Miriam López-Álvarez

Novel selenium‐doped hydroxyapatite coatings for biomedical applications

Nowadays there is a short-term need of investigating in orthopedic implants with a greater functionality, including an improved osseointegration and also antibacterial properties. The coating of metallic implants with hydroxyapatite (HA) remains to be the main proposal, but superior quality HA coatings with compositions closer to natural bone apatites, including carbonates, trace elements are required. Selenium is an essential nutrient in biological tissues and, at the same time, it also presents antibacterial properties. A pioneering study on the fabrication of selenium-doped carbonated hydroxyapatite (iHA:Se) coatings by Pulsed Laser Deposition (PLD) is presented. Different proportions of selenium were incorporated to obtain the iHA:Se coatings. Their physicochemical characterization, performed by SEM/EDS, FTIR, FT-Raman, Interferometric Profilometry and XPS, revealed typical columnar growth of HA in globular aggregates and the efficient incorporation of selenium into the HA coatings by the, most probably, substitution of SeO(3)(2-) groups in the CO(3)(2-) sites. Biological evaluation illustrated the absence of cytotoxicity when an amount of 0.6 at.% of Se was added to the iHA:Se coatings and excellent proliferation of the MC3T3-E1 preosteoblasts. Antibacterial properties were also proved with the inhibition of P. aeruginosa and S. aureus from establishing bacterial biofilms.

Three-dimensional bioactive glass implants fabricated by rapid prototyping based on CO2 laser cladding

Three-dimensional bioactive glass implants were produced by rapid prototyping based on laser cladding without using moulds. CO(2) laser radiation was employed to melt 45S5 and S520 bioactive glass particles and to deposit the material layer by layer following a desired geometry. Controlled thermal input and cooling rate by fine tuning of the processing parameters allowed the production of crack-free fully dense implants. Microstructural characterization revealed chemical composition stability, but crystallization during processing was extensive when 45S5 bioactive glass was used. Improved results were obtained using the S520 bioactive glass, which showed limited surface crystallization due to an expanded sintering window (the difference between the glass transition temperature and crystallization onset temperature). Ion release from the S520 implants in Tris buffer was similar to that of amorphous 45S5 bioactive glass prepared by casting in graphite moulds. Laser processed S520 scaffolds were not cytotoxic in vitro when osteoblast-like MC3T3-E1 cells were cultured with the dissolution products of the glasses; and the MC3T3-E1 cells attached and spread well when cultured on the surface of the materials.

Cytocompatibility of bio‐inspired silicon carbide ceramics

Due to its good mechanical and biochemical properties and, also, because of its unique interconnected porosity, bio-inspired silicon carbide (bioSiC) can be considered as a promising material for biomedical applications, including controlled drug delivery devices and tissue engineering scaffolds. This innovative material is produced by molten-Si infiltration of carbon templates, obtained by controlled pyrolysis of vegetable precursors. The final SiC ceramic presents a porous-interconnected microstructure that mimics the natural hierarchical structure of bone tissue and allows the internal growth of tissue, as well as favors angiogenesis. In the present work, the in vitro cytocompatibility of the bio-inspired SiC ceramics obtained, in this case, from the tree sapelli (Entandrophragma cylindricum) was evaluated. The attachment, spreading, cytoskeleton organization, proliferation, and mineralization of the preosteoblastic cell line MC3T3-E1 were analyzed for up to 28 days of incubation by scanning electron microscopy, interferometric profilometry, confocal laser scanning microscopy, MTT assay, as well as red alizarin staining and quantification. Cells seeded onto these ceramics were able to attach, spread, and proliferate properly with the maintenance of the typical preosteoblastic morphology throughout the time of culture. A certain level of mineralization on the surface of the sapelli-based SiC ceramics is observed. These results demonstrated the cytocompatibility of this porous and hierarchical material.

