Tailoring of TiAl6V4 Surface Nanostructure for Enhanced In Vitro Osteoblast Response via Gas/Solid (Non-Line-of-Sight) Oxidation/Reduction Reactions.

An aging global population is accelerating the need for better, longer-lasting orthopaedic and dental implants. Additive manufacturing can provide patient-specific, titanium-alloy-based implants with tailored, three-dimensional, bone-like architecture. Studies using two-dimensional substrates have demonstrated that osteoblastic differentiation of bone marrow stromal cells (MSCs) is enhanced on surfaces possessing hierarchical macro/micro/nano-scale roughness that mimics the topography of osteoclast resorption pits on the bone surface. Conventional machined implants with these surfaces exhibit successful osseointegration, but the complex architectures produced by 3D printing make consistent nanoscale surface texturing difficult to achieve, and current line-of-sight methods used to roughen titanium alloy surfaces cannot reach all internal surfaces. Here, we demonstrate a new, non-line-of-sight, gas/solid-reaction-based process capable of generating well-controlled nanotopographies on all open (gas-exposed) surfaces of titanium alloy implants. Dense 3D-printed titanium-aluminum-vanadium (TiAl6V4) substrates were used to evaluate the evolution of surface nanostructure for development of this process. Substrates were either polished to be smooth (for easier evaluation of surface nanostructure evolution) or grit-blasted and acid-etched to present a microrough biomimetic topography. An ultrathin (90 ± 16 nm) conformal, titania-based surface layer was first formed by thermal oxidation (600 °C, 6 h, air). A calciothermic reduction (CaR) reaction (700 °C, 1 h) was then used to convert the surface titania (TiO2) into thin layers of calcia (CaO, 77 ± 16 nm) and titanium (Ti, 51 ± 20 nm). Selective dissolution of the CaO layer (3 M acetic acid, 40 min) then yielded a thin nanoporous/nanorough Ti-based surface layer. The changes in surface nanostructure/chemistry after each step were confirmed by scanning and transmission electron microscopies with energy-dispersive X-ray analysis, X-ray diffraction, selected area electron diffraction, atomic force microscopy, and mass change analyses. In vitro studies indicated that human MSCs on CaR-modified microrough surfaces exhibited increased protein expression associated with osteoblast differentiation and promoted osteogenesis compared to unmodified microrough surfaces (increases of 387% in osteopontin, 210% in osteocalcin, 282% in bone morphogenic protein 2, 150% in bone morphogenic protein 4, 265% in osteoprotegerin, and 191% in vascular endothelial growth factor). This work suggests that this CaR-based technique can provide biomimetic topography on all biologically facing surfaces of complex, porous, additively manufactured TiAl6V4 implants.

Production of osteogenic and angiogenic factors by microencapsulated adipose stem cells varies with culture conditions.

Growth factors produced by stem cells aid in the bone repair process. We investigated the ability of encapsulated rat adipose-derived stem cells (rASCs) treated with osteogenic media (OM) to produce growth factors, and determined the optimal combination of OM components that will lead to the production of both osteogenic and angiogenic factors. Our results demonstrate that microencapsulated stem cells were able to produce vascular endothelial growth factor (VEGF), fibroblast growth factor-2, and bone morphogenetic protein-2 (BMP2) necessary for bone regeneration. OM led to the reduction of angiogenic factors; however, the removal of dexamethasone restored angiogenic factor production. Additionally, we determined whether the effect of dexamethasone on VEGF and BMP2 varied among rat, rabbit, mouse, and humans. Dexamethasone led to a reduction in VEGF levels in ASCs derived from rats, mice, and humans, while this reduction was absent in rabbit ASCs (rbASCs). Human ASCs (hASCs) from donors of different race and sex showed a similar response to dexamethasone with secreted VEGF levels. BMP2 levels secreted by rbASCs, mouse ASCs (mASCs), and hASCs were independent of the media treatments, while rASCs responded differently in the surrounding media and within the microbeads. In conclusion, microencapsulated ASCs can be treated to produce osteogenic and angiogenic factors for tissue regeneration applications, but outcomes may vary with culture conditions.

Effects of Tunable Keratin Hydrogel Erosion on Recombinant Human Bone Morphogenetic Protein 2 Release, Bioactivity, and Bone Induction.

IMPACT STATEMENT: Recombinant human bone morphogenetic protein 2 (rhBMP-2) delivery from collagen sponges for bone formation is an important clinical example of growth factors in tissue engineering. Side effects from rhBMP-2 burst release and rapid collagen resorption have led to investigation of alternative carriers. Here, keratin carriers with tunable erosion rates were formulated by varying disulfide crosslinking via ratios of oxidatively (keratose) to reductively (kerateine) extracted keratin. In vitro rhBMP-2 bioactivity increased with kerateine content, reaching levels greater than with collagen. Heterotopic bone formation in a mouse model depended on the keratin formulation, highlighting the importance of the growth factor carrier.