A new osteogenic protein isolated from Dioscorea opposita Thunb accelerates bone defect healing through the mTOR signaling axis

Abstract

Delayed bone defect repairs lead to severe health and socioeconomic impacts on patients. Hence, there are increasing demands for medical interventions to promote bone defect healing. Recombinant proteins such as BMP-2 have been recognized as one of the powerful osteogenic substances that promote mesenchymal stem cells (MSCs) to osteoblast differentiation and are widely applied clinically for bone defect repairs. However, recent reports show that BMP-2 treatment has been associated with clinical adverse side effects such as ectopic bone formation, osteolysis and stimulation of inflammation. Here, we have identified one new osteogenic protein, named 'HKUOT-S2' protein, from Dioscorea opposita Thunb. Using the bone defect model, we have shown that the HKUOT-S2 protein can accelerate bone defect repair by activating the mTOR signaling axis of MSCs-derived osteoblasts and increasing osteoblastic biomineralization. The HKUOT-S2 protein can also modulate the transcriptomic changes of macrophages, stem cells, and osteoblasts, thereby enhancing the crosstalk between the polarized macrophages and MSCs-osteoblast differentiation to facilitate osteogenesis. Furthermore, this protein had no toxic effects in vivo. We have also identified HKUOT-S2 peptide sequence TKSSLPGQTK as a functional osteogenic unit that can promote osteoblast differentiation in vitro. The HKUOT-S2 protein with robust osteogenic activity could be a potential alternative osteoanabolic agent for promoting osteogenesis and bone defect repairs. We believe that the HKUOT-S2 protein may potentially be applied clinically as a new class of osteogenic agent for bone defect healing.

Keywords: Bone defect repair; Bone mineral density (BMD); Dioscorea spp protein; Mesenchymal stem cells (MSCs); Osteoblast differentiation; mTOR signaling pathway.

Graphical abstract

Fig. 1

HKUOT-S2 significantly enhanced bone defect repairs in vivo. A) Representative μCT scans of the bone defect sites of the sham control and HKUOT-S2 treatment groups. B) 3D reconstructed images of the μCT scans of the bone defect sites of the sham control and HKUOT-S2 treatment groups. C–H) μCT analysis of BV/TV, BMD, TMD, Tb.th, Tb. N, and BS/TV ratio showing bone defect healing process. The values were shown as mean ± SEM, n = 5. X, 2X, and 4X = 1.09 mg/kg, 2.18 mg/kg, and 4.36 mg/kg HKUOT-S2 treatments respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 2

HKUOT-S2 enhanced new bone formation. A) Representative images of fluorochrome labeling (red for xylenol orange and green for Calcein green) for new bone formation at bone defect sites of the sham control and HKUOT-S2 treatment groups. B) Quantification of xylenol orange fluorescence intensity at bone defect sites of the sham control and HKUOT-S2 treatment groups. C) Quantification of calcein green fluorescence intensity at bone defect sites of the sham control and HKUOT-S2 treatment groups. D) Representative images of Giemsa staining of bone defect sites of the sham control and HKUOT-S2 treatment groups. E) Representative images of Masson-Goldner trichrome staining of bone defect sites of the sham control and HKUOT-S2 treatment groups. F) Representative images of H&E staining of bone defect sites of the sham control and HKUOT-S2 treatment groups. The values were shown as mean ± SEM. n = 4, X, 2X, and 4X = 1.09 mg/kg, 2.18 mg/kg, and 4.36 mg/kg HKUOT-S2 treatments respectively. **p < 0.01, ****p < 0.0001.

Fig. 3

HKUOT-S2 enhanced osteoblast activity in vivo. A) Representative images of TRACP staining showing the TRAP⁺ cells at bone defect sites of the sham control and HKUOT-S2 treatment groups. B) Representative images of ALP staining showing the ALP⁺ cells at bone defect sites of the sham control and HKUOT-S2 treatment groups. C) Quantification of TRAP⁺ cells at bone defect sites of the sham control and HKUOT-S2 treatment groups. D) Quantification of ALP⁺ cells at bone defect region of the sham control and HKUOT-S2 treatment groups. The values were shown as mean ± SEM. n = 4, X, 2X, and 4X = 1.09 mg/kg, 2.18 mg/kg, and 4.36 mg/kg HKUOT-S2 treatments respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 4

HKUOT-S2-induced osteoblast activity enhanced bone defect repairs. A-D) Relative expression of osteogenic markers, Alp, Bglap1, Bglap1, and Runx2 expressions in the bone defect sites of the sham control and HKUOT-S2 treatment groups. E-H) ELISA analysis of BALP and OCN proteins levels in both serum and bone lysates from the bone defect site of the sham control and HKUOT-S2 treatment groups. I–K) Representative immunofluorescent and measurement of ALP, OCN, and RUNX2 expression at the bone defect sites of the sham control and HKUOT-S2 treatment groups. The values were shown as mean ± SEM. X, 2X, and 4X = 1.09 mg/kg, 2.18 mg/kg, and 4.36 mg/kg HKUOT-S2 treatments respectively. *p < 0.05, **p < 0.01.

Fig. 5

HKUOT-S2 treatments induced transcriptomic changes to promote bone defect healing. A) Venn diagrams of HKUOT-S2-induced differentially expressed genes (left), differentially upregulated (middle), and downregulated genes (right). B) Pie chart showing the distribution of HKUOT-S2-induced differentially expressed genes. C) Heatmap of general differentially expressed genes induced by HKUOT-S2 protein treatments. D) Heatmap of HKUOT-S2-induced most common differentially expressed genes.

