Jordi Guillem-Marti

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.

Collagen-functionalised titanium surfaces for biological sealing of dental implants: Effect of immobilisation process on fibroblasts response

The clinical success of a dental implant requires not only an optimum osseointegration, but also the development of a biological sealing; i.e., a soft tissue seal around the transmucosal part of the implant. A promising approach to improve the biological seal of dental implants is the biomimetic modification of titanium surfaces with proteins or peptides that have specific cell-binding moieties. In this work we investigated the process of immobilising collagen on smooth and rough titanium surfaces and its effect on human dermal fibroblast (HDF) cell response. Titanium samples were activated by either oxygen plasma or acid etching to generate a smooth or nanorough surface, respectively. Subsequently, collagen grafting was achieved by either physisorption or covalent bonding through organosilane chemistry. The biofunctionalised titanium samples were then tested for stability and characterised by fluorescent labelling, wettability, OWLS and XPS studies. Biological characterisation was also performed through HDF adhesion, proliferation and gene expression. Covalent-bonded collagen showed higher stability than physisorbed collagen. A significant overexpression of the genes involved in fibroblast activation and extracellular matrix remodelling was observed in the collagen-coated surfaces. This effect was more pronounced on smooth than on rough surfaces. Immobilised collagen on the smooth plasma-treated surfaces favoured both fibroblast adhesion and activation. This study provides essential information for the design of implants with optimal biological sealing, a key aspect to avoid peri-implantitis and ensure long-lasting implant fixation.

Osteoinduction by foamed and 3D-printed calcium phosphate scaffolds: effect of nanostructure and pore architecture

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and β-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.

Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation.

Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria-cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties.

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 differentiationThe 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.

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.

Osteoblastic cell response to spark plasma-sintered zirconia/titanium cermets

Ceramic/metal composites, cermets, arise from the idea to combine the dissimilar properties in the pure materials. This work aims to study the biocompatibility of new micro-nanostructured 3 Y-TZP/Ti materials with 25, 50 and 75 vol.% Ti, which have been successfully obtained by spark slasma sintering technology, as well as to correlate their surface properties (roughness, wettability and chemical composition) with the osteoblastic cell response. All samples had isotropic and slightly waved microstructure, with sub-micrometric average roughness. Composites with 75 vol.% Ti had the highest surface hydrophilicity. Surface chemical composition of the cermets correlated well with the relative amounts used for their fabrication. A cell viability rate over 80% dismissed any cytotoxicity risk due to manufacturing. Cell adhesion and early differentiation were significantly enhanced on materials containing the nanostructured 3 Y-TZP phase. Proliferation and differentiation of SaOS-2 were significantly improved in their late-stage on the composite with 75 vol.% Ti that, from the osseointegration standpoint, is presented as an excellent biomaterial for bone replacement. Thus, spark plasma sintering is consolidated as a suitable technology for manufacturing nanostructured biomaterials with enhanced bioactivity.

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.

Plasma-induced selectivity in bone cancer cells death

Background Current therapies for bone cancers ‐ either primary or metastatic – are difficult to implement and unfortunately not completely effective. An alternative therapy could be found in cold plasmas generated at atmospheric pressure which have already demonstrated selective anti‐tumor action in a number of carcinomas and in more relatively rare brain tumors. However, its effects on bone cancer are still unknown. Methods Herein, we employed an atmospheric pressure plasma jet (APPJ) to validate its selectivity towards osteosarcoma cell line vs. osteoblasts & human mesenchymal stem cells. Results Cytotoxicity following direct interaction of APPJ with cells is comparable to indirect interaction when only liquid medium is treated and subsequently added to the cells, especially on the long‐term (72 h of cell culture). Moreover, following contact of the APPJ treated medium with cells, delayed effects are observed which lead to 100% bone cancer cell death through apoptosis (decreased cell viability with incubation time in contact with APPJ treated medium from 24 h to 72 h), while healthy cells remain fully viable and unaffected by the treatment. Conclusions The high efficiency of the indirect treatment indicates that an important role is played by the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the gaseous plasma stage and then transmitted to the liquid phase, which overall lead to lethal and selective action towards osteosarcoma cells. These findings open new pathways for treatment of metastatic bone disease with a minimally invasive approach. Graphical abstract Figure. No caption available. HighlightsCold atmospheric pressure plasmas generate various ROS & RNS that can diffuse to liquid media.The plasma treatment unveiled effective lethal and selective action towards osteosarcoma cells.Indirect effects through plasma‐treated liquid media point out the important role of the ROS and RNS.Osteosarcoma cells undergo apoptosis following cold plasma treatment.Healthy Mesenchymal Stem Cells and Osteoblasts remain fully viable after the treatment.

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.