Multidimensional analysis for the correlation of physico-chemical attributes to osteoblastogenesis in TiNbZrSnTa alloys.

Data-enabled approaches that complement experimental testing offer new capabilities to investigate the interplay between chemical, physical and mechanical attributes of alloys and elucidate their effect on biological behaviours. Reported here, instead of physical causation, statistical correlations were used to study the factors responsible for the adhesion, proliferation and maturation of pre-osteoblasts MC3T3-E1 cultured on Titanium alloys. Eight alloys with varying wt% of Niobium, Zirconium, Tin and Tantalum (Ti- (2-22 wt%)Nb- (5-20 wt%)Zr- (0-18 wt%)Sn- (0-14 wt%)Ta) were designed to achieve exemplars of allotropes (incl., metastable-ß, ß + α', α″). Following confirmation of their compositions (ICP, EDX) and their crystal structure (XRD, SEM), their compressive bulk properties were measured and their surface features characterised (XPS, SFE). Because these alloys are intended for the manufacture of implantable orthopaedic devices, the correlation focuses on the effect of surface properties on cellular behaviour. Physico-chemical attributes were paired to biological performance, and these highlight the positive interdependencies between oxide composition and proliferation (esp. Ti4+), and maturation (esp. Zr4+). The correlation reveals the negative effect of oxide thickness, esp. TiOx and TaOx on osteoblastogenesis. This study also shows that the characterisation of the chemical state and elemental electronic structure of the alloys' surface is more predictive than physical properties, namely SFE and roughness.

In-silico design and experimental validation of TiNbTaZrMoSn to assess accuracy of mechanical and biocompatibility predictive models.

Numerical design of TiNbTaZrMoSn alloy preceded its manufacture and mechanical, physico-chemical and in vitro characterisation. The specifications of the alloy required a multi-objective optimisation including lower modulus of elasticity than c.p.Ti, high strength, stabilised ß crystal structure with a low martensitic start temperature, a narrow solidification range and high biocompatibility. The results reveal that there was a good match between the bulk mechanical properties exhibited by the alloy experimentally and those predicted. Regarding surface properties, independent of roughness effects, the oxide thickness and surface zeta-potential, measured in biologically relevant electrolytes and at physiological pH, arose as important factors in osteoblastic activity (i.e., cell proliferation, measured via DNA, protein and metabolite content, and differentiation, via ALP levels), but not in cell adhesion and viability. The thinner oxide layer and lower absolute value of surface zeta-potential on the TiNbTaZrMoSn alloy explain its lesser osteogenic properties (i.e., inhibition of ALP activity) compared to the c.p. Ti. This study demonstrates that the numerical models to predict microstructure and bulk mechanical properties of ß-Ti alloys are robust, but that the prediction of cellular bioactivity lags behind and still requires parameterisation to account for features such as oxide layer composition and thickness, electro-chemical properties and surface charge, and topography to optimise cell response in silico before committing to the costly manufacture and deployment of these alloys in regenerative medicine.

The impact of multimodal pore size considered independently from porosity on mechanical performance and osteogenic behaviour of titanium scaffolds.

Titanium porous scaffolds comprising multimodal pore ranges (i.e., uni-, bi-, tri-modal and random) were studied to evaluate the effect of pore size on osteoblastogenesis. The scaffolds were manufactured using spaceholder-powder metallurgy, and porosity and pore size were kept independent. Their mechanical and physical properties (i.e., stiffness, strength, total and open porosity) were determined. In a first step, unimodal porous samples were tested with a mouse osteoblastic clonal cell line to ascertain pore size and porosity effects on cellular behaviour. Their proliferation (via cell number and total protein content), differentiation (via ALP enzyme levels) and maturation potency (with gene markers (Runx2, osteocalcin) and cytoplasmatic calcium) were investigated. In a second step informed by the previous results, multimodal scaffolds were shortlisted according to a set of criteria that included stiffness similar to that of cortical or trabecular bone, high strength and high open porosity. Their bioactivity performance was then studied to assess the benefits of mixing different pore ranges. The study concludes that pre-osteoblasts cultivated in unimodal microstructures with a pore range 106-212 µm of 36% total (actual) porosity and 300-500 µm of 55% total (actual) porosity achieved the largest extent of maturation. Bimodal microstructures comprising small (106-212 µm) and large (300-500 µm) pore ranges, distinctively distributed within the volume, and 40% (actual) porosity outperformed others, including multimodal (i.e. three or more pore ranges) and non-porous samples. They displayed a synergistic effect over the unimodal distributions. This should be a consideration in the design of scaffolds for implantation and bioengineering applications.

Addition of Sn to TiNb alloys to improve mechanical performance and surface properties conducive to enhanced cell activity.

Titanium (Ti) alloys with Niobium (Nb) and Tin (Sn) were prepared in order to conduct a systematic study on the bulk and surface properties of as-cast c.p.Ti, binary Ti-40Nb and Ti-10Sn, and ternary Ti-10Nb-5Sn (at.%) to ascertain whether Sn content can be used as an enhancer for cell activity. From a metallurgy viewpoint, a range of binary and ternary alloys displaying distinctive Ti phases (i.e. ß, α', α") were achieved at room temperature. Their surface (oxide thickness and composition, roughness, contact angle) and bulk (compressive stiffness, strength, elongation, microhardness, electrical resistance) features were characterised. The same surface roughness was imparted on all the alloys, therefore substrate-cell interactions were evaluated independently from this variable. The physico-mechanical properties of the ternary alloy presented the highest strength to stiffness ratio and thereby proved the most suitable for load-bearing orthopaedic applications. From a cellular response viewpoint, their cytotoxicity, ability to adsorb proteins, to support cell growth and to promote proliferation were studied. Metabolic activity using a mouse model was monitored for a period of 12 days to elucidate the mechanism behind an enhanced proliferation rate observed in the Sn-containing alloys. It was hypothesised that the complex passivating surface oxide layer and the bulk inhomogeneity with two dominant Ti phases were responsible for this phenomenon.

The effect of pore size and porosity on mechanical properties and biological response of porous titanium scaffolds.

The effect of pore size and porosity on elastic modulus, strength, cell attachment and cell proliferation was studied for Ti porous scaffolds manufactured via powder metallurgy and sintering. Porous scaffolds were prepared in two ranges of porosities so that their mechanical properties could mimic those of cortical and trabecular bone respectively. Space-holder engineered pore size distributions were carefully determined to study the impact that small changes in pore size may have on mechanical and biological behaviour. The Young's moduli and compressive strengths were correlated with the relative porosity. Linear, power and exponential regressions were studied to confirm the predictability in the characterisation of the manufactured scaffolds and therefore establish them as a design tool for customisation of devices to suit patients' needs. The correlations were stronger for the linear and the power law regressions and poor for the exponential regressions. The optimal pore microarchitecture (i.e. pore size and porosity) for scaffolds to be used in bone grafting for cortical bone was set to <212µm with volumetric porosity values of 27-37%, and for trabecular tissues to 300-500µm with volumetric porosity values of 54-58%. The pore size range 212-300µm with volumetric porosity values of 38-56% was reported as the least favourable to cell proliferation in the longitudinal study of 12days of incubation.