Exosomes from conditioned media of bone marrow-derived mesenchymal stem cells promote bone regeneration by enhancing angiogenesis

Growth factors in serum-free conditioned media from human bone marrow-derived mesenchymal stem cells (MSC-CM) are known to be effective in bone regeneration. However, the secretomes in MSC-CM that act as active ingredients for bone regeneration, as well as their mechanisms, remains unclear. Exosomes, components of MSC-CM, provide the recipient cells with genetic information and enhance the recipient cellular paracrine stimulation, which contributes to tissue regeneration. We hypothesized that MSC-CM-derived exosomes (MSC-Exo) promoted bone regeneration, and that angiogenesis was a key step. Here, we prepared an MSC-Exo group, MSC-CM group, and Exo-antiVEGF group (MSC-Exo with angiogenesis inhibitor), and examined the osteogenic and angiogenic potential in MSCs. Furthermore, we used a rat model of calvaria bone defect and implanted each sample to evaluate bone formation weekly, until week 4 after treatment. Results showed that MSC-Exo enhanced cellular migration and osteogenic and angiogenic gene expression in MSCs compared to that in other groups. In vivo, early bone formation by MSC-Exo was also confirmed. Two weeks after implantation, the newly formed bone area was 31.5 ± 6.5% in the MSC-Exo group while those in the control and Exo-antiVEGF groups were 15.4 ± 4.4% and 8.7 ± 1.1%, respectively. Four weeks after implantation, differences in the area between the MSC-Exo group and the Exo-antiVEGF or control groups were further broadened. Histologically, notable accumulation of osteoblast-like cells and vascular endothelial cells was observed in the MSC-Exo group; however, fewer cells were found in the Exo-antiVEGF and control groups. In conclusion, MSC-Exo promoted bone regeneration during early stages, as well as enhanced angiogenesis. Considering the tissue regeneration with transplanted cells and their secretomes, this study suggests that exosomes might play an important role, especially in angiogenesis.


Introduction
In this study, we investigated the effects of MSC-CM-derived exosomes (MSC-Exo) on bone regeneration, especially from the point of inducing angiogenesis.

Materials and methods
All animal experiments were carried out in accordance with the protocols that were reviewed and approved by the Institutional Animal Care and Use Committee of Niigata University (approval No. SA00456). All efforts were made to minimize pain and distress during experiments. All animals were maintained on a 12-hours light/ dark cycle with free access to food and water. In order to confirm that no adverse events occurred, animals were visually checked every day during the experimental period. Animals were euthanized by overdose of sevoflurane at the end of the study.

Preparation of MSC-CM
Human bone marrow-derived mesenchymal stem cells (hMSCs) were purchased from Lonza Inc. (Walkersville, MD, USA) and cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, Rockville, MD, USA) with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France). Cells were maintained at 37˚C in a humidified 5% CO 2 incubator, and media was refreshed every 3 days. The cells were trypsinized and subcultured, and hMSCs at the 3rd to 6th passages were used for experiments.
When hMSCs reached 80% confluence, the medium was replaced by serum-free DMEM (DMEM(-)). The cell-cultured conditioned medium was collected after an additional 48 hours of incubation and filtered through a 0.22 μm filter sterilizer. The collected medium was defined as hMSC cultured conditioned medium (MSC-CM) and was stored at 4 or -80˚C before use in the subsequent experiments.

Isolation and identification of MSC-Exo
For MSC-Exo isolation, exosomes-depleted FBS was obtained by subjecting FBS to ultracentrifugation at 100 000 g for 16 hours to remove exosomes. Then, hMSCs were cultured in DMEM with 10% exosome-depleted FBS. MSC-CM was ultra-centrifuged at 100 000 g for 70 minutes. The supernatants were transferred to PBS and further ultra-centrifuged for 70 minutes. The pelleted extracellular vesicles (MSC-Exo) were resuspended and stored at -80˚C. All procedures were performed at 4˚C.
Purified MSC-Exo morphologies were observed using a transmission electron microscope (TEM; JEM-1400 Plus, JEOL Ltd., Tokyo, Japan). A drop of MSC-Exo suspension was pipetted onto a carbon-coated grid which had been treated to be hydrophilic. After a few minutes of standing, the excess fluid was removed, and then the sample was negative-stained with uranyl acetate for a few minutes and analyzed by TEM.

