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Cited by 211Extracellular vesicles in diseaseMesenchymal stem cell researchBone Tissue Engineering Materials

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

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Takeuchi, Ryoko, Katagiri, Wataru, Endo, Satoshi, Kobayashi, Tadaharu·2019·PLOS ONE
DOI 10.1371/journal.pone.0225472PMC PMC6872157
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Abstract

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.

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Figures

Fig 1 Characterization of MSC-Exo. (a) Morphology observed by TEM. Scale bar: 100 μm. (b) Western blot analysis of the exosomal surface markers CD9, CD63, and CD81. (c) The size distribution and particle concentration of MSC-Exo measured using nanoparticle tracking analysis.
Fig 1 Characterization of MSC-Exo. (a) Morphology observed by TEM. Scale bar: 100 μm. (b) Western blot analysis of the exosomal surface markers CD9, CD63, and CD81. (c) The size distribution and particle concentration of MSC-Exo measured using nanoparticle tracking analysis.CC BY · PubMed Central
Fig 2 The effect of MSC-Exo on hMSC migration. (a) The experimental design of the transwell chamber assay. (b) The number of hMSCs that migrated in the DMEM(-), MSC-Exo, Exo-antiVEGF, and MSC-CM groups. (n = 6 per group. *, p < 0.05; **, p < 0.01).
Fig 2 The effect of MSC-Exo on hMSC migration. (a) The experimental design of the transwell chamber assay. (b) The number of hMSCs that migrated in the DMEM(-), MSC-Exo, Exo-antiVEGF, and MSC-CM groups. (n = 6 per group. *, p < 0.05; **, p < 0.01).CC BY · PubMed Central
Fig 3 The osteogenesis and angiogenesis effects of MSC-Exo on hMSCs. (a) Views of Alizarin red S stained hMSCs incubated with MSC-Exo, Exo-antiVEGF, MSC-CM, and DMEM with 5% FBS for 14 days. (b-h) mRNA expression of osteogenesis and angiogenesis-related genes ALP (b), COL1 (c), OCN (d), OPN (e), VEGF (f), ANG1 (g), and ANG2 (h) treated with MSC-Exo, Exo-antiVEGF, MSC-CM, and DMEM(-). (n = 5 per group. *, p < 0.05; **, p < 0.01).
Fig 3 The osteogenesis and angiogenesis effects of MSC-Exo on hMSCs. (a) Views of Alizarin red S stained hMSCs incubated with MSC-Exo, Exo-antiVEGF, MSC-CM, and DMEM with 5% FBS for 14 days. (b-h) mRNA expression of osteogenesis and angiogenesis-related genes ALP (b), COL1 (c), OCN (d), OPN (e), VEGF (f), ANG1 (g), and ANG2 (h) treated with MSC-Exo, Exo-antiVEGF, MSC-CM, and DMEM(-). (n = 5 per group. *, p < 0.05; **, p < 0.01).CC BY · PubMed Central
Fig 4 The effects of bone formation of MSC-Exo measured by Micro-CT in vivo. Rats were scanned every week until week 4 after implantation. (a) 3D reconstruction images at superficial side of rat calvarial bone defects implanted with PBS, MSC-Exo, Exo-antiVEGF, MSC-CM, and no implants (defect only). The new bones started to form from 2 weeks after implantation in the PBS, MSC-Exo, and MSC-CM groups. Four weeks after implantation, the newly formed bone was covering half of the defect in the MSC-Exo group. (b-e) Quantitative analysis of the new bone area in each group over a period of 1 to 4 weeks after implantation. (f) The time course of the newly formed bone area in each group.
Fig 4 The effects of bone formation of MSC-Exo measured by Micro-CT in vivo. Rats were scanned every week until week 4 after implantation. (a) 3D reconstruction images at superficial side of rat calvarial bone defects implanted with PBS, MSC-Exo, Exo-antiVEGF, MSC-CM, and no implants (defect only). The new bones started to form from 2 weeks after implantation in the PBS, MSC-Exo, and MSC-CM groups. Four weeks after implantation, the newly formed bone was covering half of the defect in the MSC-Exo group. (b-e) Quantitative analysis of the new bone area in each group over a period of 1 to 4 weeks after implantation. (f) The time course of the newly formed bone area in each group.CC BY · PubMed Central
Fig 5 Histological analysis of the newly formed bone. The undecalcified specimens were stained with hematoxylin-eosin. (a) The coronal cross sections of calvarial bone defect 2 weeks after implantation. The immature new bones were observed in the MSC-Exo and MSC-CM groups. (b) The coronal cross sections of calvarial bone defect 4 weeks after implantation. The bone bridge was observed in the MSC-Exo and MSC-CM groups. (c) The newly formed bone area at the coronal cross section (mm2). The newly formed bone area was significantly larger in the MSC-Exo group compared to the defect, PBS, and Exo-antiVEGF groups. (n = 5 per group. *, p < 0.05; **, p < 0.01).
Fig 5 Histological analysis of the newly formed bone. The undecalcified specimens were stained with hematoxylin-eosin. (a) The coronal cross sections of calvarial bone defect 2 weeks after implantation. The immature new bones were observed in the MSC-Exo and MSC-CM groups. (b) The coronal cross sections of calvarial bone defect 4 weeks after implantation. The bone bridge was observed in the MSC-Exo and MSC-CM groups. (c) The newly formed bone area at the coronal cross section (mm2). The newly formed bone area was significantly larger in the MSC-Exo group compared to the defect, PBS, and Exo-antiVEGF groups. (n = 5 per group. *, p < 0.05; **, p < 0.01).CC BY · PubMed Central
Fig 6 Immunohistochemical analysis of osteogenesis, angiogenesis, rat stem cells, and vascular endothelial cells. (a) The immunohistochemical staining and hematoxylin-eosin (H-E) staining showed that osteoblast-like cells around the edge of the newly formed bone were stained OCN-positive brown in the MSC-Exo group, while there were no marked findings in the defect, PBS, and Exo-antiVEGF groups. (b) The immunohistochemical staining and H-E staining showed that the VEGF-positive brown staining was apparent in vascular endothelial cells at the center area of the whole defect in the MSC-Exo and MSC-CM groups. Left side: H-E staining, right side: immunohistochemical staining. Scale bar: 100 μm. (c) The CD44-positive green-stained cells were clearly observed in the MSC-Exo group. (d) The CD31-positive red-stained cells were observed, as well as the vascular structures in the MSC-Exo group. Scale bar: 50 μm.
Fig 6 Immunohistochemical analysis of osteogenesis, angiogenesis, rat stem cells, and vascular endothelial cells. (a) The immunohistochemical staining and hematoxylin-eosin (H-E) staining showed that osteoblast-like cells around the edge of the newly formed bone were stained OCN-positive brown in the MSC-Exo group, while there were no marked findings in the defect, PBS, and Exo-antiVEGF groups. (b) The immunohistochemical staining and H-E staining showed that the VEGF-positive brown staining was apparent in vascular endothelial cells at the center area of the whole defect in the MSC-Exo and MSC-CM groups. Left side: H-E staining, right side: immunohistochemical staining. Scale bar: 100 μm. (c) The CD44-positive green-stained cells were clearly observed in the MSC-Exo group. (d) The CD31-positive red-stained cells were observed, as well as the vascular structures in the MSC-Exo group. Scale bar: 50 μm.CC BY · PubMed Central

