Role of Hedgehog-Gli1 signaling in the enhanced proliferation and differentiation of MG63 cells enabled by hierarchical micro-/nanotextured topography

Abstract

Hedgehog-Gli1 signaling is evolutionarily conserved and plays an essential role in osteoblast proliferation and differentiation as well as bone formation. To evaluate the role of the Hedgehog-Gli1 pathway in the response of osteoblasts to hierarchical biomaterial topographies, human MG63 osteoblasts were seeded onto smooth, microstructured, and micro-/nanotextured topography (MNT) titanium to assess osteoblast proliferation and differentiation in terms of proliferative activity, alkaline phosphatase (ALP) production, and osteogenesis-related gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of Sonic hedgehog (Shh), Smoothened (Smo), and Gli1, and the protein levels were assayed by Western blotting. MG63 cells treated with the Smo inhibitor cyclopamine were seeded onto the titanium specimens, and the cell proliferation and differentiation were studied in the presence or absence of cyclopamine. Our results showed that compared to the smooth and microstructured surfaces, the MNTs induced a higher gene expression and protein production of Shh, Smo, and Gli1 as well as the activation of Hedgehog signaling. The enhanced proliferative activity, ALP production, and expression of the osteogenesis-related genes (bone morphogenetic protein-2, ALP, and runt-related transcription factor 2) enabled by the MNTs were significantly downregulated by the presence of cyclopamine to a similar level as those on the smooth and acid-etched microstructured surfaces in the absence of cyclopamine. This evidence explicitly demonstrates pivotal roles of Hedgehog-Gli1 signaling pathway in mediating the enhanced effect of MNTs on MG63 proliferation and differentiation, which greatly advances our understanding of the mechanism involved in the biological responsiveness of biomaterial topographies. These findings may aid in the optimization of hierarchical biomaterial topographies targeting Hedgehog-Gli1 signaling.

Keywords: Hedgehog–Gli1; MG63; differentiation; micro-/nanotextured topography; proliferation.

Figure 1

Representative FE-SEM pictures of the smooth (S), acid-etched microstructured (R), and hierarchical micro-/nanotextured surfaces (R10 and R20). Notes: The nanotubes were found to have 25 and 70 nm outer diameters. Nanotubes of different sizes were formed on the microstructured surface and were distributed relatively uniformly. Abbreviation: FE-SEM, field emission scanning electron microscopy.

Figure 2

Micro-XAM-3D noncontact profiler surface roughness analysis of the titanium disks. Notes: (A) Representative Micro-XAM-3D noncontact profiler pictures of the smooth (S), acid-etched microstructured (R), and hierarchical micro-/nanotextured surfaces (R10 and R20) on large scanning areas of 1.8 mm ×1.4 mm and 450 μm ×350 μm, respectively. (B) Comparisons of the roughness parameter (Ra and Rq) of different titanium surfaces. ***P<0.001. Abbreviations: Ra, average roughness; Rq, root mean square roughness.

Figure 3

The initial adhesion of MG63 osteoblasts evaluated by counting the number of cell nuclei stained by DAPI under a fluorescence microscope after 30 and 60 minutes of incubation. Notes: (A) The fluorescence microscope images of stained cells. (B) Mean ± SEM cell numbers were calculated (400× magnification, counts performed over an area of 1,020×800 μm²; n=3, a total of 30 fields of view for each group of the smooth [S], acid-etched microstructured [R], and hierarchical micro-/nanotextured surfaces [R10 and R20]). *P<0.05; **P<0.01. Abbreviations: DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride; SEM, standard error of the mean.

Figure 4

Representative fluorescence microscope images of cells stained with rhodamine phalloidin for actin filaments (red) and DAPI for cell nucleus (blue) after 8 hours of incubation onto the smooth (S), acid-etched microstructured (R), and hierarchical micro-/nanotextured surfaces (R10 and R20). Notes: The cells with spindle shape spread on the smooth surface, while the cells on the other surfaces showed a rather larger and flattened morphology. The cells on the TiO₂ micro-/nanotextured titanium surfaces (R10 and R20) extended numerous pseudopodia of filament or sheet shape and spread properly on these surfaces. Abbreviation: DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride.