Enzyme-linked immunosorbent assay (ELISA)
The concentration of exosomes in MSC-CM was measured using Human CD9/CD63 Exosome ELISA Kit (Cosmo Bio Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions. Also, the concentrations of VEGF in cultured conditioned medium of 80% confluent hMSCs with MSC-Exo (5 μg/mL) + DMEM(-) (Exo-CM) or with MSC-CM for 48 hours were measured using Human VEGF Quantikine ELISA Kit (R&D Systems, Inc., MN, USA) according to the manufacturer's instructions. The concentration of each sample was determined by measurement of optical density at 450 nm with a microplate spectrophotometer (Multiskan FC, Thermo Fisher Scientific, Inc., MA, USA).

Rat calvaria bone defect model
To evaluate the bone regeneration ability of MSC-Exo, critical-sized defects in the rat parietal bones were created. For this study, 10-week-old male Wistar rats were anesthetized by intraperitoneal injection of 20 mg/kg pentobarbital. After shaving the parietal region of rats, a mucosal periosteal incision was made on the line connecting the two ear bases and peeled forward from the incision to clearly show the sagittal suture of the parietal bone. Two defects of 5 mm in diameter were carefully made in the calvarial bone using a trephine drill (Micro Tech. Corp., Tokyo, Japan), while avoiding damage to the dura mater. The defects were rinsed with PBS to remove the bone debris. Atelocollagen sponges (Terudermis1, Olympus Terumo Biomaterials Corp., Tokyo, Japan) were used as a scaffold, and were soaked with MSC-Exo (30 μg), MSC-Exo (30 μg) + anti-VEGFA antibody (1 μg), MSC-CM, or PBS. Sponges were implanted into the defects in calvarial bones. Finally, the mucoperiosteal flaps were sutured with 4-0 nylon thread. Two or four weeks after implantation, the specimens were harvested.
The rats were randomly divided into four groups (n = 6 in each group) and each of the experimental solutions soaked with atelocollagen sponges were implanted. The experimental groups were as follows: MSC-Exo group, Exo-antiVEGF group, MSC-CM group, PBS group, or defect group (defect only).

Microcomputed tomography (Micro-CT) analysis
Samples from all groups were examined using a micro-CT system (CosmoScan Gx, Rigaku Co., Tokyo, Japan). The calvarial bones were observed by micro CT every week after implantation until harvesting specimens 2 or 4 weeks later. Rats were scanned under anesthesia by intraperitoneal injection of 4% chloral hydrate. Three-dimensional (3D) images were Table 1. Primer sequences used for qRT-PCR.

Gene
Sequence Accession no. reconstructed using Analyze 12.0 software (AnalyzeDirect Inc., KS, USA). The newly formed bone area was evaluated as a percentage of the total bone defect.

Histological analysis
Samples from all groups were harvested 2 or 4 weeks after implantation. The specimens were fixed in 10% neutral formalin, and then decalcified with 10% EDTA (pH 7.4). After 4 weeks of decalcification, samples were embedded in paraffin and sectioned at 4 μm thickness in the coronal plane using a microtome (REM-710, YAMATO KOHKI Industrial Co., Ltd., Saitama, Japan). Sections were dewaxed, rehydrated, stained with hematoxylin-eosin, and analyzed using a light microscope (FX630, OLYMPUS Co., Tokyo, Japan) and FLVFS-LS software (OLYMPUS Co.).