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Metadata

Authors
Takeuchi, R., Katagiri, W., Endo, S., & Kobayashi, T.
Affiliations
Niigata University (JP)
Corresponding author
Katagiri, Wataru
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2019-11-21
Volume
14
Issue
11
Pages
e0225472
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0225472
PMC
PMC6872157
OpenAlex
W2989744940
Cited by
210
References
54
Citations by year
2026: 15 · 2025: 26 · 2024: 33 · 2023: 32 · 2022: 37 · 2021: 51 · 2020: 16
FWCI
8.60
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • Institutional Repositories DataBase (IRDB) (submittedVersion)
Funders
Japan Society for the Promotion of Science (18K09785)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Physical Sciences › Engineering › Biomedical Engineering › Bone Tissue Engineering Materials
Keywords
exosomes, bone marrow-derived mesenchymal stem cells, conditioned medium, bone regeneration, angiogenesis, VEGF inhibitors, osteogenic differentiation, paracrine signaling, cell transplantation, secretome
MeSH terms
Animals, Bone Regeneration, Cell Movement, Cells, Cultured, Disease Models, Animal, Humans, Skull, Culture Media, Conditioned, Neovascularization, Physiologic, Cell Culture Techniques, Paracrine Communication, Angiogenesis Inhibitors, Mesenchymal Stem Cell Transplantation, Cell Proliferation, Rats, Exosomes, Mesenchymal Stem Cells

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