Osteoblast-like cell response to macro- and micro-patterned carbon scaffolds obtained from the sea rush Juncus maritimus

Carbon scaffolds with a directional patterned surface were obtained by pyrolysis of the sea rush Juncus maritimus. The structure of the scaffolds was investigated using scanning electron microscopy, mercury porosimetry and interferometric profilometry. X-ray diffraction and x-ray fluorescence were the techniques used for their chemical characterization. The alignment and differentiation of pre-osteoblasts (MC3T3-E1 cell line) incubated on the patterned scaffolds were evaluated by scanning electron microscopy, confocal laser scanning microscopy and by the quantification of the phosphatase alkaline activity and the osteocalcin synthesis. It was found that pyrolysis at 500 °C preserved and even enhanced the natural macro- and micro-patterning of the plant. The results obtained for porosity and chemical composition validated these structures as viable scaffolds for tissue engineering applications. Finally, the patterned surface was confirmed to promote the oriented growth of the pre-osteoblasts MC3T3-E1, not only after short periods of incubation (hours) but also after longer ones (several weeks). The quantification of the cell differentiation markers together with the evaluation of the cell layer morphology up to 28 days of incubation confirmed the differentiation of MC3T3-E1 cells to osteoblasts.

In vitro response of pre-osteoblastic cells to laser microgrooved PEEK

Polyetheretherketone (PEEK) is currently being used in implants as an alternative to titanium, due to its mechanical properties, cytocompatibility and inertness. Several studies have demonstrated that certain patterning on the implants promotes the oriented cell growth of osteoblasts, favouring the formation of bone tissue. This patterning improves the implants osteointegration in the bone and its mechanical stability. Therefore, the objective of this work is to micro-structure PEEK by laser radiation and to carry out an exhaustive study of the orientation of pre-osteoblast cells that grow on this material. Parallel microgrooves were obtained using an ArF excimer laser coupled with a mask projection unit with distances of 25, 50, 75 and 100 µm between grooves. The cell growth on these PEEK surfaces was studied, in order to compare the effect of different distances between grooves on the biological response of MC3T3-E1 pre-osteoblastic cells. Preferential cell orientation was observed for all studied distances, which was more pronounced in the 25 and 50 µm ones.

Human mesenchymal stem cells response to multi-doped silicon-strontium calcium phosphate coatings

The search for apatitic calcium phosphate coatings to improve implants osteointegration is, nowadays, preferentially focused in the obtaining of compositions closer to that of the inorganic phase of bone. Silicon and strontium are both present in trace concentrations in natural bone and have been demonstrated, by separate, to significantly improve osteoblastic response on calcium phosphate bioceramics. This work aims the controlled and simultaneous multi-doping of carbonated calcium phosphate coatings with both elements, Si and Sr, by pulsed laser deposition technique and the biological response of human mesenchymal stem cells to them. A complete physicochemical characterization has been also performed to analyze the coatings and significant positive effect was obtained at the osteogenic differentiation of cells, confirming the enormous potential of this multi-doping coating approach.

Marine Precursors-Based Biomorphic SiC Ceramics

Biomorphic silicon carbide ceramics is very promising as a natural base material for biomedical applications due to their excellent mechanical-biochemical properties and biocompatible behaviour. This innovative material is produced by molten-Si infiltration of carbon templates obtained by controlled pyrolysis of biological precursors. The final product is a light, tough and high-strength material with predictable microstructure. In this study the possibility to produce biomorphic silicon carbide ceramics using marine precursors is demonstrated. Due to the great biodiversity offered by the marine medium, a previous selection of algae (Laminaria ochroleuca Bachelot de la Pylaie, Undaria pinnatifida (Harvey) Suringar, Saccorhiza polyschides (Lightfoot) Batters and Cystoseira baccata (Gmelin) Silva) and marine plants (Zostera marina L. and Juncus maritimus L.) was carried out, taking into account its microstructure, porosity and interconnectivity of each species. The bioceramization process was evaluated in three phases: original material analysis, pyrolysis process and reactive melt Si-infiltration. For each marine precursor, a detailed study by Scanning Electron Microscopy (SEM) of the natural material, the carbon preform and the final SiC biomorphic product is described. The viability to obtain biomorphic SiC ceramic material for all the selected marine precursors is discussed.