Immunohistochemical analysis
Immunohistochemical staining was performed for osteocalcin (OCN) (1:1000; ABOC-5021, Haematologic Technologies Inc., VT, USA) to evaluate osteogenesis, while staining for VEGF (1:1000; ab39250, Abcam) was performed to detect angiogenesis. The sections were rehydrated, subjected to antigen retrieval using citrate buffer (pH 6.0) for 10 minutes at 121˚C, and blocked for endogenous peroxidase with 0.3% H 2 O 2 in methanol and incubated for 30 minutes. After washing with PBS, the sections were blocked for non-specific binding using 5% skim milk solution for 1 hour 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 hour and developed with 3,3 0 -Diaminobenzidine (DAB) solution. Hematoxylin counterstaining was performed following the DAB reaction. Immunofluorescence staining for CD44 (1:2000; ab189524, Abcam) was performed to measure rat stem cells, and CD31 (1:50; ab28364, Abcam) was performed to measure new blood vessels in each group. Briefly, the sections were rehydrated, subjected to antigen retrieval using citrate buffer (pH 6.0), and blocked with casein solution. The sections were incubated with the primary antibody overnight at 4˚C, and subsequently incubated with the secondary antibody. Alexa Fluor 488 (1:1000; ab150077, Abcam) and Alexa Fluor 647 (1:1000; ab150079, Abcam) were used as secondary antibodies. The sections were incubated with DAPI solution (Abcam) to stain the nucleus and observed with a fluorescence microscope (Axioplan 2, Carl Zeiss AG, Oberkochen, Germany).

Statistical analysis
All data were analyzed as mean ± standard deviation (SD). Comparisons between experimental groups and control groups were analyzed by means of Tukey's honestly significant difference test. The differences were considered statistically significant when p < 0.05.

Isolation and characterization of MSC-Exo
The exosomes were isolated from the cultured conditioned media of hMSCs by ultracentrifugation. In the purified materials, typical exosome structures were observed by TEM, whereby a large majority of particles exhibited a round-shaped morphology (Fig 1A). Expression of the characteristic surface markers on isolated particles was confirmed, and CD9, CD63, and CD81 were detected by western blotting; these biomarkers are conserved in exosomes and widely used for testing exosomes (Fig 1B). Moreover, the size distribution and particle concentration of MSC-Exo were determined by nanoparticle tracking analysis. The exosomes had a narrow size distribution, which peaked at 80-100 mm, indicating that these particles were exosomes (Fig 1C).

The presence of MSC-Exo in MSC-CM, and VEGF concentrations in Exo-CM and MSC-CM
The concentration of exosome in MSC-CM was determined. MSC-CM contained CD9 and CD63 at a total concentration of 42.6 ± 1.5 pg/mL. The concentration of VEGF in Exo-CM was 1140.3 ± 90.4 pg/mL, whereas the concentration of VEGF in MSC-CM was 1219.9 ± 126.6 pg/mL, supporting that Exo-CM contains equivalent amounts of VEGF when compared to MSC-CM.

MSC-Exo increased hMSC migration
The effect of MSC-Exo on hMSC migration was analyzed by transwell chamber assay (Fig 2A). After 48 hours of cell culture with each medium, the cells that migrated were counted. The

MSC-Exo induced mineral deposition in hMSCs
To determine the effect of MSC-Exo on hMSC calcification, mineral deposition in hMSCs were observed following alizarin red S staining. The mineral deposition in the MSC-Exo group was markedly enhanced compared with that in the Exo-antiVEGF group (Fig 3A). These results indicate that MSC-Exo enhanced the differentiation potential of hMSCs into osteoblasts. However, in the Exo-antiVEGF group, the mineral deposition was insufficient, suggesting that osteoblast differentiation ability might be poor.

MSC-Exo enhanced osteogenesis and angiogenesis-related gene expression in hMSCs
The transcript expression levels of COL I, ALP, OCN, and OPN were significantly upregulated in hMSCs cultured with MSC-Exo compared with those cultured with DMEM(-) and Exo-antiVEGF (p < 0.01) (Fig 3B-3E). The expression of VEGF, ANG1, and ANG2 were also significantly upregulated in hMSCs cultured with MSC-Exo compared to that with DMEM(-) and Exo-antiVEGF (p < 0.01) (Fig 3F-3H). These results indicate that MSC-Exo enhanced the osteogenic and angiogenic potentials of hMSCs in vitro.

