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. 2019 Oct 3:14:7987-8000.
doi: 10.2147/IJN.S216182. eCollection 2019.

Nano-Hydroxyapatite Coating Promotes Porous Calcium Phosphate Ceramic-Induced Osteogenesis Via BMP/Smad Signaling Pathway

Jing Wang  1 , Menglu Wang  1 , Fuying Chen  1 , Yihang Wei  1 , Xuening Chen  1 , Yong Zhou  2 , Xiao Yang  1 , Xiangdong Zhu  1 , Chongqi Tu  2 , Xingdong Zhang  1
Affiliations

Nano-Hydroxyapatite Coating Promotes Porous Calcium Phosphate Ceramic-Induced Osteogenesis Via BMP/Smad Signaling Pathway

Jing Wang et al. Int J Nanomedicine. .

Abstract

Background: The hierarchical porous structure and surface topography of calcium phosphate (CaP) bioceramics have a crucial impact on their osteoinductivity.

Purpose: To fabricate a biomimetic bone graft with an interconnected porous structure analogous to that of trabecular bone and a bioactive nanostructured surface with excellent osteoinductive potential.

Materials and methods: A biphasic CaP (BCP) substrate with highly porous structure was fabricated by an improved sponge replication method. Surface modification was performed by uniformly depositing a hydroxyapatite (HA) nanoparticle layer to create nHA-coated BCP scaffolds. The effects of these scaffolds on osteogenic differentiation of murine bone marrow-derived stem cells (BMSCs) were investigated in vitro, and their osteoinductivity was further assessed in vivo.

Results: The BCP and nHA-coated BCP scaffolds had similar trabecular bone-like architectures but different surface structures, with mean grain sizes of ~55 nm and ~1 μm, respectively. Compared with the BCP substrate, the nHA-coated BCP scaffolds favored cell adhesion and promoted osteogenic differentiation of BMSCs, as evidenced by upregulated expression of osteogenic genes, enhanced alkaline phosphatase activity, and increased osteocalcin production. This could be attributed to activation of the BMP/Smad signaling pathway, as significantly higher expression levels of BMPRI, Smad1, Smad4, and Smad5 were observed in the nHA-coated BCP group. The nHA-coated BCP scaffold not only maintained scaffold integrity but also induced ectopic bone formation when implanted into rabbit dorsal muscle in vivo for 90 days, whereas the BCP substrate underwent marked biodegradation that led to severe inflammation with no sign of osteogenesis.

Conclusion: The present study demonstrates the potential of this biomimetic bone graft with a trabecular framework and nanotopography for use in orthopedic applications.

Keywords: MSCs; calcium phosphate ceramics; nano-hydroxyapatite; osteoblastic differentiation; osteoinduction; porous scaffolds.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Scheme illustration for the fabrication process of BCP substrate and nHA-coated BCP scaffold (A). Stereo microscopy photos, SEM images of macroporous structure, cross-section, and surface morphology for BCP substrate (B) and nHA-coated BCP (C).
Figure 2
Figure 2
AFM topographic images for 3D view (A) and top view (B) of BCP substrate; 3D view (C) and top view (D) of nHA-coated BCP scaffold.
Figure 3
Figure 3
SEM micrographs of BMSC attachment onto the surface of BCP substrates (A–C) and nHA-coated BCP scaffolds (D–F) at day 3.
Figure 4
Figure 4
The expression of specific osteogenic genes (BMP-2, Runx2, OSX, ALP, BSP and OPN) in BMSCs cultured in BCP, nHA-coated BCP and control group for 1, 4 and 7 days. * refers to p<0.05, ** refers to p<0.01.
Figure 5
Figure 5
Intracellular ALP activity (A) and OCN production (B) for BMSCs cultured in BCP, nHA-coated BCP and control group. * refers to p<0.05, ** refers to p<0.01.
Figure 6
Figure 6
The expression of genes related to BMP/Smad signals – BMPRI (A), Smad1 (B), Smad4 (C), and Smad5 (D). Schematic illustration of the possible involvement of BMP/Smad signaling pathway in pro-osteogenic effects of nanostructured surface (E).  * refers to p<0.05, ** refers to p<0.01.
Figure 7
Figure 7
A rabbit intramuscular implantation model for the evaluation of material’s osteoinductivity (A); μ-CT images for structural changes of BCP (B) and nHA-coated BCP scaffolds (C) before and after implantation.
Figure 8
Figure 8
HE histological analysis of in vivo ectopic bone formation ability for BCP (A) and nHA-coated BCP (B) scaffolds after implanted in back muscles of rabbits for 90 days. Yellow arrows: inflammatory cells; black arrows: bone formation as evidenced by osteocytes settled in bone lacuna. Immunofluorescent staining of osteocalcin (OCN) for BCP (C) and nHA-coated BCP (D) groups. Green and blue colors represented osteocalcin and DAPI-stained nuclei, respectively.
Figure 9
Figure 9
Scheme illustration demonstrated that nHA-coated BCP scaffolds could stimulate osteogenic differentiation via BMP/Smad signaling pathway in vitro, and induce ectopic bone formation in vivo.
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