Elsevier

Materials & Design

Volume 145, 5 May 2018, Pages 65-73
Materials & Design

Effect of HF/HNO3-treatment on the porous structure and cell penetrability of titanium (Ti) scaffold

https://doi.org/10.1016/j.matdes.2018.02.059Get rights and content

Highlights

  • Hydrofluoric acid and nitric acid mixture is introduced for tunable modification of the pore structure of a titanium scaffold

  • The porosity, pore and pore neck sizes, and mechanical strength can be easily controlled by varying the acid treatment time

  • The relationship between pore structure and actual cell penetrability is demonstrated using the cell perfusion technique

  • Increases in internal pore interconnectivity and cell penetrability are conformed astreatment time increased up to 12 min

Abstract

Porous titanium (Ti) implants have been used in orthopedic and dental applications because of their superior mechanical properties. Sufficient pore interconnectivity is required for effective bone regeneration and growth inside the Ti scaffold pore structure. We proposed post-treatment with HF/HNO3 to efficiently modify the internal pore structure of a Ti scaffold and achieve controllable mechanical properties with a pore neck structure. The porosity, pore size, wall thickness, and pore neck size were easily controlled by varying the acid treatment time, which produced a Ti scaffold with mechanical properties that were suitable for bone tissue engineering. As the mixed acid treatment time increased, internal isolated pores were gradually interconnected with adjacent pores. After 10 min of treatment, nearly all the pores were interconnected. The post-treatment with HF/HNO3 also affected the surface properties. Surface carbon contaminants were significantly reduced after treatment with no hydride formation. Micron-scale surface roughness was uniformly generated across the whole surface. The actual cell penetrability of the Ti scaffold was evaluated using a perfusion-based in vitro cell test. Over 90% of the surface pores depict cell penetrability with a sufficient number of cells attached to the wall surface of the pore after performing acid treatment for 12 min.

Introduction

Titanium (Ti) is widely used as a biomedical material in orthopedic and dental applications because of its high corrosion resistance, low density, extraordinary mechanical properties, and satisfactory biocompatibility. Its inherent osseointegration ability enables the formation of direct bone contact on the implant surface for mechanically stable anchorage of implants to the surrounding living bone [[1], [2], [3]]. However, mismatch of the Ti implant rigidity with that of the host bone tissue creates a stress shielding effect at the bone–Ti implant interface, which can diminish the long-term clinical performance and stability of Ti implants [4,5]. The introduction of an internal pore structure inside Ti implants is a promising strategy to overcome this problem. By controlling the pore size, distribution, and interconnectivity, porous Ti was designed to match the mechanical properties of the native bone tissue [6,7]. Additionally, the porous structure can influence the cellular activity of a Ti scaffold; the surface pore size of several hundreds of microns allows sufficient free space for inward bone growth as well as body fluid transmission of oxygen and nutrients and metabolic waste excretion, which is essential for the strong fixation of a Ti scaffold with long-term reliability [8,9]. Therefore, the generation and precise control of an internal pore architecture have been thoroughly investigated for Ti implants using polymer replication [10], space holding [11,12], and freeze casting [13,14] methods.

Dynamic freeze casting based on a Ti/camphene slurry has recently emerged as a promising technique for producing a homogenous porous Ti structure. The slurry was continuously frozen under dynamic rotational conditions for 12 h, creating stable spherical growth of camphene crystals and maintaining a uniform distribution inside the slurry [13]. Furthermore, the sedimentation of the coarse Ti particles which is one of the major problems associated with conventional freeze casting techniques, that generates undesirable density and porosity gradients can be prevented by the rotational movement during the freezing process. In our previous study, spherical-like pore structures were surrounded by dense Ti walls with a smooth surface finish, which successfully maintained the mechanical behavior of the Ti powder after sintering [15,16]. However, the dynamic freeze casting method still produced pore structures lacking interconnectivity because of the spherical growth of camphene crystals. Pores were easily isolated by the separated camphene particles in the slurry, and the coalesced spherical camphene particles could only form a small number of interconnected pores by cold isostatic pressing (CIP). Increasing the volume fraction of camphene in Ti slurry can increase coalesced camphene particle formation. However, even above 70% total porosity, surface pores were partially connected by small pore neck sizes and cell attachments were restricted within the isolated surface pores [17]. Therefore, further enhancement of pore interconnectivity is necessary for uniform cell distribution, facilitating cell migration inside the scaffold, and for achieving regenerated bone tissue homogeneity both inside and outside the porous Ti scaffold.

