Vascularization converts the lineage fate of bone mesenchymal stem cells to endothelial cells in tissue-engineered bone grafts by modulating FGF2-RhoA/ROCK signaling

Abstract

The prevascularization of tissue-engineered bone grafts (TEBGs) has been shown to accelerate capillary vessel ingrowth in bone defect remodeling and to enhance new bone formation. However, the exact mechanisms behind this positive effect remain unknown. Here, we report that basic fibroblast growth factor (FGF2)-Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling functions as a molecular switch to regulate the lineage fate of bone mesenchymal stem cells (BMSCs) and that prevascularization promotes the cell fate switch, which contributes to increased bone regeneration with the use of prevascularized TEBGs compared with control TEBGs. Prevascularized TEBGs enhanced the in vivo endothelial differentiation of BMSCs by inhibiting RhoA/ROCK signaling. In vitro data more clearly showed that BMSCs differentiated into von Willebrand factor (vWF)-positive endothelial cells, and FGF2-induced inhibition of RhoA/ROCK signaling played a key role. Our novel findings uncovered a new mechanism that stimulates the increased vascularization of engineered bone and enhanced regeneration by promoting the endothelial differentiation of BMSCs implanted in TEBGs. These results offer a new molecular target to regulate TEBG-induced bone regeneration.

Fig. 1. The large bone defect model in rat femur. Third-passage BMSCs were analyzed by flow cytometry.

Most cells were CD31^- and CD11b/c^- (a, left panel), and a further representative image shows that the percentage of the CD31^-, CD11b/c^- and CD45⁻ cells that express CD90 was 93.6% (a, right panel). b Multi-lineage differentiation assay of rat BMSCs: osteogenic, adipogenic and chondrogenic differentiation. c β-TCP scaffolds (5 mm in height and 4 mm in diameter, 70% porosity, 400 µm pore diameter) were incubated with GFP⁺ BMSCs for a week. d The femur bone defect was made in the left femur and fixed by internal fixation. After surgery, all rats were examined using X-ray imaging to confirm that the model was successful. e, f Two types of femur defect models were used: WT rats matched with GFP⁺ BMSCs-TEBGs and GFP⁺ rats matched with WT BMSCs-TEBGs

Fig. 2. Prevascularized TEBGs promoted repair of bone defects.

a After surgery, all rats were exposed to X-ray to affirm the model was successful. The images were collected at 1, 4 and 8 weeks after implantation. b, d Micro-CT 3D reconstruction images of the new bone formation and β-TCP matrix at 1, 4 and 8 weeks after surgery. c BV/TV was used to evaluate new bone formation. e RSV/SV was used to evaluate scaffold degradation. n = 10 samples per group. Data are represented as the mean ± s.e.m. *P < 0.05, **P < 0.01 as determined by Student’s t-tests. f H&E staining of rat femur sections at 4 and 8 weeks after the surgery

Fig. 3. Prevascularization of TEBGs promoted proliferation and differentiation of seed cells.

Image was the center region of transection. Double-immunofluorescence images of BMSCs (green) and Osterix (red; a) or vWF (red; b) from TEBG sections, scale bar = 50 μm. White arrows represent double-positive staining cells. Hoechst33342 stained the nuclei blue. a, c Seeding cells continued to survive after implantation, and the number of seeding cells reached the peak at 4 weeks. c The number of GFP⁺ cells per area (10⁵ μm²), n = 10. d Percentage of Osterix⁺ cells overlapped with GFP⁺ cells, n = 10. e Percentage of vWF⁺ cells overlapped with GFP⁺ cells, n = 10. Data are represented as the mean ± s.e.m. *P < 0.05, **P < 0.01 as determined by Student’s t-tests

Fig. 4. Prevascularization of TEBGs promoted osteogenic and endothelial differentiation of host cells.

