rays and micro-CT examinations were used to monitor the process of fracture healing. Mice treated with M2D-Exos exhibited a larger callus volume and smaller fracture gap compared with control animals (Fig. 3a, b). Furthermore, on day 21, bone samples were collected for qRT-PCR analysis of osteogenic genes, revealing that levels of collagen I, ALP, OCN, and Runx2 were increased significantly in M2D-Exos-treated animals relative to those in the other treatment groups (Fig. 3c). In addition, bone samples were further collected for Hematoxylin and Eosin (H&E)/Alcian-blue staining on days 21post-surgery. H&E/Alcian-blue staining results revealed a reduced cartilage area and increased bone area at the fracture junction in the M2D-Exo group, suggesting more rapid remodeling for these animals (Fig. 3d, e). Moreover, M2D-Exo-treated animals exhibited significantly higher bone volume (BV), total volume (TV), BV/TV, and bone mineral density (BMD) relative to other study animals (Fig. 3f). Together these results suggested that M2D-Exos mediated accelerated fracture healing.

Fig. 3
figure 3

M2D-Exos accelerates fracture healing in vivo. a X-rays comparison of fracture healing between control, M1D-Exos, and M2D-Exos groups on day 3, 7, 14 and 21 post-injury; b micro-CT three dimensional construction and cross sections of the fracture site among the three groups on day 14 and 21; c Osteogenic expression levels were measured by qRT-PCR analysis, n = 6 mice/group; d The H&E/Alcian-blue staining images on day 21 post-operation. e The statistical results of H&E/Alcian-blue staining; f BV and TV of the callus, BV/TV, and BMD datas on days 14 and 21 post-operation were assessed via micro-CT. n = 6 mice/group. Data are mean ± SD of triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001

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Microarray-mediated identification of miRNAs differentially expressed in M1D- and M2D-Exos

To identify miRNAs differentially expressed between M1D-Exos and M2D-Exos, we first extracted M1D-Exos and M2D-Exos via ultracentrifugation method, and then used a miRNA microarray approach to compare the miRNA expression profiles therein. Unsupervised clustering of the microarray results revealed significantly distinct miRNA profiles between M2D-Exos and M1D-Exos (Fig. 4a–b). We then identified miRNAs with a mean fold change > 5 or < − 5 and a p value < 0.05 for further analyses. Based on the criteria, miR-709, miR-3112-3p, miR-3069-3p, miR-6406, miR-6389, miR-5106, miR-8103, miR-1934-3p, miR-22-3p, and miR-505-5p were the top ten overexpressed miRNAs. The expression of these top 10 miRNAs was then validated via qRT-PCR in six independent M1D-Exo and M2D-Exo samples, revealing miR-5106 to be significantly upregulated in M2D-Exos relative to M1D-Exos, leading us to study it further (Fig. 4c). We compared miR-5106 expression between M1 and M2 macrophage via qRT-PCR analysis, confirming its overexpression in the M2 macrophage group (Fig. 4d). Furthermore, we co-cultured M1 or M2 macrophages with BMSCs in a transwell system as above, and a significant increase in miR-5106 abundance in the BMSCs was detectable following M2 macrophage co-culture (Fig. 4e). In addition, BMSCs were divided into four groups and subjected to four different treatments (PBS only, M2D-Exos, antagomiR-5106, antagomiR-5106 + M2D-Exos), after which miR-5106 expression was assessed via qRT-PCR analysis, revealing that M2D-Exos were able to partially reverse the antagomiR-5106-mediated reduction in miR-5106 levels in these cells (Fig. 4f). As demonstrated above, MD-Exos can be internalized by BMSCs. Thus, these results suggested that miR-5106 is enriched in M2D-Exos and can be delivered to BMSCs by containing in M2D-Exos.

