Functionalization of a clustered TiO2 nanotubular surface with platelet derived growth factor-BB covalent modification enhances osteogenic differentiation of bone marrow mesenchymal stem cells
Introduction
Due to excellent bio-compatibility, titanium (Ti) endosseous implantable medical devices are widely used in orthopedic and dental applications [1]. Successful osseointegration is the decisive factor underlying Ti implant performance [2]. Unfortunately, clinical applications of endosseous implants are often accompanied by a series of osseointegration-related complications, such as impeded formation of implant osseointegration and infection-related implant failure due to bone loss around implants. The revisions of failed hip implants alone cost billions of Euros per year solely in the Euro zone [3]. Infection-related loss of osseointegration affects almost half of all patients and accounts for approximately 28% of failed implants [4]. According to the “adhesion racing theory” between bacteria and host cells, rapid adhesion and proliferation of host osteoblastic lineage cells can favor host cell occupation of binding sites on the Ti implant surface, which facilitates osseointegration as well as inhibits adhesion of bacteria [5]. Therefore, achieving faster, better, stable, and controllable implant osseointegration is an important goal of implantable material research and necessitates long-term study and novel resolutions [6].
It has been shown that the surface characteristics of implantable materials profoundly determine their clinical performances pertaining to host-material interaction [7,8]. In this regard, modifying the surface of implantable Ti materials to improve osteoinductivity by mimicking the spatial structure of the bone extracellular matrix (ECM) and developing a “niche” to facilitate the behavior and function of bone formation-related cells may be a promising strategy. Based on these theories, a multitude of strategies, including sandblasting, acid etching, and micro-arc oxidation (such as SLA, SLActive, TiUnite) have been used to alter the surface topography of Ti implants at a micron-scale [9]. Compared with machined Ti implants, these strategies have gained better implant osteointegration, as well as higher rates of successful implantation in the clinic [[10], [11], [12]]. Dalbly et al., reported that nanopits, with a diameter of ~120 nm on polymethylmethacrylate, induced osteogenic differentiation of mesenchymal stem cells (MSCs) [13]. In addition, titania nanopillars with a height of 15 nm engineered by Terje et al., induced greater extracellular matrix (ECM) mineralization of MSCs compared to polished Ti controls and their counterparts with heights of 55 and 100 nm [14]. Patterned silicon surfaces with parallel nanogrooves from 400 to 4000 nm showed different regulation of osteogenic differentiation of human MSCs compared to the planar control [15]. Moreover, other nanostructure surfaces such as nanoribbons, nanodots, and nanosheets with specific-scales could directly influence the fate of MSCs without exogenous inducers [[16], [17], [18]]. Previously, we fabricated a TiO2 nanotube array with a tube size of 30–80 nm on the Ti implant surface to mimic the cross sectional structure of type I collagen, a main component of bone ECM, and found that this surface nanostructure facilitated ECM deposition and mineralization of bone marrow MSCs (bMSCs) [19,20]. Using a method of anodization plus acid etch, we also constructed an etched Ti surface with a nanotube array [21]. Unfortunately, the surface topographies failed to represent the spatial hierarchical structure of natural bone tissue due to limitations in either the sole micron-scale or sole nano-scale, respectively [22]. Considering the fact that natural bone tissue is composed of micron-scale functional units that are assembled of nano-scale components, a bone-like surface structure with integrated micro- and nano-scale elements should be optimal for a Ti implant.
In addition to topographic modification and physicochemical treatments, biochemical methods have offered alternative approaches to enhance bone-implant interactions and reduce the risk of inflammation by directly eliciting an advantageous response of the tissue around the implant. To control specific cell and tissue responses at the bone-implant interface, bio-active molecules such as proteins and peptides are immobilized on the surfaces of implantable materials via biochemical modification [23]. Several cytokines and growth factors, such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and platelet derived growth factor (PDGF), have been shown to play critical roles in bone regeneration after injury. In particular, PDGF significantly contributes to all stages of bone regeneration after trauma [24,25].
