Surface engineering of titanium alloy substrates with multilayered biomimetic hierarchical films to regulate the growth behaviors of osteoblasts
Graphical abstract
Introduction
Titanium (Ti) and its alloys have been widely applied as orthopedic implants in the clinical setting, mainly due to their excellent mechanical properties and biocompatibility [1], [2]. However, Ti-based implants without superficial modifications generally lack osseointegration with the surrounding bone tissue, leading to a short lifespan or even implantation failure [3]. Many efforts have been made to improve the biological performance of a Ti-based implant, including physical, chemical and biological modifications of Ti substrates. As an artificial extracellular support, Ti substrates would provide desired surface structures for cell anchorage, such as microstructures [4], nanostructures (e.g. Ti nanotubes [5], Ti nanofibers [6]) and micro/nanostructures [7]. More importantly, Ti materials are expected to communicate with cells [8]. From this point of view, it is essentially to restore in biomimetic terms a desirable extracellular microenvironment that regulates cell fates and biological functions, such as cell adhesion, migration, proliferation and differentiation [9]. For instance, hydrogels with bioactive motifs [10] biopolymers [11], multilayer embedding proteins [12] and extracellular matrix mimetic [13] were employed to regulate the cells’ fates.
Inspired by these studies, an orthopedic implant should provide an appropriate extracellular microenvironment to natural bone as much as possible, not only the macroscopic structures but also the components at the nanoscale. Natural bone can be divided into two categories: cancellous bone and cortical bone, depending on their density or porosity. The cancellous bone has a porous structure composed of bone trabecula and cavities, whereas the cortical bone is composed of cylindrically shaped osteons with high density [14]. It is well known that natural cortical bone (e.g. long bone) has a hierarchical organization on different dimensions. The outside of a cortical bone is a compact and calcified layer, consisting of cylindrical osteons. Furthermore, on a sub-hierarchical level, an osteon is composed of nanosized hydroxyapatite (HA) and self-assembling collagen nanofibers, which organizes into a lamellar structure [15].
Previously, from a biomimetic aspect, many strategies have been developed for the fabrication of bone regenerative materials from three directions. The first strategy was directly mixing nanoscale inorganic substances with organic components to form nanocomposites [16]. The second one was to use pre-formed polymer scaffolds as biomineralization templates [17]. The third one was to form inorganic/organic hybrid nanocomposites in situ [18]. Nevertheless, those strategies have disadvantages for controlling the inside hierarchical structures on a nanoscale.
In the study, we intend to construct spatial intercalated nanostructures onto the surfaces of Ti6Al7Nb substrates. The intercalated nanostructures would well mimic the natural bone in both components and lamellar structures. The nanostructure was constructed by embedding HA nanofibers in between multilayers of gelatin (Gel) and chitosan (Chi) with a spin-assisted layer-by-layer (LbL) assembly technique (Fig. 1). Along with the degradation of organic layer components of Chi and Gel, the leaving cavities would induce osteoblast migration and then the cells secreting extracellular substances, such as collagen, glycosaminoglycan and proteoglycan, would fill the cavities, finally leading to in-growth bone formation and bone remodeling.
The LbL assembly technique is an efficient tool for the construction of multilayered structures on a solid substrate [19]. In this study, LBL was employed to construct HA nanofibers embedding a multilayered structure onto the surfaces of Ti6Al7Nb substrates. HA is the main inorganic component of natural bone, which was commonly used as a coating material for orthopedic implants, due to its excellent osteoinduction and biocompatibility [20], [21]. Gel is a denatured biopolymer derived from collagen, having a composition and immunogenicity similar to collagen [22]. Chi was reported to speed up bone reformation with osteoinductive properties [23]. In our previous studies, we constructed Chi and Gel multilayers and bilayers of Chi/Gel embedding bone morphogenetic protein 2 (BMP2) and fibronectin (FN) onto the surfaces of Ti substrates. The biological results indicated that those multilayers improved the biocompatibility of Ti substrates [13], [24].
In this study, we took nanoscale details into consideration. The HA nanofiber used in this study has a diameter of ∼20–30 nm and is hundreds of nanometers in length. A previous study proved that HA, less than 100 nm at least in one direction, was similar to the mineral component of bone tissue [25], since natural bone consists of ∼70% HA with a needle-like shape [26]. Moreover, nanosized HA demonstrated enhanced resorbability and bioactivity [27], [28]. Thus, we hypothesized that the biomimetic hierarchical structure deposited onto Ti6Al7Nb substrates would improve the biological functions of osteoblasts.
The objective of this study was to fabricate a biomimetic lamellar structure on Ti6Al7Nb substrates with HA nanofibers as the intercalated materials, and the Gel and Chi as the polyelectrolytes, via a spin-assisted LbL assembly technique. Furthermore, we investigated the effect of such a structure on the biological functions of osteoblasts with regard to cell adhesion, viability, alkaline phosphatase activity and mineralization, as well as bone formation related genes expressions of osteocalcin and osteopontin in vitro.
Section snippets
Materials
Ti6Al7Nb disks (diameter: 15 mm; thickness: 2 mm) were purchased from the Northwest Institute for Non-ferrous Metal Research, China. Chitosan, gelatin polyethylenimine (PEI) (MW 70 kD), 3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide) (MTT) and Hoechst 33258 were obtained from Sigma Chemical Co. (MO, USA). BCA kit was obtained from Beyotime Co. (Beijing, China). Rhodamine-phalloidin was supplied by Invitrogen Co. (USA). All other reagents were purchased from Oriental Chemical Co.
Characterization of HA nanofibers
The morphologies of xonotlite and HA nanofibers were observed by FESEM and TEM, respectively. HA nanofibers were obtained by the thermal treatment of xonotlite nanofibers in Na3PO4 solution. The high temperature and high pressure eroded the surfaces of xonotlite nanofibers, resulting in cavities, and offered plenty of Ca2+ ions. When the concentrations of Ca2+ and PO43− ions reached oversaturation, the nucleation of HA occurred and filled the surface cavities of the eroded xonotlite nanofibers
Conclusion
In summary, we designed and fabricated a biomimetic hierarchical structure on titanium alloy substrates by embedding HA nanofibers in between Chi/Gel pair layers via a spin-assisted LbL assembly technique, which demonstrated great potential for osteoinduction. The hierarchical structure promoted the migration, proliferation and bone-formation-related genes expressions of OC and OPN. The study affords a biomimetic approach for the fabrication of biofunctionalized titanium alloy implants for
Acknowledgements
We gratefully acknowledge the financial support from the Natural Science Foundation of Chongqing Municipal Government (CSTC, 2011JJJQ10004), the Natural Science Foundation of China (11032012 and 51173216), the National Key Technology R&D Program of the Ministry of Science and Technology (2012BAI18B04) and Fundamental Research Funds for the Central Universities (CQDXWL-2013-Z002).
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