Fig. 4
figure 4

Identification of differentially expressed miRNAs between M1D-Exos and M2D-Exos. a Heat map identified the differently expressed miRNAs (fold change > 5 or < − 5, Benjamini-Hochberg-corrected p); b The GO (gene ontology) enrichment of the differently expressed miRNAs; c Expression of the top ten differentially expressed miRNAs between M1D-Exos and M2D-Exos; d The miR-5106 level between M1 and M2 macrophages; e The miR-5106 level among groups following co-cultured BMSCs with M1 or M2 macrophages; f The miR-5106 level among the groups with different treatments. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

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MiR-5106 induces BMSCs differentiation in vitro

Next, we explored the ability of miR-5106 to directly affect BMSCs by treating cells with PBS, control agomiR construct (agomiR-NC), agomiR-5106, control antagomiR construct (antagomiR-NC), or antagomiR-5106 respectively, and the results indicated that the agomiR-5106 treatment significantly increasing the miR-5106 expression in BMSCs (Fig. 5a). Osteogenic related genes, including collagen I, ALP, OCN, and Runx2, were then measured by western blotting and qRT-PCR analysis, revealing a clear increase in these genes in cells in the agomiR-5106 group (Fig. 5b–d). Moreover, we treated M2 macrophages with miR-5106 inhibitor (antagomiR-5106) to suppress the expression of miR-5106 in the cells, and then extract the exosomes from these cells to investigate the effect of M2D-Exos with miR-5106-silence on the osteoblastic differentiation of BMSCs. The results showed that antagomiR-5106 can significantly decrease the miR-5106 expression in M2D-Exos, and decreased osteogenic genes expression can be detected in M2D-ExosantagomiR−5106 group compared with PBS and M2D-Exos groups (Fig. 5e–f). Additionally, to explore the roles of miR-5106 on extracellular matrix mineralization, cells were continuously cultured for 21 days and then analyzed via alizarin red staining, revealing enhanced mineral deposition in agomiR-5106-treated cells (Fig. 5g). Similarly, miR-5106 induced more pronounced ALP activity and staining (Fig. 5h). Together these results thus confirmed the ability of miR-5106 to directly induce BMSCs differentiation in vitro.

Fig. 5
figure 5

M2D-Exos induce osteoblastic differentiation of BMSCs in vitro. a miRNA-5106 was upregulated in agomiR-5106 treated BMSCs following transfection using lipofecamine control, 200 µM agomiR-5106, agomiR-NC, antagomiR-5106 or antagomiR-NC for 48 h; b Western blotting analysis of Collagen I, ALP, OCN and Runx2 protein levels in BMSCs treated using lipofecamine control, agomiR-NC, agomiR-5106, antagomiR-NC or antagomiR-5106 for 48 h; c The relative intensity of the western blotting analysis; d qRT-PCR analysis was used to assess expression of osteoblast differentiation genes including Col1a1, ALP, OCN, and Runx2 in transfected BMSCs; e M2D-Exos were treated with miR-5106 inhibitor (antagomiR-5106) to investigate the effect of M2D-Exos with miR-5106-silence on the osteoblastic differentiation of BMSCs. The results showed that antagomiR-5106 can significantly decrease the miR-5106 expression in M2D-Exos; f Decreased osteogenic genes expression can be detected in M2D-ExosantagomiR−5106 group compared with PBS and M2D-Exos groups; g Alizarin red-mediated calcium staining in BMSCs following differently transfeced for 21 days, Scale bar = 10 mm; h ALP staining in BMSCs following different treatments for 14 days. Scale bar = 10 mm. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

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SIK2 and SIK3 are involved in BMSCs differentiation

