Elsevier

Biomaterials

Volume 167, June 2018, Pages 44-57
Biomaterials

Osteogenesis potential of different titania nanotubes in oxidative stress microenvironment

https://doi.org/10.1016/j.biomaterials.2018.03.024Get rights and content

Abstract

Oxidative stress is commonly existed in bone degenerative disease (osteoarthritis, osteoporosis etc.) and some antioxidants had great potential to enhance osteogenesis. In this study, we aim to investigate the anti-oxidative properties of various TiO2 nanotubes (TNTs) so to screen the desirable size for improved osteogenesis and reveal the underlying molecular mechanism in vitro. Comparing cellular behaviors under normal and oxidative stress conditions, an interesting conclusion was obtained. In normal microenvironment, small TNTs were beneficial for adhesion and proliferation of osteoblasts, but large TNTs greatly increased osteogenic differentiation. However, after H2O2 (300 μM) treatment (mimicking oxidative stress), only large TNTs samples demonstrated superior cellular behaviors of increased osteoblasts' adhesion, survival and differentiation when comparing with those of native titanium (control). Molecular results revealed that oxidative stress resistance of large nanotubes was closely related to the high expression of integrin α5β1 (ITG α5β1), which further up-regulated the production of anti-apoptotic proteins (p-FAK, p-Akt, p-FoxO3a and Bcl2) and down-regulated the expression of pro-apoptotic protein (Bax). Moreover, we found that Wnt signals (Wnt3a, Wnt5a, Lrp5, Lrp6 and β-catenin) played an important role in promoting osteogenic differentiation of osteoblasts under oxidative condition.

Introduction

As for orthopedic implantation in elderly population suffering from degenerative diseases (osteoarthritis, osteoporosis etc.), the osseointegration of an implant with surrounding bone is generally limited [1,2]. For those patients, their cellular microenvironments in bone tissue change significantly, such as reactive oxygen species (ROS) accumulation, stem cell aging, etc. [2,3]. Due to the opposite effects on osteoclasts (promoting activity) and osteoblasts (inhibiting initial cells adhesion, proliferation, osteogenic differentiation, etc.), oxidative stress damage caused by the ROS accumulation attract much attentions in recent decades [[4], [5], [6]].

Oxidative stress, deriving from the imbalance between oxidation and anti-oxidation due to the accumulation of ROS (including .O2, .OH, H2O2, etc.), play a major role in the genesis and development of bone degenerative diseases, such as osteoporosis [[7], [8], [9]]. Oxidative stress is capable of inhibiting osteogenesis through various pathways. For example, FoxOs expression can be up-regulated via PI3K/Akt/FoxOs signals under oxidative stress, which could enter cells nuclei and competitively bind with TCF/LEF to block the osteogenic differentiation of pre-osteoblasts [5,6]. Meanwhile, ROS would activate osteoclasts and accelerate bone loss by regulating the expressions of OPG, RANKL, PPARy, etc. [4]. Moreover, under oxidative stress, the apoptosis rate of mature bone cells significantly increased, which is another important cause of osteoporosis [10].

Recent studies revealed that the modulation of antioxidant through surface modification could substantially improve osteogenesis potential of titanium (Ti) based orthopedic implants. Lei et al. reported that chitosan coating on porous titanium alloy implants dramatically promoted the integration between implant and bone tissue due to the antioxidant ability of chitosan [11]. Ogawa et al. proved that ultraviolet-treated titanium substrates significantly reduced oxidative stress, which effectively protected the viability of osteoblasts via reducing the production of endogenous ROS and inflammatory factors [12]. Moreover, our recent study also showed that antioxidant coating on the surface of titanium implants significantly promoted the new bone formation surrounding implants [13]. Therefore, the development of antioxidant orthopedic implants has great potential for fracture fixation in patients suffering bone degenerative diseases.

