Rapid evaluation of bioactive Ti-based surfaces using an in vitro titration method
Weitian Zhao, David Michalik, Stephen Ferguson, Willy Hofstetter, Jacques Lemaître, Brigitte von Rechenberg & Paul Bowen
Nature Communicationsvolume 10, Article number: 2062 (2019) | Download Citation
Abstract
The prediction of implant behavior in vivo by the use of easy-to-perform in vitro methods is of great interest in biomaterials research. Simulated body fluids (SBFs) have been proposed and widely used to evaluate the bone-bonding ability of implant materials. In view of its limitations, we report here a rapid in vitro method based on calcium titration for the evaluation of in vivo bioactivity. Using four different titanium surfaces, this method identifies that alkaline treatment is the key process to confer bioactivity to titanium whereas no significant effect from heat treatment is observed. The presence of bioactive titanium surfaces in the solution during calcium titration induces an earlier nucleation of crystalline calcium phosphates and changes the crystallization pathway. The conclusions from this method are also supported by the standard SBF test (ISO 23317), in vitro cell culture tests using osteoblasts and in vivo animal experiments employing a pelvic sheep model.
Introduction
Understanding biomineralization processes carries an important medical significance. As one of its applications, the in vivo deposited calcium phosphate layer on bone implants can help to diminish the typical adverse host response and facilitate the formation of a direct bonding with the bone1,2,3,4,5. Generally, the biocompatibility and/or the bioactivity study of medical implants is a critical component in biomaterials research and well-established in vitro methods are highly desirable compared with animal experiments for their economic and ethical advantages.
Previously, Kokubo proposed an in vitro method using simulated body fluids (SBFs) with ion concentration similar to biological fluids to test implant materials (ISO 23317)6,7,8. According to this method, materials that are able to induce hydroxyapatite formation on its surface after a 4-week immersion in the otherwise stable SBF are considered bioactive and would result in a better bonding with the bone after implantation7. This method is relatively simple to perform and is widely used in the research of bioglass and Ti-based materials9,10,11,12.
However, despite its success and the following efforts to improve the current testing protocol13, the method is fundamentally limited due to its inability to produce a quantitative measure for the bioactivity of a material. After a testing duration of 1–4 weeks, the bioactivity is simply categorized into “yes” (with apatite observed on material’s surface) and “no” (no apatite observed). Comparison between different materials are difficult. Although the amount of precipitated hydroxyapatite can be quantified, it is often also a function of other factors, such as the available surface area. In fact, after the initial nucleation stage, the growth of hydroxyapatite crystals is dominated by secondary nucleation14,15,16. This contribution from secondary nucleation makes the isolation of the effect from surface chemistry a challenging task. In addition, although the method can easily distinguish inert materials (such as untreated titanium surface) and bioactive materials (such as bioglass), it struggles with materials having moderate activity. The use of a metastable solution for up to a month of testing duration as recommended in the ISO protocol amplifies the subtle changes during the test, sometimes producing inconsistent results13,17,18. Factors such as the vicinity to other surfaces in the static SBF test was also reported to influence the result17,19, meaning that a material can be either inert or bioactive depending on slight differences in operational procedures.
In this paper, we try to address the above-mentioned issue and propose a different in vitro protocol based on a titration method. A calcium solution is continuously added to a phosphate solution containing the testing material until calcium phosphate nucleation takes place, as monitored by a calcium selective electrode and a pH electrode. The degree of supersaturation of the solution required for the nucleation event is used as an indication of the bioactivity of the material, i.e., the lower the supersaturation, the higher the bioactivity. Four titanium-based surfaces with different chemical treatments are tested. The solution behavior in the absence of a testing sample is first of all discussed and compared with the results from titrations in the presence of four surfaces. Using the same surfaces, this method is also directly compared with the standard static SBF test. To verify the proposed in vitro method, animal experiments using a sheep pelvic model are carried out. Experimental designs using a total of 96 implants in 8 sheep are performed followed by biomechanical tests and histological studies. Bone-implant-contact area as well as torque values after implantation are measured and used as an indication of osseointegration for the evaluation of the bioactivity of different implants. In addition, cell culture tests using osteoblasts are conducted. Both cell viability and biosynthetic activity as measured by alkaline phosphatase (ALP) activity are determined. These studies aim to corroborate the proposed in vitro titration method for the evaluation of bioactive materials.
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