We further assessed the molecular mechanisms whereby miR-5106 promotes BMSCs functionality. To do so, TargetScan (http://www.targetscan.org/vert_70/) was used to identify putative miR-5106 targets. Following a literature review, we found SIK2 and SIK3 to be the most associated with osteogenic differentiation among these candidate genes [20]. Furthermore, a luciferase reporter assay was used to confirm that miR-5106 was able to specifically bind to the predicted target region of the SIK2 and SIK3 mRNAs such that when these regions were mutated, the miRNAs were no longer able to bind to suppress luciferase activity (Fig. 6a). Moreover, we found that miR-5106 overexpression led to a clear suppression of SIK2 and SIK3 expressions (Fig. 6b, c, Additional file 1: Figure S4A). To explore the dependence of osteoblastogenesis on SIK2 and SIK3 genes, we used a SIK2-specific and SIK3-specific siRNA to examine its role in this process (Fig. 6d, e, Additional file 1: Figure S4B). SIK2 and SIK3 knockdown resulted in elevated collagen I, ALP, OCN, and Runx2 expression (Fig. 6f–g, Additional file 1: Figure S4C). Moreover, SIK2 and SIK3 expressions in mice callus decreased significantly after receiving the injection of agomiR-5106 compared with PBS and antagomiR-5106 groups (Fig. 6h, i, Additional file 1: Figure S4D). However, when over-expression of SIK2/SIK3, decreased levels of osterogenic genes can be detected in BMSCs, and their negative effects on osteogenic differentiation can be partly rescued by agomiR-5106 (Fig. 6j–l). In addition, we further explored the impact of SIK2 and SIK3 genes on extracellular matrix mineralization. After growth in for 21 days in osteoblast inducing conditional media, stronger mineral deposition was evident for cells treated with SIK2-specific siRNA and SIK3-specific siRNA relative to controls (Fig. 6m). Similarly, SIK2-specific siRNA and SIK3-specific enhanced ALP activity and staining (Fig. 6n).

Fig. 6
figure 6

miRNA-5106 targets SIK2 and SIK3 to induce osteoblastic activity in vitro. a Luciferase reporter constructs containing either a WT SIK2 and SIK3 3′ UTR (WT 3′ UTR) or this same region after site-directed mutagenesis (3′ UTR-Mut). Luc, luciferase. Endogenous miRNA-5106 effects in BMSCs on WT SIK2 and SIK3 3′ UTR (luc-UTR), the SIK2 and SIK3 3′ UTR mutant (luc-UTR-Mut), as assessed following agomiR-NC or agomiR-5106 treatment; b The levels of mRNA SIK2 and SIK3 were western blotting analysis in agomiR-5106 treated BMSCs following transfection using lipofecamine control, 200 µM agomiR-NC, agomiR-5106, antagomiR-NC or antagomiR-5106 for 48 h; c The expression of SIK2 and SIK3 was measured by qRT-PCR analysis; d, e Western blotting and qRT-PCR analysis were used following control, siRNA-NC, siRNA-SIK2, and siRNA-SIK3 transfection to assess SIK2 and SIK3 expression, with a scrambled siRNA utilized as a negative control (siRNA-NC); f, g The levels of osteogenic genes were measured by western qRT-PCR and blotting analysis; h, i SIK2 and SIK3 expressions in mice callus decreased significantly on day 14 after receiving the injections of agomiR-5106 compared with PBS and antagomiR-5106 groups; j, k SIK2 and SIK3 expressions were measures by qRT-PCR analysis following different treatments; l Osteogenic related genes level was assessed by qRT-PCR analysis; m Alizarin red-mediated calcium staining and n ALP staining in BMSCs following differently transfected, Scale bar = 10 mm. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

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M2D-exos-derived miR-5106 accelerates fracture healing in vivo

To investigate how M2D-Exos-derived miR-5106 affects fracture healing, we administered PBS, M2D-Exos, or M2D-ExosantagomiR−5106 directly to the fracture site in our murine model system on days 1, 4, and 7 post-injury. Animals were then monitored via X-rays and micro-CT approaches, we observed a significantly reduced fracture gap and a larger callus volume in mice treated using M2D-Exos, but not M2D-ExosantagomiR−5106, compared with control group. And on day 21 post-fracture, animals treated using M2D-Exos no longer exhibited a clear boundary between hardened callus and cortical bone, and remodeling had clearly taken place. However, when silencing the expression of miR-5106 in M2D-Exos, the positive effect was mainly reversed (Fig. 7a, b). Furthermore, on day 14, bone samples were collected for qRT-PCR analysis of miR-5106, revealing that levels of miR-5106 was increased significantly in M2D-Exos-treated animals relative to those in the other treatment groups (Fig. 7c). In addition, H&E/Alcian-blue staining on days 21 further indicated reduced cartilage area at the fracture site for mice treated with M2D-Exos group and knocked out miR-5106 can impair this po-fracture-healing effect, consistent with this miRNA facilitating more rapid remodeling (Fig. 7d, e). Moreover, on day 14 in M2D-ExosantagomiR−5106-treated animals the total and bone callus volume was smaller relative to mice in M2D-Exos groups, and this remained significant when comparing M2D-ExosantagomiR−5106 and M2D-Exos mice on day 21 post-injury (Fig. 7f). BMD was also significantly higher for mice treated with M2D-Exos relative to animals in other groups (Fig. 7f). This suggested that miR-5106 derived from M2D-Exos positively regulates fracture healing.

