Regulation of osteoblast differentiation by osteocytes cultured on sclerostin antibody conjugated TiO2 nanotube array

https://doi.org/10.1016/j.colsurfb.2018.12.023Get rights and content

Highlights

  • Sclerostin antibody can be conjugated onto TiO2 nanotube in a simple process.

  • The TiO2 nanotube and sclerostin antibody could promote osteoblast differentiation.

  • Sclerostin antibody could improve the biomedical performance of the substrate.

  • Reducing sclerostin was beneficial for bone healing under osteoporotic conditions.

Abstract

Sclerostin is a negative regulator of the Wnt signaling pathway for osteoblast differentiation. In this study, osteoblasts were co-cultured with osteocytes (MLO-Y4 cells) on the surface of sclerostin antibody-conjugated TiO2 nanotube arrays (TNTs-scl). Field emission scanning electron microscopy (SEM), contact angle measurement and confocal laser scanning microscope (CLSM) were employed to characterize the conjugation of sclerostin antibody onto the surface of TiO2 nanotube arrays. The cellular viability and morphology results displayed TNTs-scl (TNT30-scl and TNT70-scl) were beneficial to the growth of MLO-Y4 cells. There was no apparent change in sclerostin gene expression between MLO-Y4 cells grown on TNTs and TNTs-scl. However, TNTs-scl significantly reduced the amount of sclerostin in the medium. In comparison with the control groups, osteoblasts displayed higher differentiation capability when co-cultured with MLO-Y4 cells on the surface TNTs-scl, which was indicated by the ALP activity, mineralization capability as well as expression levels of key proteins in Wnt signaling. This study provides a simple strategy to engineer titanium surface for bone fracture recovery, especially in osteoporotic conditions.

Introduction

Titanium is widely used in the clinical field of orthopedics due to its good physical and chemical properties [1]. However, its bioinertness and poor ability of inducing bone formation may limit their use in biomedical applications [2]. In order to enhance the biological activity of titanium, some studies have focused on mimicking the nanostructure property of bone tissue on the titanium surface [3], which can provide a stable and controllable microenvironment for cell growth [4]. Numerous studies have shown that TiO2 nanotubes was beneficial for cell proliferation and differentiation [5]. This nanoscale topology could promote interaction between cell and substrates via various biological mechanisms (integrin, regulated cell adhesion, cytoskeleton spreading and intracellular signaling) to control cell proliferation and differentiation [5,6].

In addition to mimicking the nanostructure of natural bone tissue, scientists have also developed other strategies to improve osteogenesis especially in osteoporotic condition through surface modification. Most of these design rely on the conjugation of anti-osteoporotic drugs (such as bisphosphonates, calcitonin, fluoride, estrogens and some hormonal drugs) onto the surface of titanium substrates to inhibit osteoclast activity and bone resorption [5,7]. However, the bioavailability of drugs delivered via this approach is usually very low. Moreover, it also has high cytotoxicity to the peripheral cells [8]. Alternatively, biological molecules (such as extracellular matrix proteins, growth factor) were introduced onto titanium surface to stimulate osteoblast differentiation and osteogenesis [8].

Previous research mainly focuses on regulating the biological behavior of osteoclasts and osteoblasts around titanium implants to repair the osteoporotic fracture. However, osteoclasts and osteoblasts only occupy 5% of the total cells within bone tissue, where the rest of cells are all osteocytes [9,10]. Osteocytes are embedded in the mineralized bone matrix and connected with other cells and blood vessels through the lacune-tubule network. Therefore, those negative regulators such as sclerostin and dikkopf1 secreted by osteocytes could transport through the lacune-tubule network to act on osteoblasts [11]. It was reported that sclerostin played an important role during osteogenic differentiation through canonical Wnt signaling [12,13]. It can bind with Wnt co-receptor LRP5/6 and LRP4 and inhibit the binding between Wnt and its receptor (a specific frizzled transmembrane receptors), resulting in the phosphorylation of β-catenin by GSK-3β and degradation of β-catenin by ubiquitin-mediated proteolysis [14,15].The phosphorylation and degradation of β-catenin decrease the expression of osteogenic related genes. Therefore, high level of sclerostin expression may suppress bone formation and accelerate bone resorption, resulting the imbalance between bone formation and bone resorption. Previous studies reported that serumic levels of sclerostin or dikkopf1 would increase with age, especially in the patient with osteoporosis. It was confirmed that level of sclerotin in postmenopausal women are two times higher than that in premenopausal women [16,17]. Therefore, the inhibition of sclerostin and other bone negative regulatory factors in the molecular level could be an efficient way to treat osteoporosis.

In this study, we firstly prepared TiO2 nanotube arrays on titanium substrate to mimic the nano-scale structure of natural bone and then conjugated sclerostin antibody onto their surface via dopamine (Fig. 1). Ti is biolofically inert and cannot directly bind to sclerostin antibody. Dopamine hydrochloride is a biomolecule that contains catechol and quinone functional groups. TNTs soaked into dopamine hydrochloride solution overnight can form a polydopamine films which exploited to covalently immobilize biomolecules onto surfaces through a reaction between nucleophiles and the polydopamine surface [18]. We hypothesized that TNTs-scl could decrease the sclerostin level in the medium secreted by the adhered osteocytes. Reducing ambient sclerostin could be beneficial for osteoblast differentiation via the Wnt signaling way. To confirm the hypothesis, the osteocytes were seeded onto the surface of sclerostin antibody-conjugated TiO2 nanotube arrays and co-cultured with osteoblasts (Fig. 1). The behavior of both osteocytes and osteoblasts was investigated at cellular and molecular level.

Section snippets

Materials

Titanium foils (0.25 mm thickness, 99.5%) were purchased from Alfa Aesar Co. (Tianjin, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP) and bicinchoninic acid (BCA) assay kit were provided by Sigma-Aldrich Co. (MO, USA). Dopamine hydrochloride was obtained from Aladdin Industrial Co. (Shanghai, China). Sclerostin antibody was purchased from Santa Cruz Biotechnology Co. (Shanghai, China).

TiO2 nanotube fabrication

Titanium foils (10 mm × 10 mm) was washed by acetone,

Sample fabrication and characterization

SEM and AFM were used to characterize the surface morphology and roughness of different TiO2 nanotubes. As shown in Fig. 2A and Fig. 2B, native titanium displayed high surface roughness due to the scratching during production. After the anodization treatment, highly ordered TiO2 nanotube (TNT) arrays of 30 nm and 70 nm were observed onto the surface of titanium substrates (Fig. 2). There was no obvious difference for the surface morphology of different TNTs before and after sclerostin antibody

Conclusion

In this study, osteoblasts were co-cultured with MLO-Y4 cells on the surface of sclerostin antibody-conjugated TiO2 nanotube arrays (TNTs-scl). TNTs-scl not only promoted the cyto-compatibility to MLO-Y4 cells but also reduced the amount of sclerostin secreted by the MLO-Y4 cells. The lowered sclerostin secretion greatly stimulated Wnt signaling to promote the differentiation of osteoblasts. Therefore, this study provides a simple and effective approach for surface engineering of titanium to

Acknowledgements

The work was financially supported by National Key R&D Program of China (2016YFC1100300), National Natural Science Foundation of China (51773023 and 51173216), Natural Science Foundation of Chongqing Municipal Government (cstc2018jcyjAX0368), Fundamental Research Funds for the Central Universities (2018CDPTCG0001/21) and China Postdoctoral Science Foundation Grant (2016M592644).

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