Surface nano-topological fabrication has been proved as an effective approach to improve the biological performance of Ti based implants in recent years [[14], [15], [16]]. It not only preserves the excellent mechanical properties of Ti substrates, but also promotes the interactions between implant and surrounding cells through exposing the active domains of those adsorbed proteins and/or activating some special membrane signals (e.g., integrin, Wnt) [16,17]. Moreover, previous studies also proved that various properties (e.g., composition, shape and size) of nanostructures affected their biological functions. Chien et al. reported that large titania nanotubes could greatly improve the differentiation of MSCs with reduced cell adhesion, however, small titania nanotubes promoted cell adhesion and proliferation while with reduced differentiation in vitro [18]. Moreover, Zhang et al. demonstrated that the optimal size of nanotubes was 70 nm to obtain favorable osteoconductivity and osseointegration in vivo when comparing with 30 and 110 nm nanotubes [19]. Our previous study proved that only large TiO2 nanoparticles (about 80 nm) on micro-structured surface significantly increased the proliferation and differentiation of osteoblasts [20]. Although previous studies claimed that specific surface nanostructures effectively improved osteogenesis, it remains unknown whether these conclusions are consistent under pathological oxidative stress condition.

Thus, in this study we investigated the oxidation resistance and osteogenesis capacities of different titania nanotubes with H2O2 (300 μM) treatment (mimicking pathological oxidative stress) for screening superior antioxidant materials. Taking advantage of the highly ordered surface structures and simple preparation process, we chose TNTs as substrate materials in this study [21,22]. There are two issues to be addressed in this study as follows: (1) to investigate the size effect of TNTs on antioxidant and osteogenesis under oxidative condition; (2) to preliminarily elucidate the molecular mechanism for relieving the oxidative stress.

Section snippets

Materials

Titanium foils (purity: 99.5%; thickness: 0.25 mm) was purchased from Alfa Aesar Co. (Tianjin, China). Aladdin Industrial Co. (Shanghai, China) provided glycerol and NH4F. Alizarin red, H2O2 solution (3%), Hoechst 33258, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fluorescein diacetate (FDA), propidium iodide (PI), RNAase and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were bought from Sigma Chemical Co. (MO, USA). Commercial testing kits of bicinchoninic acid (BCA),

Surface characterization

From SEM images (Fig. 1A), it was found that there were many scratches on Ti substrates surface, which was attributed to the pretreatment of sanding [13,22]. After anodizing, regular tubular structures were observed. Through quantitative statistical analysis, it was determined that the nanotube sizes of TNT30, TNT70 and TNT110 were around 30, 70 and 110 nm, respectively. The AFM result showed that the surface roughness (Rq) of substrates obviously decreased after oxidation treatment (about

Discussion

In this study, we evaluated the antioxidant activities of different TiO2 nanotubes at both cellular and molecular levels. The results demonstrated that large nanotubes (110 nm) could more effectively attenuate the negative effects of oxidative damage via ITG α5β1 and Wnt signals activation than nanotubes with a size of 30 nm.

Due to the superior mechanical properties and biocompatibility, Ti-based implants were widely applied in orthopedic and dental applications [28]. However, implant loosing

Conclusion

In this study, we evaluated the anti-oxidative potential of various TiO2 nanotubes at cellular and molecular levels. Large nanotubes displayed strong capacities to improve cell adhesion, survival and differentiation of osteoblasts after H2O2 treatment. Meanwhile, the synergetic effect of ITG α5β1 and Wnt signals directly contributed to the superior antioxidant ability of TNT110 substrates. Overall, large nanotubes (TNT110) owned superior antioxidant potential and would be beneficial for the

Acknowledgments

This work was financially supported by State Key Project of Research and Development (Grant No. 2016YFC1100300 & 2017YFB0702603), National Natural Science Foundation of China (51673032, 21734002 & 31700827), Natural Science Foundation of Chongqing Municipal Government (CXTDX201601002) and China Postdoctoral Science Foundation Grant (2017M622971), Innovation Team in University of Chongqing Municipal Government (CXTDX201601002), and Fundamental Research Funds for the Central Universities (

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