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. 2020 Sep 3;21(17):6425.
doi: 10.3390/ijms21176425.

Umbilical Cord Mesenchymal Stem Cell-Derived Nanovesicles Potentiate the Bone-Formation Efficacy of Bone Morphogenetic Protein 2

Songhyun Lim  1 , Hao-Zhen Lyu  2 , Ju-Ro Lee  1 , Shi Huan Han  2 , Jae Hyup Lee  2   3 , Byung-Soo Kim  1   4
Affiliations

Umbilical Cord Mesenchymal Stem Cell-Derived Nanovesicles Potentiate the Bone-Formation Efficacy of Bone Morphogenetic Protein 2

Songhyun Lim et al. Int J Mol Sci. .

Abstract

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is one of the most potent osteogenic factors used to treat bone loss. However, at higher doses, rhBMP-2 does not necessarily increase bone formation but rather increases the incidence of adverse side effects. Here, we investigated whether umbilical cord mesenchymal stem cell (UCMSC)-derived nanovesicles (NVs) further increase the in vivo bone formation at high doses of rhBMP-2. In the presence of UCMSC-derived NVs, proliferation, migration, and tube formation of human umbilical vein endothelial cells were stimulated in vitro. Furthermore, migration and osteogenesis of human bone marrow-derived mesenchymal stem cells were stimulated. To examine the efficacy of UCMSC-derived NVs on in vivo bone formation, collagen sponges soaked with rhBMP-2 and UCMSC-derived NVs were used in athymic nude mice with calvarial defects. At a high rhBMP-2 dosage (500 ng/mL), UCMSC-derived NVs significantly promoted bone formation in calvarial defects; however, the UCMSC-derived NVs alone did not induce in vivo bone formation. Our results indicate that UCMSC-derived NVs can potentiate the bone formation efficacy of rhBMP-2 at a high dosage.

Keywords: angiogenesis; bone formation; osteogenesis; recombinant human bone morphogenetic protein 2; umbilical cord mesenchymal stem cell-derived nanovesicles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of umbilical cord mesenchymal stem cell (UCMSC)-derived nanovesicles (NVs). (a) Schematic illustration of UCMSC-derived NV fabrication from UCMSCs. (b) TEM image of UCMSC-derived NVs. Scale bar, 50 nm. (c) Size distribution of UCMSC-derived NVs. (d) Representative plots and (e) quantification of UCMSC surface marker expression for UCMSCs and UCMSC-derived NVs analyzed by flow cytometry. Blue: cells, red: NVs. Data are presented as means; n = 3 per group. The Mann–Whitney test was used for statistical analysis. ns for not significant.
Figure 2
Figure 2
UCMSC-derived NVs promote migration but not proliferation of human bone marrow-derived MSCs (hBMSCs) in vitro. (a) Migration and (b) proliferation of hBMSCs in the presence or absence of UCMSC-derived NVs at different concentrations of recombinant human bone morphogenetic protein 2 (rhBMP-2). (a) Scale bars, 100 µm. Veh indicates vehicle treatment. (a,b) Data are presented as means; n = 6 per group. Black: Veh, Red: NVs. The Mann–Whitney test was used for statistical analysis. * p < 0.05. ns for not significant.
Figure 3
Figure 3
UCMSC-derived NVs promote osteogenesis of hBMSCs, but not MC3T3-E1 cells in the presence or absence of rhBMP-2 in vitro. (a) mRNA levels 11 days and (b) 21 days after, and (c) Alizarin red S staining 18 days after inducing osteogenesis of hBMSCs in the presence or absence of UCMSC-derived NVs at different concentrations of rhBMP-2. (d) mRNA levels 7 days after and (e) alkaline phosphatase (ALP) staining 10 days after inducing osteogenesis of MC3T3-E1 cells in the presence or absence of UCMSC-derived NVs at different concentrations of rhBMP-2. (a,b,d) Black: Veh, Red: NVs. Data are presented as means; n = 4 per group. The Mann–Whitney test was used for statistical analysis. * p < 0.05. ns for not significant. (c,e) Scale bars, 50 µm. Veh indicates vehicle treatment.
Figure 4
Figure 4
UCMSC-derived NVs promote tube formation and proliferation of human umbilical vein endothelial cells (HUVECs) in the presence or absence of rhBMP-2 in vitro. (a) Migration, (b) tube formation, and (c) proliferation of HUVECs in the presence or absence of UCMSC-derived NVs at different concentrations of rhBMP-2. (a) Scale bars, 500 µm. n = 10 per group. (b) Scale bars, 100 µm. n = 12 per group. (c) n = 6 per group. (a,b) Veh indicates vehicle treatment. (a–c) Black: Veh, Red: NVs. Data are presented as means. The Mann–Whitney test was used for statistical analysis. * p < 0.05. ns for not significant.
Figure 5
Figure 5
UCMSC-derived NVs promote bone formation in calvaria defects of mice at 6 weeks. (a) Image of collagen sponge with 4 mm diameter used for implantation. (b) The calvaria scanned by micro-CT. Veh indicates vehicle treatment. (c) Bone volume (BV), bone volume to tissue volume ratio (BV/TV) and trabecular number (Tb.N) evaluated with micro-CT. Black: Veh, Red: NVs. Data are presented as means; n = 8 for w/col-Veh-0. n = 9 for w/col-Veh-0.7. n = 10 for the other groups. One-way ANOVA with Tukey’s post-test was used for statistical analysis. * p < 0.05. ns for not significant. (d) Cross-sectional views of the calvaria defects stained with hematoxylin and eosin (H&E). Arrows indicate defect margin. Scale bars, 1 mm. (b–d) w/o col: without collagen sponge, w/col: with collagen sponge.
Figure 6
Figure 6
UCMSC-derived NVs promote angiogenesis in calvaria defects of mice at 6 weeks. (a) Cross-sectional views of the calvaria defects stained with H&E. Veh indicates vehicle treatment. Arrows indicate vessel structures. Scale bars, 50 µm. (b) Quantification of vessel structures in the defects. Black: Veh, Red: NVs. Data are presented as means; n = 5 per group. One-way ANOVA with Tukey’s post-test was used for statistical analysis. * p < 0.05. (a,b) w/o col: without collagen sponge, w/col: with collagen sponge.
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