Smoothened agonist sterosome immobilized hybrid scaffold for bone regeneration

Abstract

Biomaterial delivery of bioactive agents and manipulation of stem cell fate are an attractive approach to promote tissue regeneration. Here, smoothened agonist sterosome is developed using small-molecule activators [20S-hydroxycholesterol (OHC) and purmorphamine (PUR)] of the smoothened protein in the hedgehog pathway as carrier and cargo. Sterosome presents inherent osteoinductive property even without drug loading. Sterosome is covalently immobilized onto three-dimensional scaffolds via a bioinspired polydopamine intermediate to fabricate a hybrid scaffold for bone regeneration. Sterosome-immobilized hybrid scaffold not only provides a favorable substrate for cell adhesion and proliferation but also delivers bioactive agents in a sustained and spatially targeted manner. Furthermore, this scaffold significantly improves osteogenic differentiation of bone marrow stem cells through OHC/PUR-mediated synergistic activation of the hedgehog pathway and also enhances bone repair in a mouse calvarial defect model. This system serves as a versatile biomaterial platform for many applications, including therapeutic delivery and endogenous regenerative medicine.

Fig. 1. Schematic illustration of smoothened agonist sterosome and its hybrid scaffold for bone regeneration.

(A) Smoothened agonist sterosome consisted of SA, OHC (osteogenic cholesterol), and PUR (smoothened agonist). (B) Fabrication process for PLGA 3D hybrid scaffold with sterosome by layer-by-layer modification via bioinspired dopamine chemistry, and the stepwise bone repair strategy through hedgehog signaling pathway. Smo, smoothened; PTCH, patched.

Fig. 2. Bioactivity of smoothened agonist sterosomes in BMSCs.

(A) ALP staining and (B) colorimetric quantification of ALP activity at day 4. (C) Mineralization stained with Alizarin red S and (D) colorimetric quantification of mineralization activity at day 14. Scale bars, 200 μm. Data were presented as means ± SD. Statistical analysis was determined by one-way ANOVA with Tukey’s post hoc test; **P < 0.01, ***P < 0.001. CM, culture medium; OM, osteogenic medium.

Fig. 3. Characterization of surface-modified PLGA 3D scaffold with smoothened agonist sterosomes via bioinspired dopamine chemistry.

(A) Quantification of immobilized sterosome on surface of PLGA and PDA-coated scaffold via PDA for 1 hour. Nile red–loaded sterosome was used as a model sterosome. The concentration of nile red–loaded sterosome is 1.0 g liter⁻¹. Data were presented as means ± SD. ***P < 0.001, two-tailed t tests. (B) Confocal laser scanning microscopy images of sterosome-immobilized scaffold (scale bar, 100 μm). Sterosome is labeled by FITC. Nile red is used as model cargo. (C) XPS spectra of (i) PLGA scaffold, (ii) PDA-coated scaffold, and sterosome-immobilized scaffold (iii) without second PDA layer or (iv) with second PDA layer. (D) XPS C 1s spectra of (i) PLGA scaffold, (ii) PDA-coated scaffold, and sterosome-immobilized scaffold (iii) without second PDA layer or (iv) with second PDA layer.

Fig. 4. Cell adherence, proliferation, and bioactivity evaluation of scaffolds.

(A) In vitro cell proliferation after 1, 4, and 7 days. The value was normalized by PLGA scaffold at day 1. (B) Representative confocal fluorescence images of BMSCs stained with calcein AM (live cells, green fluorescence) and ethidium homodimer (dead cells, red fluorescence) for day 7. Scale bar, 200 μm. (C) ALP staining and (D) colorimetric quantification of BMSCs on scaffolds at day 4. (E) Alizarin red S staining and (F) quantified mineralized extracellular matrix of BMSCs on scaffolds at day 14. Data were presented as means ± SD. **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey’s post hoc test. ns, not significant in (D) and (F). (G) Representative confocal laser scanning microscopy images of BMSCs incubated on FITC-labeled, nile red–loaded sterosome-immobilized hybrid scaffold for 4 and 24 hours. Nucleus is stained with Hoechst 33342 (blue). Scale bar, 100 μm. Sterosomes are labeled by FITC.

Fig. 5. Gene expression related to osteogenesis and hedgehog signaling pathway in scaffolds.

(A) ALP, (B) Runx2, and (C) Col1a1 were evaluated to assess the osteogenic differentiation after 4 days of incubation. (D) OCN and (E) OPN were evaluated to assess the osteogenic differentiation after 14 days of incubation. (F) Schematic illustration of hedgehog signaling pathway in the presence or absence of sterosomes. (G) PTCH and (H) Gli1 were evaluated after 24 hours of incubation. The results are normalized by expression level of GAPDH and then presented as relative ratios to the PLGA scaffold. ALP, Runx2, Col1a1, OCN, and OPN are osteogenic markers. Gli1 and PTCH are hedgehog signaling pathway markers. Data were presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey’s post hoc test.

Fig. 6. In vivo bone regeneration after 6 weeks of implantation of smoothened agonist sterosome-immobilized hybrid scaffolds on mouse calvarial defect.

(A) Representative 3D reconstructed images of calvarial defect at 6 weeks after implantation taken a superficial view. Quantified parameters of the regenerated bone including (B) bone fractional area, (C) bone volume density (BV/TV), (D) bone mineral density, and (E) trabecular number taken at the 3-mm-diameter cylindrical defect. All data were presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey’s post hoc test. (F) H&E-stained (top) and Masson’s trichrome–stained (bottom) histologic sections of calvarial decalcified sections after 6 weeks of implantation. S, remained scaffold; CT, connective tissue; NB, new bone. Scale bars, 100 μm.