Double-immunofluorescence images of BMSCs (green) and Osterix (red; a) or vWF (red; b) from TEBG sections, scale bar = 50 μm. White arrows represent double-positive staining cells. Hoechst33342 stained the nuclei blue. a, c Host cells migrated into the TEBG pores. c The number of GFP⁺ cells per area (10⁵μm²), n = 10. d Percentage of Osterix⁺ cells overlapped with NOT-GFP⁺ cells, n = 10. e Percentage of vWF⁺ cells overlapped with NOT-GFP⁺ cells, n = 10. Data are represented as the mean ± s.e.m. *P < 0.05 as determined by Student’s t-tests

Fig. 5. BMSCs were morphologically altered after being co-cultured with RAOECs.

Immunofluorescence images of CD31 (red: a left panel) and vWF (red; a right panel); Hoechst33342 stained the nuclei blue, scale bar = 50 μm. b Schematic drawing of co-culture system. Representative images of BMSCs under an optical microscope (100 × ) (c) and an SEM (×500, ×1000) (d). BMSCs in the control group and the co-culture group were stained by ALP after 14 days of culture (e, left panel). BMSCs in the control group and the co-culture group were stained by Alizarin Red S after 21 days of culture (f, left panel). n = 3. Data are represented as the mean ± s.e.m. **P < 0.01 as determined by Student’s t-tests

Fig. 6. BMSCs differentiated into endothelial-like cells in the co-culture system.

Immunofluorescence staining of BMSCs using antibodies against Osterix (a, shown in red, scale bar = 200 μm) and vWF (b, shown in red, scale bar = 100 μm). Ratio of Osterix⁺ cells (%), n = 3 (a, right panel). Ratio of vWF + cells (%), n = 3 (b, right panel). c Relative mRNA expression of Osterix, RUNX2, CD31 and VEGF, n = 3. Data are represented as the mean ± s.e.m. **P < 0.01 as determined by Student’s t-tests. d WB analysis of CD31, RUNX2 and β-actin expression in BMSCs between the control and co-culture groups. e Representative images of the tube-like structures for the two groups. Images were taken at ×100 magnification. Tube formation assay analysis was carried out using the Wimasis platform, plotted as the number of tube length and branching points. Results are expressed relative to control. n = 3. Data are represented as the mean ± s.e.m. *P < 0.05; **P < 0.01 as determined by Student’s t-tests. f Representative images of the Crystal Violet staining for the two groups. Cells couting was using Image J. Results are expressed relative to control. n = 3. Data are represented as the mean ± s.e.m. *P < 0.05 as determined by Student’s t-tests

Fig. 7. RAOECs inhibited the RhoA/ROCK signaling pathway in BMSCs.

Double-immunofluorescence images of BMSCs (green) with RhoA (red; a) or p-RhoA (red; b); Hoechst33342 stained the nuclei blue, scale bar = 50 μm. Percentage of RhoA⁺ cells overlapped with GFP⁺ cells (a, right panel). Percentage of p-RhoA⁺ cells overlapped with GFP⁺ cells (b, right panel). (c) qRT-PCR analysis of RhoA, ROCK-1 and ROCK-2 in BMSCs in the control group and the co-culture group, n = 3. Data are represented as the mean ± s.e.m. **P < 0.01 as determined by Student’s t-tests. d WB analysis of RhoA expression in BMSCs. e Immunofluorescence images of BMSCs (green) and RhoA (red) in TEBG sections, scale bar = 50 μm

Fig. 8. RAOECs regulate BMSC differentiation into endothelial cells via the FGF2-RhoA/ROCK signaling pathway.

a Level of FGF2 in the control group and the co-culture group, n = 3. Data are represented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001as determined by Student’s t-tests. b Relative FGF2 mRNA expression in the control group and the co-culture group, n = 3. Data are represented as the mean ± s.e.m. **P < 0.01; ***P < 0.001 as determined by Student’s t-tests. c, e qRT-PCR analysis of RhoA, ROCK-1, ROCK-2 and CD31 in BMSCs from groups with different treatments, n = 3. Data are represented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 as determined by Student’s t-tests. d, f WB analysis of RhoA, ROCK-1, ROCK-2 and CD31 in BMSCs from groups with different treatments. g FGF2, secreted by endothelial cells, binds with FGFRs on BMSC membranes and promotes GDP-RhoA translation into inactive p-RhoA but not active GTP-RhoA