Currently, while most of the post-treatment methods for Ti mainly focus on surface modification in order to accelerate osseointegration, e.g., apatite blasting [18], micro-arc oxidation (MAO) [19], and anodizing [20,21], acid etching treatment is the only adopted approach that can modify both the overall pore structure and its surface because of the high reactivity of Ti with acidic solutions [[22], [23], [24], [25]]. In the tissue engineering field, hydrochloric acid, sulfuric acid, hydrofluoric acid (HF), and nitric acid (HNO3) are extensively used to conduct post-acid etching treatment of Ti that induces micron-scale surface roughness, which directly affects the hydrophilicity, cytotoxicity, and biocompatibility. In particular, HF treatment can uniformly etch the Ti surface and generate an irregular complex surface morphology that enhances osteoblastic cell attachment and differentiation by increasing bone-specific gene expression. However, the etching rate of pure HF is too slow to modify the overall pore structure inside the Ti scaffold. Flammable and explosive hydrogen gases are generated during the reaction, which can also induce the formation of titanium hydride at the metal surface with consequent embrittlement [26]. Therefore, additives are required.

In this study, we adopted a mixed acid solution of HF and HNO3. HNO3 by itself is not able to etch the Ti scaffold because of the formation of a protective TiO2 layer on top of the Ti surface in pure HNO3 solution [26]. However, with this combination, HF is observed to become more stable, and the evolution of hydrogen gas can be prevented by the production of hexafluorotitanic acid, nitrogen dioxide, and water [26,27]. Therefore, the etching rate of the Ti scaffold can increase with the increasing concentration of HF without facing the aforementioned problems.

The effects of acid post-treatments on the internal pore structure of prefabricated Ti scaffolds and the degree of cell penetration through interconnected pores were evaluated. For maximization of the etching rate of the Ti scaffold, we developed extremely strong HF and HNO3 formulas with different etching times from one to 12 min. The surface crystalline phase and chemical composition changes were investigated using X-ray diffraction (XRD) and X-ray photoemission spectroscopy (XPS), respectively. The effect of the mixed acid treatment on the surface and pore structure of the Ti scaffold was monitored by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). We established a novel in vitro experimental model based on a cell perfusion method and evaluation protocol to evaluate the cell penetration capacity of HF/HNO3-treated Ti scaffolds and assess the practical performance of the Ti scaffold in the human body.

Section snippets

Preparation of flat Ti substrates and porous Ti scaffolds

Commercially available pure Ti plates (10 mm × 10 mm × 2 mm) were gradually polished with SiC papers from P400 to P2000. The plates were washed with ethanol for 20 min in an ultrasonic bath after polishing. A dynamic freeze casting method was used to prepare porous Ti scaffolds [13]. Commercially available Ti powder (−325 mesh, Alfa Aesar, Ward Hill, MA, USA) and camphene (C10H16, Sigma-Aldrich, St. Louis, MO, USA) were used as the starting materials. Ti/camphene slurries with 15 vol% Ti powder were

Surface characterization of Ti treated with HF/HNO3

We employed new extreme conditions with HF/HNO3 mixed acid as an etching solution for Ti to effectively modify closed or isolated pore openings and enhance the porous Ti scaffold interconnectivity. Before direct application to the Ti scaffolds, the chemical and crystallographic effects of the mixed acid solution on a flat Ti surface were investigated for treatment times up to 12 min. Fig. 2 shows representative XRD patterns of each Ti specimen before and after HF/HNO3 treatment. The precipitated

Conclusion

HF/HNO3 post-treatment was successfully performed on a Ti scaffold with tunable modification of the pore structure and mechanical properties. The porosity, pore size, and pore neck size also increased as the treatment time increased due to the high etching rate of the Ti pore walls. The amount of surface C contaminants decreased with increasing surface wettability after HF/HNO3 treatment. The cellular responses to the treated Ti scaffold were enhanced due to the roughened surface and altered

Acknowledgments

This research was supported by Basic Science Research Program (2015R1D1A1A01057311 and 2017R1A6A3A03008914) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education and Ministry of Trade, and Technology Innovation Program (Grant No. 10037915, WPM Biomedical Materials-Implant Materials) funded by the Ministry of Trade, Industry & Energy (MI, Korea).

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