Tuning Mesenchymal Stem Cell Response onto Titanium-Niobium-Hafnium Alloy by Recombinant Fibronectin Fragments.

Since metallic biomaterials used for bone replacement possess low bioactivity, the use of cell adhesive moieties is a common strategy to improve cellular response onto these surfaces. In recent years, the use of recombinant proteins has emerged as an alternative to native proteins and short peptides owing to the fact that they retain the biological potency of native proteins, while improving their stability. In the present study, we investigated the biological effect of two different recombinant fragments of fibronectin, spanning the 8-10th and 12-14th type III repeats, covalently attached to a new TiNbHf alloy using APTES silanization. The fragments were studied separately and mixed at different concentrations and compared to a linear RGD, a cyclic RGD and the full-length fibronectin protein. Cell culture studies using rat mesenchymal stem cells demonstrated that low to medium concentrations (30% and 50%) of type III 8-10th fragment mixed with type III 12-14th fragment stimulated cell spreading and proliferation compared to RGD peptides and the fragments separately. On the other hand, type III 12-14th fragment alone or mixed at low volume percentages ≤50% with type III 8-10th fragment increased alkaline phosphatase levels compared to the other molecules. These results are significant for the understanding of the role of fibronectin recombinant fragments in cell responses and thus to design bioactive coatings for biomedical applications.

Ion-doping as a strategy to modulate hydroxyapatite nanoparticle internalization.

Although it is widely acknowledged that ionic substitutions on bulk hydroxyapatite substrates have a strong impact on their biological performance, little is known of their effect on nanoparticles (NPs) especially when used for gene transfection or drug delivery. The fact that NPs would be internalized poses many questions but also opens up many new possibilities. The objective of the present work is to synthesize and assess the effect of a series of hydroxyapatite-like (HA) NPs doped with various ions on cell behavior, i.e. carbonate, magnesium and co-addition. We synthesized NPs under similar conditions to allow comparison of results and different aspects in addition to assessing the effect of the doping ion(s) were investigated: (1) the effect of performing the cell culture study on citrate-dispersed NPs and on agglomerated NPs, (2) the effect of adding/excluding 10% of foetal bovine serum (FBS) in the cell culture media and (3) the type of cell, i.e. MG-63 versus rat mesenchymal stem cells (rMSCs). The results clearly demonstrated that Mg-doping had a major effect on MG-63 cells with high cytotoxicity but not to rMSCs. This was a very important finding because it proved that doping could be a tool to modify NP internalization. The results also suggest that NP surface charge had a large impact on MG-63 cells and prevents their internalization if it is too negative-this effect was less critical for rMSCs.

Bioactive compounds immobilized on Ti and TiNbHf: AFM-based investigations of biofunctionalization efficiency and cell adhesion.

Implant materials require optimal biointegration, including strong and stable cell-material interactions from the early stages of implantation. Ti-based alloys with low elastic modulus are attracting a lot of interest for avoiding stress shielding, but their osseointegration potential is still very low. In this study, we report on how cell adhesion is influenced by linear RGD, cyclic RGD, and recombinant fibronectin fragment III8-10 coated on titanium versus a novel low-modulus TiNbHf alloy. The bioactive molecules were either physisorbed or covalently coupled to the substrates and their conformation on the surfaces was investigated with atomic force microscopy (AFM). The influence of the different bioactive coatings on the adhesion of rat mesenchymal stem cells was evaluated using cell culture assays and quantitatively analyzed at the single cell level by AFM-based single-cell force spectroscopy. Our results show that bioactive moieties, particularly fibronectin fragment III8-10, improve cell adhesion on titanium and TiNbHf and that the covalent tethering of such molecules provides the most promising strategy to biofunctionalize these materials. Therefore, the use of recombinant protein fragments is of high importance for improving the osseointegration potential of implant materials.

Mechanical and physicochemical characterization along with biological interactions of a new Ti25Nb21Hf alloy for bone tissue engineering.

Nowadays, one of the main challenges in metal implants for bone substitution is the achievement of an elastic modulus close to that of human cortical bone as well as to provide an adequate interaction with the surrounding tissue avoiding in vivo foreign body reaction. From this perspective, a new Ti-based alloy has been developed with Nb and Hf as alloying elements which are known as non-toxic and with good corrosion properties. The microstructure, mechanical behaviour and the physicochemical properties of this novel titanium alloy have been studied. Relationship of surface chemistry and surface electric charge with protein adsorption and cell adhesion has been evaluated due to its role for understanding the mechanism of biological interactions with tissues. The Ti25Nb21Hf alloy presented a lower elastic modulus than commercial alloys with a superior ultimate strength and yield strength than CP-Ti and very close to Ti6Al4V. It also exhibited good corrosion resistance. Furthermore, the results revealed that it had no cytotoxic effect on rat mesenchymal stem cells and allowed protein adsorption and cell adhesion. The experimental results make this alloy a promising material for bone substitution or for biomedical devices.

Mechanical properties and in vitro biological response to porous titanium alloys prepared for use in intervertebral implants.