Figure 5

The proliferation and differentiation of MG63 osteoblasts on the smooth (S), acid-etched microstructured (R), and hierarchical micro-/nanotextured surfaces (R10 and R20). Notes: (A) The proliferation of MG63 cells seeded onto the titanium surfaces was measured by the MTT assay. (B) A quantitative ALP activity standardized relative to the intracellular total protein content after 7 days of incubation is presented. (C) ALP activity evaluated by the ALP staining after 7 days of incubation is also presented. The expression of BMP-2 (D), ALP (E), Runx2 (F), and OCN (G) in MG63 osteoblasts after 3 and 7 days of incubation was detected by quantitative real-time PCR. *P<0.05; **P<0.01. Abbreviations: ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; OCN, osteocalcin; PCR, polymerase chain reaction; Runx2, runt-related transcription factor 2.

Figure 5

The proliferation and differentiation of MG63 osteoblasts on the smooth (S), acid-etched microstructured (R), and hierarchical micro-/nanotextured surfaces (R10 and R20). Notes: (A) The proliferation of MG63 cells seeded onto the titanium surfaces was measured by the MTT assay. (B) A quantitative ALP activity standardized relative to the intracellular total protein content after 7 days of incubation is presented. (C) ALP activity evaluated by the ALP staining after 7 days of incubation is also presented. The expression of BMP-2 (D), ALP (E), Runx2 (F), and OCN (G) in MG63 osteoblasts after 3 and 7 days of incubation was detected by quantitative real-time PCR. *P<0.05; **P<0.01. Abbreviations: ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; OCN, osteocalcin; PCR, polymerase chain reaction; Runx2, runt-related transcription factor 2.

Figure 6

The activity of Hedgehog–Gli1 signaling pathway on the smooth (S), acid-etched microstructured (R), and hierarchical micro-/nanotextured surfaces (R10 and R20). Notes: Quantitative real-time PCR analysis of Shh (A), Smo (B), and Gli1 (C) gene expressions in MG63 osteoblasts after 3 and 7 days of incubation; Western blot analysis of Shh (D), Smo (E), and Gli1 (F) protein levels in MG63 osteoblasts after 7 days of incubation. *P<0.05; **P<0.01. Abbreviations: PCR, polymerase chain reaction; Shh, Sonic hedgehog; Smo, Smoothened.

Figure 7

The Smo inhibitor cyclopamine suppressed the activity of Hedgehog–Gli1 signaling. Notes: (A and B) The gene expression and protein production of Smo in MG63 osteoblasts after 7 days of incubation on the different titanium surfaces. (C and D) The gene expression and protein production of Gli1 in MG63 osteoblasts after 7 days of incubation on the different titanium surfaces. *P<0.05; **P<0.01. Abbreviation: Smo, Smoothened.

Figure 8

The effect of cyclopamine on the proliferation and differentiation of MG63 osteoblasts on the smooth (S), acid-etched microstructured (R), and hierarchical micro-/nanotextured surfaces (R10 and R20). Notes: (A) The proliferation of cyclopamine-treated and untreated cells seeded onto the titanium surfaces as measured by the MTT assay after 7 days of incubation. (B) A quantitative ALP activity standardized relative to the intracellular total protein content after 7 days of incubation is presented. Quantitative real-time PCR analysis of BMP-2 (C), ALP (D), Runx2 (E), and OCN (F) expressions in the cyclopamine-treated and untreated cells after 7 days of incubation on the different titanium surfaces. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; OCN, osteocalcin; PCR, polymerase chain reaction; Runx2, runt-related transcription factor 2.