Fig. 7
figure 7

miR-5106 accelerates fracture healing in vivo. a X-rays comparison of fracture healing between control, M2D-Exos, and M2D-ExosantagomiR−5106 groups on day 3, 7, 14 and 21 post-injury; b micro-CT three dimensional construction and cross sections of the fracture site among the three groups on day 14 and 21; c miR-5106 level in callus was measured by qRT-PCR analysis, n = 6 mice/group; d The H&E/Alcian-blue staining images on day 21 post-operation. e The statistical results of H&E/Alcian-blue staining; f BV and TV of the callus, BV/TV, and BMD datas on days 14 and 21 post-operation were assessed via micro-CT. n = 6 mice/group. Data are mean ± SD of triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001

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Discussion

Macrophage is a cell type that can readily undergo differentiation into a range of effector subtypes depending on local cellular and secreted signals. The positive regulatory role of M2 macrophages during bone fracture healing is already well known [19,20,21]. However, the underlying mechanisms for this beneficial role remain elusive. To more fully explore the role of M2 macrophages in bone formation and to explore the potential underlying mechanisms, we herein focused on the effects of M2D-Exos on osteoblast differentiation in vitro and in vivo. Exosomes are small vesicles that are secreted into circulation by a range of cell types in vivo, whereupon they can be internalized by proximal or distal cells [22]. The small molecules within these exosomes (including proteins and nucleic acids) can then regulate the functionality of recipient cells upon internalization, and thereby conducting a communication among various cells and organs [23]. In addition, exosomes have been demonstrated to be one of the ideal nanomaterials for delivering the regulatory substances to the targets and acted as a protective role in this process [24]. Our results suggested that M2D-Exos were able to stimulate osteoblast differentiation and bone mineral deposition, as well as to promote fracture healing in murine models. All these data thus suggest that the ability of M2D-Exos to induce osteoblast function may explain the previously reported benefits of M2 macrophages in the context of fracture healing, as well as providing a novel therapeutic strategy for the treatment of fracture.

miRNAs are small RNA molecules that cannot encode protein but play vital roles in diseases such as cancer and processes such as immunoregulation and inflammation [24,25,26]. In immune system, miRNA expression patterns can be used as a biomarker for particular disease states such as cancer or endocrine events [27, 28]. Evidence also reported that exosomes can be taken up into neighboring or distant cell types to modulate the function of recipient cells [29]. Therefore, to explore the relationship between macrophage-derived exosomal miRNAs and BMSCs, we co-cultured these two cell types, revealing that exosomal miRNAs from macrophages could be internalized by BMSCs, providing a strong basis for our further research. In the present research, we searched out miR-5106 to be the key miRNA enriched in M2D-Exos by using microarray analysis. There is few research had reported the functionality of miR-5106 previously. To get the knowledge of this miRNA, we screened the NCBI database (https://www.ncbi.nlm.nih.gov/gene/100628618). It reported that miR-5106 is involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. Therefore, we explored the target mRNA of miR-5106 by using TargetScan system, and the genes SIK2 and SIK3 were selected to be the potential target genes for miR-5106. SIK2 and SIK3 were demonstrated involved in various processes such as cell cycle regulation, gluconeogenesis and lipogenesis regulation, muscle growth and differentiation and tumor suppression [30, 31]. And a recent research had indicated that SIK2 and SIK3 inhibition is associated with the enhance of anti-inflammatory phenotype of macrophages [31]. In the present study, we demonstrated the relationship of M2D-Exos-derived miR-5106 and SIK2 and SIK3 genes by using luciferase assays, and the negative regulatory effect of miR-5106 on SIK2 and SIK3 expression was then verified in vitro.