MSC-Exo promoted bone formation in vivo
The newly formed bone in the rat calvarial bone defect was evaluated with micro-CT scanning every week after implantation. Three-dimensional micro-CT reconstruction images revealed the morphology of the newly formed bone (Fig 4A). Quantitative analysis of the newly formed bone area was performed as an evaluation of percentage of the total graft area (Fig 4B-3F). One week after implantation, the newly formed bone areas in the defect (1.7 ± 0.4%), PBS (5.9 ± 3.3%), and Exo-antiVEGF (4.3 ± 0.6%) groups were less than 10% of the defect area; in contrast, the areas in the MSC-Exo (13.5 ± 1.3%) and MSC-CM (16.3 ± 2.8%) groups were significantly higher and over 10%.
The new trabecular bone started to form from 2 weeks after implantation in the MSC-Exo and MSC-CM groups. Two and three weeks after implantation, the newly formed bone areas in the MSC-Exo (2 w: 31.5 ± 6.5%, 3 w: 38.8 ± 6.2%) and MSC-CM (2 w: 27.2 ± 4.9%, 3 w: 44.3 ± 6.9%) groups were also higher than that in the defect (2 w: 3.6 ± 0.6%, 3 w: 5.5 ± 0.9%), PBS (2 w: 15.4 ± 4.5%, 3 w: 23.6 ± 6.5%) and, Exo-antiVEGF groups (2 w: 8.7 ± 1.1%, 3 w: 10.9 ± 1.0%). The newly formed bones at 1-3 weeks after implantation in the Exo-antiVEGF group were significantly decreased in terms of size, compared with the MSC-Exo group (p < 0.01). This indicated that the anti-VEGF antibody affected bone formation at an early stage. Four weeks after implantation, the newly formed bone covered almost half of the defect in the MSC-Exo group (48.9 ± 6.2%), and a wide range of the defect area was occupied with Exosomes from mesenchymal stem cell conditioned media promote bone regeneration the newly formed bone in the MSC-CM group (56.6 ± 6.0%). On the other hand, newly formed bones were not sufficiently found in the defect (7.0 ± 0.9%), PBS (27.3 ± 7.7%), and Exo-anti-VEGF (13.4 ± 1.5%) groups. The newly formed bone area in the MSC-Exo group was significantly higher than that in the above mentioned three groups (p < 0.01). Moreover, the Exo-antiVEGF group significantly decreased bone formation when compared to the PBS group.
Furthermore, histological findings also showed that the undecalcified specimens in the MSC-Exo group had a larger area of new bone formation compared with that in the defect, PBS, and Exo-antiVEGF groups (Fig 5B). Two weeks after implantation, accumulation of osteoblast-like cells and infiltration of neutrophils were observed in the bone defect margin, and immature trabecular bones were formed along with the osteoid in the MSC-Exo group. Four weeks after implantation, the bone bridge was observed clearly in the MSC-Exo group. Several osteocytes could be observed in the bone bridge, which indicated that the newly formed bone was maturing. In addition, cells were accumulated in the newly formed bone edge, and vasculature structures were observed around it. These results showed the same trend as the results of Micro-CT analysis, and further support the findings of bone formation. Moreover, the newly formed bone areas at the coronal cross section were measured in square millimeters. The newly formed bone areas were 0.16 ± 0.03 mm 2 , 0.51 ± 0.14 mm 2 , 1.96 ± 0.1 mm 2 , 1.39 ± 0.34 mm 2 , and 0.26 ± 0.08 mm 2 in the defect, PBS, MSC-Exo, MSC-CM, and Exo-anti-VEGF groups, respectively (Fig 5C). The newly formed bone area in the MSC-Exo group was significantly larger compared to that in the defect, PBS, and Exo-antiVEGF groups.

Immunohistochemical staining of the newly formed bone
The osteogenic marker OCN and the angiogenic marker VEGF were detected using immunohistochemical staining, with results showing that there was no obvious positive staining for OCN in the defect, PBS, and Exo-antiVEGF groups, but positive brown staining for OCN was apparent in the MSC-Exo group (Fig 6A). Moreover, VEGF-positive brown staining was clear in the vascular endothelial cells at the center area of whole defects in the MSC-Exo and MSC-CM groups, while there were no marked findings in the defect, PBS, and Exo-antiVEGF groups (Fig 6B). The number of OCN and VEGF positive stained cells were counted and averaged from 3 random 100 μm 2 fields around the edge of the newly formed bone (S2 Fig). Immunohistochemical analysis of CD44 was performed to confirm the mobilization of endogenous MSC to rat calvarial defects by MSC-Exo. CD44 was used to detect rat MSCs in the newly formed bone area of specimens 2 weeks after implantation. The CD44-positive cells were observed around the edge of the newly formed bone in the MSC-Exo and MSC-CM groups (Fig 6C). Similarly, CD31 was used to detect vascular endothelial cells. The CD31-positive cells were also clearly observed around the newly formed bone area in the MSC-Exo and MSC-CM groups, indicating that MSC-Exo and MSC-CM enhanced endogenous stem cell migration and angiogenesis in the rat calvarial bone defect (Fig 6D).