As a potent chemoattractant and mitogen participating in bone healing, PDGF consists of at least three types of dimerism: PDGF-AA, -BB, and –AB [25]. Among all the dimer isoforms, PDGF-BB exerts the most potent chemotactic effects on bMSCs and mesenchymal progenitor cells [26,27], and its expression has been shown to be elevated during bone healing [25,28]. Several studies have reported that recombinant human PDGF-BB (rhPDGF-BB) not only improves proliferation of various cell types but also stimulates osteogenesis following bone fracture or at defective sites in vivo [[29], [30], [31], [32]]. A multi-center randomized controlled trial showed that local administration of rhPDGF-BB was safe and effective for treating periodontal osseous defects [33]. Remarkably, even in the diabetic rat model, rhPDGF-BB accelerated the healing process of bone fracture [34].
In the present work, using modified anodization with a perchloric acid electrolyte and programmed voltage alteration, TiO2 nanotubes were rapidly constructed and self-assembled to form a cluster-like topography at the micron-scale on the Ti implant surface. rhPDGF-BB was immobilized on this micro-nano hierarchical structure through covalent modification utilizing carbonyldiimidazole (CDI) chemistry. Since increasing the distance between the bonded protein and the substrate is beneficial for the activity of biomolecules and cell growth [35], the anodized Ti surface was pretreated with 11-hydroxyundecylphosphonic acid (PhoA) before CDI activation to reduce the negative effects of unwanted steric hindrance on the immobilized protein [36]. We conducted comprehensive in vitro and in vivo experiments to systematically examine the surface characteristics, cytotoxicity, and osteogenic activity of the modified Ti surfaces with rhPDGF-BB immobilization.
Section snippets
Fabrication and characterization of the nanostructured TiO2 surface with PDGF-BB modification
Nanostructured Ti samples were prepared using a multi-step procedure. Medical-grade pure titanium (99.9%, Grade 1, donated by Northwest Institute for Nonferrous Metal Research, Xi'an, China) was first machined to form circular disks (15 mm in diameter and 1 mm in thickness, for cell culture) and Ti screw implants (2.8 mm in diameter, 5 mm in length and 400 μm in screw pitch) for implantation in vivo. The Ti samples were polished using 500- to 8000-grit SiC sandpapers (MATADOR, Germany) and
Anodization leads to the formation of a clustered TiO2 nanotubular surface
Our previous work demonstrated that a TiO2 nanotubular surface could be built via anodization in F− containing electrolytes [19]. Due to the fact that this surface nanostructure was constituted with a vertically arranged nanotube array, the host tissue can only make contact with the upper side of the TiO2 nanotubular surface. In this study, we modified the anodization protocol, such that anodization was performed in a perchloric acid/glycol electrolyte solution. Under this condition, as
Conclusions
In this study, we used anodization followed by PhoA/CDI chemistry mediated immobilization of PDGF-BB at a physiological relevant concentration to establish a novel clustered TiO2 nanotubular surface structure on a pure Ti implant surface. Our analysis of C1s, N1s, and O1s peaks by XPS validated that PDGF-BB was successfully covalently bonded to the nanostructured Ti surface. Such bioactive/biomimetic surface modification effectively enhanced cell attachment, proliferation, osteogenic human bMSC
Ethical approval
For human research: The medical ethics committee of the School of Stomatology of the Fourth Military Medical University approved this study [IRB-REV-2014004]. The study adhered to the tenets of the Declaration of Helsinki. All bMSC donors provided written informed consent.
For animal studies: The animal experiment ethics committee of the No.323 Hospital of PLA (People's Liberation Army of China) approved this study and supervised all animal experiments in this study [LLWYH3232015006].
Data availability
The raw data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. The processed data required to reproduce these findings are available to download from [https://doi.org/10.17632/fz5dp4b64s.3].
Declaration of competing interest
The authors declare there are no conflicts of interest regarding the publication of this paper.
Acknowledgments
This work was supported financially by grants from the National Natural Science Foundation of China (Nos. 81971752, 81501599, 91442108, 81571531, 81530051) and a grant from the China Postdoctoral Science Foundation (No. 2016M593009).
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These authors contributed equally to this work.