The generation of titanium foams is a promising strategy for modifying the mechanical properties of intervertebral reinforcements. Thus, the aim of this study was to compare the in vitro biological response of Ti6Al4V alloys with different pore sizes for use in intervertebral implants in terms of the adhesion, proliferation, and differentiation of pre-osteoblastic cells. We studied the production of Ti6Al4V foams by powder metallurgy and the biological responses to Ti6Al4V foams were assessed in terms of different pore interconnectivities and elastic moduli. The Ti6Al4V foams obtained had similar porosities of approximately 34%, but different pore sizes (66 µm for fine Ti6Al4V and 147 µm for coarse Ti6Al4V) due to the sizes of the microsphere used. The Ti6Al4V foams had a slightly higher Young׳s modulus compared with cancellous bone. The dynamic mechanical properties of the Ti6Al4V foams were slightly low, but these materials can satisfy the requirements for intervertebral prosthesis applications. The cultured cells colonized both sizes of microspheres near the pore spaces, where they occupied almost the entire area of the microspheres when the final cell culture time was reached. No statistical differences in cell proliferation were observed; however, the cells filled the pores on fine Ti6Al4V foams but they only colonized the superficial microspheres, whereas the cells did not fill the pores on coarse Ti6Al4V foams but they were distributed throughout most of the material. In addition, the microspheres with wide pores (coarse Ti6Al4V) stimulated higher osteoblast differentiation, as demonstrated by the Alcaline Phosphatase (ALP) activity. Our in vitro results suggest that foams with wide pore facilitate internal cell colonization and stimulate osteoblast differentiation.

Robocasting of biomimetic hydroxyapatite scaffolds using self-setting inks.

Low temperature self-setting ceramic inks have been scarcely investigated for solid freeform fabrication processes. This work deals with the robocasting of alpha-tricalcium phosphate/gelatine reactive slurries as a bioinspired self-setting ink for the production of biomimetic hydroxyapatite/gelatine scaffolds. A controlled and totally interconnected pore network of ∼300 µm was obtained after ink printing and setting, with the struts consisting of a micro/nanoporous matrix of needle-shaped calcium deficient hydroxyapatite crystals, with a high specific surface area. Gelatine was effectively retained by chemical crosslinking. The setting reaction of the ink resulted in a significant increase of both the elastic modulus and the compressive strength of the scaffolds, which were within the range of the human trabecular bone. In addition to delaying the onset of the setting reaction, thus providing enough time for printing, gelatine provided the viscoelastic properties to the strands to support their own weight, and additionally enhanced mesenchymal stem cell adhesion and proliferation on the surface of the scaffold. Altogether this new processing approach opens good perspectives for the design of hydroxyapatite scaffolds for bone tissue engineering with enhanced reactivity and resorption rate.

Influence of acid-etching after grit-blasted on osseointegration of titanium dental implants: in vitro and in vivo studies.

Rough implant surfaces have shown improved osseointegration rates. In a majority of dental implants, the microrough surfaces are obtained by grit blasting and/or acid-etching. The aim of this contribution was to evaluate the effects of acid-etching, after the grit-blasted treatment in titanium dental implants, on surface wettability, surface energy, osteoblast responses and its osseointegration behavior. Four surfaces were studied: as-machined, acid-etched, micro-rough by grit-blasting and the combination grit-blasted surface with acid-etched. The surfaces with increasing roughness show more osteoblastic adhered cells. This effect was most pronounced on samples blasted and blasted with acid-etching. The roughness obtained by grit-blasting is the main factor in comparison with the acid etching treatment in the biological response. These results were confirmed in vivo tests and histological analysis. The results demonstrated that the combination of the grit-blasted and acid-etched accelerated lightly bone regeneration at the different periods of implantation in comparison with the grit-blasted implants.

Fibroblast adhesion and activation onto micro-machined titanium surfaces.

OBJECTIVES: Surface modifications performed at the neck of dental implants, in the manner of micro-grooved surfaces, can reduce fibrous tissue encapsulation and prevent bacterial colonization, thereby improving fibrointegration and the formation of a biological seal. However, the applied procedures are technically complex and/or time consuming methods. The aim of this study was to analyse the fibroblast behaviour on modified titanium surfaces obtained, applying a simple and low-cost method. MATERIAL AND METHODS: An array of titanium surfaces was obtained using a commercial computerized numerical control lathe, modifying the feed rate and the cutting depth. To elucidate the potential ability of the generated surfaces to activate connective tissue cells, a thorough gene (by real time - qPCR) and protein (by western blot or zymography) expression and cellular response characterization (cell morphology, cell adhesion and cell activation by secreting extracellular matrix (ECM) components and their enzyme regulators) was performed. RESULTS: Micro-grooved surfaces have statistically significant differences in the groove's width (approximately 10, 50 and 100 µm) depending on the applied advancing fixed speed. Field emission scanning electron microscopy images showed that fibroblasts oriented along the generated grooves, but they were only entirely accommodated on the wider grooves (≥50 µm). Micro-grooved surfaces exhibited an earlier cell attachment and activation, as seen by collagen Iα1 and fibronectin deposition and activation of ECM remodelling enzymes, compared with the other surfaces. However, fibroblasts could remain in an activated state on narrower surfaces (<50 µm) at later stages. CONCLUSIONS: The use of micro-grooved surfaces could improve implant integration at the gingival site with respect to polished surfaces. Micro-grooved surfaces enhance early fibroblast adhesion and activation, which could be critical for the formation of a biological seal and finally promote tissue integration. Surfaces with wider grooves (≥50 µm) seem to be more appropriate than surfaces with narrow grooves (<50 µm), as fibroblasts could persist in an activated state on narrower grooved surfaces, increasing the probability of producing a fibrotic response.