Osteoblast differentiation is a vital process for fracture healing, arising as a result of various intersecting factors [32]. We observed the upregulation of miR-5106 in exosomes isolated from M2 macrophages, and we found that this miRNA positively regulated osteogenic differentiation. When miR-5106 was transferred into BMSCs, their ability to undergo such differentiation was markedly enhanced. Similarly, we further observed that miR-5106 was able to accelerate fracture healing in vivo. Several regulators have been reported to mediate the osteogenic differentiation of BMSCs, [33, 34] with SIK2 and SIK3 genes being the two of the most important such factors [35, 36]. Herein, we found that SIK2 and SIK3 were the miR-5106 target genes, thus highlighting a mechanistic role for miR-5106 in mediating osteoblastic differentiation. When downregulated, SIK2 and SIK3 are no longer able to suppress the osteoblastic differentiation of BMSCs, and thereby accelerating fracture healing in vivo.

Conclusions

Taken together, our results indicate that exosomes isolated from the M2 macrophages are capable of accelarating fracture healing via interference with BMSC differentiation in vitro and in vivo. This positive effect is at least partially mediated by miR-5106, which is enriched in M2D-Exos and can promote osteogenic differentiation of BMSC via suppressing the expression of SIK2 and SIK3 (Fig. 8). Therefore, our findings suggest that local injection of M2D-Exos may represent a promising therapeutic strategy to bolster fracture healing.

Fig. 8
figure 8

Schematic diagram showing proposed mechanisms by which exosomal miR-5106 derived from M2 macrophages induce the BMSCs towards osteoblastic differentiation. Enriched level of miR-5106 in MED-Exos enhances the inhibitory effect on SIK2 and SIK3. Decreased SIK2 and SIK3 expression induces BMSCs differentiation and thereby accelerates bone remodeling

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Methods

Culture of macrophages

The Institutional Review Board at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology approved this study. C57BL/6J mice (6-week-old) from the Center of Experimental Animals, Tongji Medical College, Huazhong University of Science and Technology were used for this study. BMDMs were generated using M-CSF as in past reports [26]. Briefly, bone marrow cells isolated from these mice were grown in RPMI-1640 (#11875093, Gibco, Grand Island, USA) containing 10% FBS (#10099141, Gibco, Grand Island, USA), 1% penicillin/streptomycin (#SV30010, Hyclone, USA), and 50 ng/ml M-CSF (#216-MC, R&D System, Minneapolis, MN, USA) for 7 days. Subsequently, BMDMs were harvested for flow cytometric and transcriptomic analysis. M1 and M2 macrophages differentiation was initiated by treating BMDMs for 24 h using either 100 ng/ml LPS (#L2630, Sigma–Aldrich, St Louis, MO, USA) + 20 ng/ml IFNγ (#285-IF-100/CF, R&D Systems), or 20 ng/ml IL-4 (#204-IL-010, R&D Systems), respectively. M1 and M2 macrophages were then harvested for flow cytometric and transcriptomic analysis.

Flow cytometric analysis

This procedure was performed as descripted previously [27]. Antibodies were purchased from eBioscience company. BMDMs, M1 macrophages, and M2 macrophages were stained using lineage-specific antibodies, with F4/80 (#123107, Biolegend, San Diego, CA, USA) and CD 209 (#12-2092-80, Sigma–Aldrich) used for BMDM identification, F4/80 and CD11c (#117307, Biolegend) used for M1 macrophage identification, and F4/80 and CD206 (#141705, Biolegend) used for M2 macrophage identification. FlowJo software (version: FlowJo™ v10.6.1) was used for data analysis.