Discussion
Remarkable progress has been made in the development of regenerative medicine, in terms of creating substitutes for conventional therapies. Although cell-based regenerative medicine is widely studied, its success depends on the viability and activity of stem cells and the surrounding environment of the recipient sites [34]. Stem cells are present in various organs and tissues, and they play an important role in maintaining homeostasis in the healthy body [35]. Thus, the stem cell niche provides a source of quiescent stem cells in response to body conditions such as injury or inflammation [36]. Specifically, some events occur as a stem cell niche response, including secretion of growth factors, migration of stem cells to an inflammation site, differentiation into appropriate cell types, and tissue repair [37]. In cell transplantation, even if only healthy cells are transplanted, the cell viability is low and the regenerative therapeutic effect is not always favorable in the case of a poor surrounding environment due to tissue damage [38].
Our previous studies reported that MSC-CM contain numerous growth factors and investigated the biological effects of MSC-CM that promoted bone regeneration [18]. We showed that MSC-CM enhanced cell migration, expression of angiogenic and osteogenic genes including VEGF, ANG1, ANG2, COLI, OCN, and Runt-related transcription factor 2 (Runx2), and capillary sprout and tube formation of human umbilical cord vein endothelial cells (HUVECs) [28]. In vivo, Ogata et al. showed that MSC-CM induced early bone regeneration by accelerating migration of stem cells by observing the migration of rat MSCs toward the local area where MSC-CM was implanted using an in vivo imaging method [39]. Moreover, it was interesting to note that transplantation of MSC-CM promoted bone regeneration compared to transplantation of MSCs themselves [18]. Thus, we revealed that bone regeneration was mainly promoted by cytokines secreted from MSCs, even without cell transplantation. As mentioned Exosomes from mesenchymal stem cell conditioned media promote bone regeneration above, MSC-CM contains abundant growth factors that contribute to bone regeneration, but it is also necessary to search for other ingredients in order to clarify the paracrine mechanisms of action of MSC-CM.
Angiogenesis contributes to the progression of osteogenesis, where blood supply induces the migration of osteoblasts and mineralization of bone tissue. Since vascular endothelial cells stimulate osteoblast maturation and activity, angiogenesis is closely associated with successful bone regeneration [40]. Paino et al. reported that human dental pulp stem cells had the angiogenic potential and fabricated the vascularized woven bone tissue without scaffolds [41]. VEGF, which is a specific growth factor that acts on vascular endothelial cells, promotes revascularization, regulation of vascular endothelial cell migration, proliferation, and capillary production, and improves the cellular activity of osteoblasts, thereby promoting bone regeneration [42,43]. Local application of VEGF enhances angiogenesis at defect sites, and promotes osteogenesis [44,45]. For example, Kumar et al. showed that VEGF increased bone mineral density and bone mineral content by increasing cell recruitment using a mouse tibia model [44]. Also, Katagiri et al. revealed that the addition of anti-VEGF to MSC-CM reduced blood vessel formation at the bone defect site and resulted in insufficient bone regeneration using a rat calvaria model [21]. Many studies have reported the effect of VEGF on bone regeneration. Local VEGF delivery may couple angiogenesis with bone regeneration and remodeling. In addition, VEGF may act as a central mediator for other factors [45]. Here, the aim of this study was to investigate the effects of MSC-Exo on bone regeneration, particularly with respect to angiogenesis. In our results, MSC-Exo enhanced the expression of not only osteogenesis-related genes but also angiogenesis-related genes such as VEGF, ANG 1, and ANG2, whereas expressions of these genes were decreased in the Exo-antiVEGF group in vitro. This indicated that MSC-Exo had an angiogenic potential. In addition, we also confirmed that MSC-Exo had the ability to enhance cell migration. This was also thought to be a crucial step in tissue regeneration