The deposition of strontium-substituted hydroxyapatite coatings.

Strontium substituted hydroxyapatite (SrHA) coatings have received a lot of interest recently as strontium (Sr) has been shown to have the dual benefit of promoting bone formation and reducing bone resorption, in vivo. In this work, SrHA coatings were deposited onto polycrystalline titanium surfaces using radio frequency (RF) magnetron co-sputtering and compared to those deposited from HA alone. In particular, the influence of different levels of Sr-substitution of the sputtering targets (5 and 13% Sr-substituted HA targets) on the properties of the deposited coatings produced at a low discharge power level (150 W) were investigated using FTIR, XPS, XRD, ToFSIMS and AFM techniques (both before and after annealing at 500 °C). The results show that Sr could be successfully incorporated into the HA lattice to form SrHA coatings and that they contained no other impurities. However, the coating produced from the 13% Sr-substituted target had a higher Ca+Sr/P ratio (1.95±0.14) and Sr content when compared to the coating produced from the 5% Sr-substituted target (1.58±0.20). The deposition rate also decreased with increasing Sr content of the sputtering targets. Furthermore, as the Sr content of the coatings increased, so did the preferred 002 orientation of the coating along with increased surface roughness and heterogeneity of the surface features. Therefore, this study has shown that RF magnetron sputtering offers a means to control attendant properties of Sr-substituted HA, such as the crystallinity, stoichiometry, phase purity and surface topography.

Hydrodynamic control of titania nanotube formation on Ti-6Al-4V alloys enhances osteogenic differentiation of human mesenchymal stromal cells.

In order to obtain bioactive bone-implant interfaces with enhanced osteogenic capacity, various approaches have been developed to modify surface physicochemical properties of bio-inert titanium and titanium alloys. One promising strategy involves fabricating highly ordered nanotubes (NT) on implant surfaces via electrochemical anodization. However, few studies have applied this technique to Ti-6Al-4V alloys most commonly adopted for the fabrication of osteo-integrated surfaces on orthopedic implants. In this study, we investigated the influence of electrolyte hydrodynamics to NT fabrication on Ti-6Al-4V in ethylene glycol based electrolyte and evaluated the osteogenic differentiation capacity of human mesenchymal stromal cells (hMSCs) on different diameter NT surfaces. Computational Fluid Dynamics (CFD) analysis was used to simulate electrolyte flow profiles under various stirring conditions (e.g. stirrer bar location and flow direction) and their correlation to NT formation. Polished Ti-6Al-4V disks (240 grit) were anodized at 20 and 40 V under optimal electrolyte flow conditions for comparison of NT diameter-controlled osteogenic differentiation and mineralization potential of hMSCs over 21 days culture in osteogenic media. Ti-6Al-4V surfaces anodized with 20 and 40 V resulted with NTs diameter approx. 39 and 83 nm, respectively. Electrolyte hydrodynamics (flow profile) significantly influenced the uniformity of NT formation. Here, a uniform velocity and shear stress profile at the surface promoted homogeneous NT growth, whereas large variation in either flow velocity or shear stress to the surface impaired mature NT formation. After 21 days of culture, fluorescence staining demonstrated significantly greater osteocalcin and osteopontin expression, and increased mineralized deposits (xylenol orange staining) on fluctuating NT surfaces anodized under 20 V (Ø 39 nm) relative to flat NT layer anodized with 40 V (Ø 83 nm) and polished controls. This study provides a systematic investigation of NT formation with respect to the electrolyte hydrodynamic effects to NT growth on Ti-6Al-4V alloys, demonstrating the feasibility of a one-step anodization process for generating uniform NT under optimal hydrodynamics. Optimized wavy micro-/nano-topography with Ø 39 nm NT stimulated osteogenic differentiation capacity of hMSCs on Ti-6Al-4V alloys and confirmed the potential application of anodization to improve osteo-integrative surfaces in orthopedic implants.

Automated 3D bioassembly of micro-tissues for biofabrication of hybrid tissue engineered constructs.

Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.

Positive and negative bioimprinted polymeric substrates: new platforms for cell culture.

Bioimprinting, which involves capturing cell morphological details into a polymer matrix, provides a new class of patterned surfaces which opens an opportunity to investigate how cells respond to their own signatures and may introduce possibilities for regulating their behaviour. In this study, phenotypic details of human nasal chondrocytes (HNCs) were replicated in soft polydimethylsiloxane (PDMS) mould resulting in inverse replicas of cells, which have been termed here as 'negative bioimprint'. For the first time, the information from this negative bioimprint was then transferred into another PDMS layer resulting in surfaces which resemble cell morphology and were called 'positive bioimprints'. Soft lithography was used to transfer these details from PDMS into different polymers like polystyrene, tissue culture polystyrene and clinically used block co-polymer poly (ethylene glycol) terephthalate-poly (butylene terephthalate) (PEGT-PBT). Results obtained from surface characterization confirmed that fine details of cells were successfully replicated from cells to different polymer matrices without any significant loss of information during the different steps of pattern transfer. HNCs seeded on different polymer surfaces with positive and negative bioimprints exhibited distinct behaviour. Cells cultured on positive bioimprints were more spread out and displayed high levels of proliferation compared to those on negative bioimprints, where cells were more compact with lower proliferation.