BMSCs culture and transfection

BMSCs were kindly donated by the Huazhong University of Science and Technology, Wuhan, China. Cells were grown in a specific media designed for C57BL/6 mouse mesenchymal stem cells (#MUBMX-03011-440, Cyagen, Guangzhou, China) at 37 °C in a 5% CO2 incubator. Cells were maintained for a maximum of 3 passages. Lipofectamine 3000 (#L3000001, ThermoFisher Scientific, USA) was used to transfect cells with siRNAs or miRNAs based on provided directions. agomiR-5106, agomiR-NC, antagomiR-5106, and antagomiR-NC (GenePharma, Shanghai, China) were transfected into cells using a 20 μM final concentration, whereas SIK2 siRNA, and SIK3 siRNA constructes (RIBOBIO, Guangzhou, China) were transfected at 50 nM. Plasmid-NC, plasmid-SIK2, and plasmid-SIK3 were synthesized by GenePharma (GenePharma, Shanghai, China). After receiving the different treatments, BMSCs were resuspended with PBS, and the consistent number of cells among the different groups were collected for the qRT-PCR analysis.

Exosome purification, characterization and uptake

Following a 72 h culture step, we removed debris and dead cells from samples via a 10 min spin at 1000g, followed by filtration with a 0.2 μm filter (122-0020PK ThermoFisher Scientific). Media then underwent ultracentrifugation for 6 h at 100,000g at 4 °C. Samples were then washed with PBS and spun for an additional 20 min at 100,000g, after which PBS was used to resuspend the pellet, which contained exosomes. Transmission electron microscopy (TEM; EFI, TECNAI G2) was used to assess morphology, while a Nanosizer™ instrument (Malvern Instruments, Malvern, UK) was used for Dynamic light scattering (DLS) analyses and flow cytometry was used to assess exosome surface marker expression. BMSC uptake of these exosomes was confirmed by labeling isolated exosomes using PKH 26 (#MINI26-1KT, Sigma, MO, USA) and then collecting them via centrifugation at 110,000g for 20 min. Briefly, exosomes were suspended in 1 mL diluent C containing 5 μM PKH26 and incubated for 5 min. The labeling action was stopped after 1 min incubation with an equal volume of 1% bovine serum albumin (Bovogen, Melbourne, Australia). The exosomes were washed three times with Amicon ultrafilter (10 kDa cut-off, Millipore, MA, USA) with cold PBS and then resuspended in 200 μL PBS. These labeled exosomes were used to treat BMSCs for 12 h followed by analysis under a confocal microscope.

qRT-PCR analysis

Before extracting miRNAs, callus samples were preserved using RNA Later (#76104, QIAGEN, Germany). Trizol (#15596018, Invitrogen, USA) was used to isolate total cell RNA, after which a Verso™ cDNA Synthesis Kit (#AB-1054/A, ThermoFisher Scientific) was applied to reverse transcribe RNA. miRNA expression was assessed using the SeraMir Exosome RNA purification Kit (System Biosciences, USA) to extract exosomal miRNAs, followed by use of a TaqMan microRNA assay kit (Applied Biosystems, USA) for cDNA synthesis. All qRT-PCR reactions were performed using a Thermal Cycler C-1000 Touch system (#10021377, Bio-Rad CFX Manager, USA), and U6 or GAPDH were used to normalize target expression as appropriate. Data were expressed as fold changes over controls. Primers used herein are compiled in Table 1.

Table 1 miRNAs and mRNA primer sequence
Full size table

Luciferase reporter assays

BMSCs were added to 24-well plates (2.5 × 105 cells/well) followed by transfection with dual-luciferase vectors (SIK2 WT, SIK2 Mut, SIK3 WT, and SIK3 Mut) along with either miR-5106 agonist (AgomiR-5106) or negative control (AgomiR-NC). A Quik Change Site-Directed Mutagenesis Kit (Strata gene) was used to insert mutations in the binding-region and dual luciferase reporter assay (Promega) was performed according to the manufacturer’s instructions. Ultimately, a luminometer (Glomax, Promega) was used to quantify luminescence, normalizing each value from the firefly luciferase construct to the corresponding Renilla signal.