The improved biological response of shark tooth bioapatites in a comparative in vitro study with synthetic and bovine bone grafts.

Autologous bone is considered to be the gold standard for bone tissue regeneration, providing more highly efficient functional responses compared to synthetic materials, and avoiding the rejection risks of allogenic grafts. However, it presents limitations for certain types of surgery due to its high resorption levels and donor site morbidity. Different biphasic synthetic composites, based onnon-apatitic calcium phosphates enriched with apatitic phases-such as hydroxyapatite, and bioderived bone grafts of bovine and porcine origin-are proposed as lower resorption materials due to their higher crystalline structure. The present work proposes two new sources of bioapatites for bone filler applications obtained from the dentine and enameloid of shark teeth, respectively. These bioapatites each present a characteristic apatite-based composition and additional enrichments of specific trace elements, such as magnesium and fluorine, with proven roles in bone metabolism. Their processing and physicochemical characterization (SEM, FT-Raman and XRD) is presented, together with an in vitro evaluation of osteogenic activity compared to a commercial bovine mineralized matrix and synthetic HA/β TCP grafts. The results proved the globular morphology (0.5-1.5 μm) and porosity (~50 μm and ~0.5-1 μm) of shark dentine bioapatites with biphasic composition: apatitic (hydroxyapatite and apatite-(CaF)), non-apatitic (whitlockite), and an apatitic phase (fluorapatite), organized in oriented crystals in enameloid bioapatites. An evaluation of the pre-osteoblast MC3T3-E1 morphology revealed the colonization of pores in dentine bioapatites and an aligned cell growth in the oriented enameloid crystals. A higher proliferation (p  <  0.01) was detected at up to 21 d in both the shark bioapatites and synthetic biphasic graft with respect to the bovine mineralized matrix. Finally, the great potential of porous biphasic dentine bioapatites enriched with Mg and the aligned fluorapatite crystals of enameloid bioapatites in promoting greater osteogenic activity was confirmed with a significantly increased ALP synthesis (p  <  0.01) compared to the commercial grafts.

Bioceramic Coatings on Biomorphic SiC by Electrophoretic Deposition

In this research, bioactive glass powders were electrophoretically deposited on biomorphic SiC ceramic substrates. A post-deposition thermal treatment was carried out to improve the properties of the coatings. Particle size, surface morphology, composition and thickness of the coatings have been studied by Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS) and interferometric profilometry respectively. The analysis demonstrated that the electrophoresis parameters, such as the voltage, the distance between the electrodes and the deposition time, play an important role on the thickness of the coatings. The post-deposition thermal treatment produces glass particles cohesion and leads to obtaining a homogeneous microstructure. The excellent coverage of the porous SiC surface morphology is also demonstrated. Finally, in order to assess the bioactive character of the glass coatings, in vitro test by immersion in simulated body fluid (SBF) was carried out.

Ex vivo analysis of the oral epithelium by high-wavenumber Raman spectroscopy

Raman spectroscopy at wavenumber region of 400-1800 cm−1 is an already proved promising technology for objective clinical evaluation of biological tissues to obtain their molecular fingerprint. The study of the high-wavenumber region (2800-3100 cm−1) is also of interest to significantly shorten the diagnosis time due to the intense Raman signal in a narrower range. This work studies parameters dependence, Raman saturation in depth and repeatability of tissue measurements at this region and proposes three indices for quantitative diagnose in oral epithelium. Sixteen oral mucosa biopsies in different thicknesses were evaluated in varying laser powers. The non-saturation of Raman signal up to 120 μm in depth was demonstrated. Three peaks (2874, 2926, 3056 cm−1) attributed to functional groups of interest in oral mucosa were identified from the Raman band deconvolution. The high-wavenumber correspondence with the fingerprint region (400-1800 cm−1) was proven and three diagnose indices proposed to quantitatively evaluate oral epithelium tissue in routinely clinical practice.