by MSC-Exo. Indeed, obvious bone formation was observed in vivo in the MSC-Exo group at an early stage (31.5 ± 6.5% at 2 weeks), and the newly formed bone in the MSC-Exo group (48.9 ± 6.2%) was similar to the results in the MSC-CM group (56.6 ± 6.0%) 4 weeks after implantation. Interestingly, histological analysis revealed that the newly formed bone area of the coronal cross section was larger in the MSC-Exo group (1.96 ± 0.1 mm 2 ) than in the MSC-CM group (1.39 ± 0.34 mm 2 ). Stem cells were found to be accumulated around the bone defects and newly formed bone in the MSC-Exo and MSC-CM groups. Moreover, VEGF was highly expressed in vascular endothelial cells in the MSC-Exo group, while there were no such findings in the defect, PBS, and Exo-antiVEGF groups. Vascular endothelial cells and blood vessels were also observed well around the newly formed bone in the MSC-Exo group following immunofluorescence staining for CD31. These results indicated that MSC-Exo give the recipient cells some information related to angiogenesis and/or bone regeneration, which leads the recipient cells to activate the secretion of VEGF and osteogenesis-related paracrine factors. Therefore, MSC-Exo may partially contribute to controlling the cell niche in the local environment and activating the paracrine factors including VEGF in bone regeneration.
Exosomes contain miRNA and mediate cell-cell communication by transporting genetic information. Also, miRNAs are involved in gene regulatory networks in various signaling pathways and have been studied as target gene therapy agents [46]. Qin et al. showed that extracellular vesicles secreted from MSCs promoted bone regeneration in vivo, and three osteogenesis-related miRNAs (miR-196a, miR-27a, and miR-206) in extracellular vesicles were highly upregulated, and also that miR-196a was considered one of the most important regulators of osteogenesis [47]. Furuta et al. showed that the differentially expressed miRNA such as miR-21, miR-4532, miR-125b-5p, and miR-338-3p in exosomes secreted from MSCs might contribute to the enhancement of osteogenesis and angiogenesis [48]. Chen et al. revealed that exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells had osteogenic potential and promoted bone regeneration [49]. Therefore, early bone regeneration induced by exosomes was considered to depend on the regulation of multiple miRNAs and their relevant pathways, and miRNAs affect tissue development and homeostasis by regulating gene expression. In our study, we found that MSC-Exo contain miRNAs that may enhance VEGF secretion from the recipient cells, which contributes to bone regeneration.
Several factors contained in MSC-CM that contribute to bone regeneration have been investigated so far, but MSC-CM also contains other miscellaneous components that have not been fully elucidated. To partially address this, we isolated MSC-Exo, which was a single component type in MSC-CM, and found that it promoted bone regeneration by enhancing angiogenesis. Recent studies have reported that exosomes have various potential applications in diagnostics and treatment [50,51,52]. The application of exosomes could help to avoid the potential risks of toxicity and immunogenicity caused by biomaterial processing [53]. In addition, exosomes have a lower risk of serious complications such as tumorigenesis or emboli formation when compared to cell transplantation [54]. Hence, exosomes are suggested as a potential regenerative agent that can replace the conventional methods of bone regeneration including stem cell transplantation. However, further studies are needed to verify the active molecules of exosomes in angiogenesis, cell migration, and bone regeneration under various cell environments. It is important to identify the optimal conditions for exosomes to exert the most effective action on bone regeneration, potentially contributing to the basis of drug discovery, offering novel insights into cell-based regenerative medicine that can be applied in various medical fields.

Conclusion
Our results suggest that MSC-Exo have a remarkable osteogenic potential, as we reported previously in MSC-CM. Exosomes contained in MSC-CM may play important roles in bone regeneration by enhancing angiogenesis. Our findings also indicate that MSC-Exo can be used as bioactive agents for bone regeneration.