Western blotting

Lysis buffer (#AS1004, Aspen, South Africa) containing 1% protease inhibitor (#AS1008, Aspen) was used to lyse cells or callus samples, after which protein was separated via SDS-PAGE and transferred to NC membranes (#IPVH00010, Millipore, USA) that were blocked with 5% nonfat milk and stained overnight at 4 °C overnight with antibodies specific for collagen I (1:500, Sigma, USA,#ab34710), ALP (1:1000, Sigma, USA,#ab95462), Osteocalcin (1:500, Sigma, USA, #ab93876), RunX2 (1:500, Sigma, USA, #ab23981), SIK2 (1:1000, Sigma, USA, #SAB1302059), SIK3 (1:1000, Sigma, USA, #SAB3500695), and GAPDH (1:10,000, Sigma, USA, #ab37168). Blots were then stained with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) (#AS1058, Aspen), and proteins were detected with a chemiluminescence detection system. Each experiment was repeated three times.

CCK-8 (Cell counting kit-8) assay

BMSCs were added to 96-well plates and cultured for 24, or 48 respectively. Then, CCK-8 reagent (#96992, Sigma-Aldrich, MO, USA) was added into cells in serum-free medium for 2 h, followed by measurements of absorbance at 450 nm.

Alizarin red staining

BMSCs were grown in 6-well plates in media containing 100 nM dexamethasone, 50 mM ascorbic acid, and 10 mM b-glycerophosphate to promote osteogenesis (#HUXMA-90021, Cyagen, USA). Briefly, cells were washed twice by using PBS, after wich 10% formalin was added to fix the cells for 15 min. Subsequently, 1 mL 0.5% alizarin red staining solution was used to stain the cells at room temperature for 15 min. After rinsing with distilled water for 5 min, red mineralized nodules were analyzed via charge-coupled device microscope. Absorbance was then measured at 570 nm. Experiments were repeated in triplicate.

ALP staining

A BCIP/NBT alkaline phosphatase color development kit (#C3206, Beyotime, China) was utilized based upon provided directions. Briefly, cells were washed twice by using PBS, after wich 10% formalin was added to fix the cells for 15 min. BCIP/NBT substrate was then used to treat cells for 24 h, and colorimetric changes were analyzed using a charge-coupled microscope, with a scanner used to image stained cells. Absorbance was then measured at 405 nm. Experiments were repeated in triplicate.

Mice fracture model and treatment

Male C57BL/6J mice (8-week-old) were obtained as above, with all studies being approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology. Intraperitoneal pentobarbital sodium (50 mg/kg; Sigma-Aldrich) was used to anesthetize animals, and a model of femoral fracture was then generated via longitudinal incision and blunt separation of the underlying muscles without periosteum removal, as in previous reports28. The femur was then cut with a diamond disk, yielding a mid-diaphysis transverse osteotomy. Next, a 23-gauge intramedullary needle was used to stabilize the fracture site. On day 14 post-fracture, 50% of study animals were euthanized to harvest and analyze calluses, with the remaining mice being analyzed on day 21.

Radiographic images

On days 7, 14, and 21 post injury, all animals were subjected to X rays with an In-Vivo FX PRO imaging system (BRUKER, Karlsruhe, Germany) with a 10 s exposure time.

Micro-computer tomography (Micro-CT) analysis

A BRUKER SkyScan 1276 scanner micro-CT system (BRUKER, Karlsruhe, Germany) was used to image fracture regions in mice (2400 views, 5 frames/view, 37 kV, and 121 mA), with the platform software being used to assess segmentation, three dimensional morphometric analyses, density, and distance parameters (BRUKER, Karlsruhe, Germany). After scanning, preserved calluses were stored at − 80 °C prior to further experimental use. Measure parameters, includeding bone volume (BV), total volume (TV), BV/TV, and bone mineral density (BMD), were analyzed by CT Analyser evaluation software (Version: 1.15.4.0) on day 14 and day 21 post-operation. Micro-CT analysis was done in a completely blinded manner with all mice assigned to coded sample numbers.

Therapeutic administration in fracture mice

Control mice received a local fracture site injection of 0.1 mL PBS on days 1, 3, and 7 post-fracture, while other mice were instead administered equivalent volumes of M1D-Exos, M2D-Exos, M2D-ExosantagomiR−5106. At the end of the experiment, bone and callus samples were analyzed as above.