Fig. 6

HKUOT-S2 treatments modulated cellular components and mTOR signaling pathway to promote bone defect healing. A) Both X and 2X HKUOT-S2 enriched GO terms are associated with macrophages, stem cells, and osteoblasts functions. B) Both X and 2X HKUOT-S2 treatment enriched GO terms associated with bone formation. C) Both X and 2X HKUOT-S2 enriched the mTOR signaling pathway. The values were shown as mean ± SEM, n = 3. X, 2X, and 4X = 1.09 mg/kg, 2.18 mg/kg, and 4.36 mg/kg (4X) HKUOT-S2 treatments respectively. *p < 0.05 and **p < 0.01.

Fig. 7

HKUOT-S2 treatments activated the mTOR signaling to promote bone defect healing in vivo. A-C) Representative Western blot images and corresponding quantification of p-mTOR/total mTOR protein and p-4E-BP1/total 4E-BP1 protein in the bone defect sites of the sham control and HKUOT-S2 treatment groups. D) Relative Mtor mRNA expression in the bone defect sites of the sham control and HKUOT-S2 treatment groups. The values were shown as mean ± SEM, n = 3. *p < 0.05, **p < 0.01.

Fig. 8

HKUOT-S2 treatment significantly enhanced anti-inflammatory activity in vivo to promote bone defect repairs. A-C) Relative M1 macrophage gene expressions (iNos, Socs3, and Tnfα) in the bone defect sites of the sham control and HKUOT-S2 treatment groups. D-F) Relative M2 macrophage gene expressions (Arg-1, Cd206, and Mgl-1) in the bone defect sites of the sham control and HKUOT-S2 treatment groups. The values were shown as mean ± SEM, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 9

HKUOT-S protein modulated macrophage polarization. A, B) Relative expression of Socs3 and Tnfα in the control and HKUOT-S2 treated M1 macrophages derived from RAW267.4 cells. C, D) Relative expression of Arg-1 and Mgl-1 in the control and HKUOT-S2 treated M2 macrophages derived from RAW267.4 cells. E) Flow cytometry analysis showing expression of CD206 and MGL-1 double-positive control and HKUOT-S2 treated M1 and M2 macrophages derived from the primary bone marrow macrophage (M0 macrophage). F, G) Representative Western blot images and corresponding quantification of ARG-1/β-ACTIN in the control and HKUOT-S2 treated M1 and M2 macrophages derived from the M0 macrophage. HKUOT-S2 = 0.1 μg/ml, HKUOT-P1 = 5 μg/ml. The values were shown as mean ± SEM. *p < 0.05, **p < 0.01.

Fig. 10

HKUOT-S entered and localized in the cytoplasm of the hMSCs. A) Flow cytometry analysis showing positively stained hMSCs with Alexa Fluor 568 conjugated HKUOT-S2 protein. B) Confocal fluorescence microscope analysis showing the localization of Alexa Fluor 568 conjugated HKUOT-S2 protein in the cytoplasm and nuclei of the hMSCs.

Fig. 11

HKUOT-S2 treatment enhanced hMSCs to osteoblast differentiation. A-C) Relative expression of osteogenic markers, ALP, COL1A1, and RUNX2 in the hMSCs control, osteoblast control, rhBMP-2, and HKUOT-S2 treatment groups. D) Images of ALP activity and its corresponding quantification in the osteoblast control, rhBMP-2, and HKUOT-S2 treatment groups. E) Images of Alizarin Red staining of mineralized osteoblasts and its corresponding quantification in the osteoblast control, rhBMP-2, and HKUOT-S2 treatment groups.

Fig. 12

HKUOT-S2 treatment enhanced the crosstalk between macrophage and MSCs to promote osteoblast biomineralization. A) Image of cytokine array analysis and its corresponding quantification in HKUOT-S2 treated M1 and M2 macrophage CM. B) Representative images of Alizarin Red staining osteoblasts treated with macrophage CM with/without HKUOT-S2 C) Quantification of Alizarin Red staining osteoblasts treated with M1 macrophage CM with/without HKUOT-S2 treatment. D) Quantification of Alizarin Red staining of osteoblasts treated with M2 macrophage CM with/without HKUOT-S2 treatment. CM = conditioned media. The values were shown as mean ± SEM, n = 5. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 13

HKUOT-S2 modulated mTOR/4E-BP1/S6K1/AKT1 axis to promote osteogenesis. A-F) Relative expression of ALP, RUNX2, MTOR, 4E-BP1, AKT1, and S6K1 in the hMSCs control, osteoblast control, osteoblast + XL388 and osteoblast + XL388+HKUOT-S2 treatment groups. (G–M) Representative Western blot images of p-mTOR, mTOR, p-4E-BP1, 4E-BP1, P-p70S6K, p-AKT1, and AKT1 in the hMSCs control, osteoblast control, osteoblast + XL388, and osteoblast + XL388+HKUOT-S2 treatment groups. H-M) Quantification of Western blot images of p-mTOR/mTOR, mTOR/β-ACTIN, p-4E-BP1/4E-BP1 4E-BP1/β-ACTIN, P-p70S6K/β-ACTIN, and AKT1/β-ACTIN in the hMSCs control, osteoblast control, osteoblast + XL388 and osteoblast + XL388+HKUOT-S2 treatment groups. The values are shown as mean ± SEM, X-125 = 125 nM XL388. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 14

Schematic diagram illustrating how HKUOT-S2 protein modulates macrophage polarization, osteoblast differentiation, and activation of the mTOR signaling axis to enhance bone defect repairs.