Histological analysis

Following decalcification, 5–7 µm thick paraffin-embedded tissue samples were prepared and subjected to H&E and Alcian blue staining. Sections were then imaged and measured with an Olympus BX51 microscope and a DP73 CCD Olympus Imaging System (Olympus Corporation, Tokyo).

Microarray analysis

RNA sequencing from M1 and M2 macrophages’ supernatant was conducted using a BGISEQ 500 platform (BGI). Downstream library was constructed by using RNA samples with RNA integrity number values > 8.0. mRNAs were isolated and reverse transcription with PCR amplification was performed. Then, cDNA was obtained as the final sequencing library, and verified on a bioanalyzer (Agilent 2100). DNA nanoballs were produced by phi29 and the library was amplified, which were loaded into the patterned nanoarray followed by SE50 sequencing. Differentially expressed miRNAs (DEMs)with statistical significance between the two groups were identified by volcano plot filtering and fold change filtering was used to identify DEMs between the two samples. The Gene Cluster 3.0 software (Stanford University) was applied to make hierarchical cluster analysis. Database of annotation, visualization, and integrated discovery (DAVID 6.8; https://david.ncifcrf.gov/) was used to conduct functional analysis. The p value was set to 0.05 to denote the significance of GO enrichment of DEMs.

Statistical analysis

Data are mean ± SD, and GraphPad Prism 8.0 (GraphPad Software, CA, USA) was used for all statistical testing. Data were compared via Student’s t-tests and one-way ANOVAs with Tukey’s post hoc test as appropriate. p < 0.05 was the significance threshold.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Change history

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Acknowledgements

We thank Jing Liu, Abududilibaier Abudula, Ranyang Tao, Zexi He, Xingjie Zhan, and Chenming Zhang for assistance with data collection.

Funding

This study was supported by the National Science Foundation of China (No. 81772345), National Health Commission of the People’s Republic of China (No. ZX-01-018, ZX-01-C2016153), Ministry of Science and Technology of the People’s Republic of China (No.2018YFC2001502, 2018YFB1105705), Health Commission of Hubei Province (No. WJ2019Z009), Wuhan Science and Technology Bureau (No. 2017060201010192).

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  1. Yuan Xiong, Lang Chen, and Chenchen Yan contributed equally to this work

Authors and Affiliations

  1. Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China

    Yuan Xiong, Lang Chen, Chenchen Yan, Wu Zhou, Faqi Cao, Hang Xue, Yiqiang Hu, Dong Chen, Bobin Mi & Guohui Liu

  2. Department of Orthopedic Surgery, Tongji Hospital, Tongji University School of Medicine, Shanghai, 200065, China

    Tao Yu

  3. Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China

    Yun Sun

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Contributions

BM and GL conceived and designed the study, YX, LC, and CY carried out the molecular genetic studies, WZ, YS, TY and FC performed experiments; HX, YH and DC analyzed the data; BM and GL provided advice and technical assistance; and YX wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Bobin Mi or Guohui Liu.

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The original online version of this article was revised: Following publication of the original article [1], the authors reported that Figure 2 was not updated during the production process.

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Additional file 1: Figure 1.

(A) BMDM, (B) M1 macrophage, and (C) M2 macrophage were identified by flow cytometry. Figure 2. The biocompatibility of M1D- and M2D- Exos was tested by CCK-8 assay. Figure 3. In the control group, the appearance of Cy3 red fluorescence on BMSCs was examined after co-culture with M1/M2 macrophages treated with Cy3 dye (without miR-26a-5p mimic) for 12 hours. Figure 4. Relative intensity of western blotting analysis.

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Xiong, Y., Chen, L., Yan, C. et al. M2 Macrophagy-derived exosomal miRNA-5106 induces bone mesenchymal stem cells towards osteoblastic fate by targeting salt-inducible kinase 2 and 3. J Nanobiotechnol 18, 66 (2020). https://doi.org/10.1186/s12951-020-00622-5

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