Functionalized biomaterials for tissue engineering. Biomateriales funcionalizados para ingeniería de tejidos

Tesis de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular, leída el 14-12-2018La ingeniería de tejidos ha establecido un nuevo paradigma en la medicina: las propiedades de los biomateriales modernos han de cumplir diversas funciones relacionadas con las necesidades de la sociedad, las cuales varían según los grupos de edad específicos y las distintas enfermedades de los pacientes. La revisión bibliográfica realizada en la Introducción de esta Tesis Doctoral aborda los mecanismos de regeneración ósea y revela las estrategias que han sido empleadas en el pasado por varias generaciones de biomateriales. Además, se analizan tanto los materiales tradicionales como los recientemente desarrollados para su aplicación como implantes permanentes y temporales. La modificación de la superficie de estos implantes se ha confirmado como la principal estrategia aplicada para la funcionalización de biomateriales. Por ello, se discuten las técnicas más importantes que conducen a la modificación simultánea de la composición y la topografía de la superficie de los materiales base. Asimismo, se revisan los avances en la generación de biomateriales híbridos a través de la combinación de materiales tratados superficialmente con la adición de capas poliméricas y se relacionan con la ingeniería de monocapas celulares..Tissue engineering has introduced a new paradigm in medical care: the properties of modern biomaterials have to address multiple functions related to the needs of specific age groups of the patients and different diseases. The literature review carried out in the Introduction of the Thesis approaches the specificities of bone regeneration mechanisms and discloses how these have been addressed in the past by several generations of biomaterials. Both the traditional and newly developed materials for permanent and temporary implant applications are reviewed. Surface modification is revealed as the main strategy employed for functionalization of biomaterials. The most important surface modification techniques that lead to simultaneous modification of the composition and topography of the surface of the core materials are discussed. Advances in hybridization of biomaterials via combination of surface treated core materials with polymeric top-coat systems and cell sheet engineering are reviewed...Sección Deptal. de Bioquímica y Biología Molecular (Biológicas)Fac. de Ciencias BiológicasTRUEunpu

Dual-labeled nanoparticles based on small extracellular vesicles for tumor detection.

Small extracellular vesicles (sEVs) are emerging natural nanoplatforms in cancer diagnosis and therapy, through the incorporation of signal components or drugs in their structure. However, for their translation into the clinical field, there is still a lack of tools that enable a deeper understanding of their in vivo pharmacokinetics or their interactions with the cells of the tumor microenvironment. In this study, we have designed a dual-sEV probe based on radioactive and fluorescent labeling of goat milk sEVs. The imaging nanoprobe was tested in vitro and in vivo in a model of glioblastoma. In vitro assessment of the uptake of the dual probe in different cell populations (RAW 264.7, U87, and HeLa) by optical and nuclear techniques (gamma counter, confocal imaging, and flow cytometry) revealed the highest uptake in inflammatory cells (RAW 264.7), followed by glioblastoma U87 cells. In vivo evaluation of the pharmacokinetic properties of nanoparticles confirmed a blood circulation time of ~ 8 h and primarily hepatobiliary elimination. The diagnostic capability of the dual nanoprobe was confirmed in vivo in a glioblastoma xenograft model, which showed intense in vivo uptake of the SEV-based probe in tumor tissue. Histological assessment by confocal imaging enabled quantification of tumor populations and confirmed uptake in tumor cells and tumor-associated macrophages, followed by cancer-associated fibroblasts and endothelial cells. We have developed a chemical approach for dual radioactive and fluorescent labeling of sEVs. This methodology enables in vivo and in vitro study of these vesicles after exogenous administration. The dual nanoprobe would be a promising technology for cancer diagnosis and a powerful tool for studying the biological behavior of these nanosystems for use in drug delivery.This study was supported by Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, project “PI20/01632” and “PT20/00044”, co-funded by the European Regional Development Fund (ERDF), “A way of making Europe”, by Comunidad de Madrid, project “Y2018/NMT-4949 (NanoLiver-CM)” and “S2017/ BMD-3867 (RENIM-CM)”, co-funded by the European Structural and Invest‑ ment Fund, and by Agencia Estatal de Investigación “PID2019‐110369RB‐I00/ AEI/https://doi.org/10.13039/501100011033” grant. This work was also sup‑ ported by “Diagnosis and treatment follow‐up of severe Staphylococcal infec‑ tions with anti‐Staphylococcal antibodies and immune‐PET ‐ Grant Fundación BBVA a Equipos de Investigación Científca 2018 and Ramon Areces Grant “Imagen molecular de la infección por Clostridiodes difcile”. Grant EQC2019006674-P funded by MCIN/AEI /https://doi.org/10.13039/501100011033 and by “ERDF A way of making Europe”. A. Santos-Coquillat is grateful for fnancial support from Consejería de Educación e Investigación Comunidad de Madrid, co-fnanced by European Social Fund (ESF) grant PEJD-2018-POST/BMD-9592 and the Sara Borrell Fellowship from Ministerio de Ciencia e Innovación, Insti‑ tuto de Salud Carlos III grant CD19/00136. M.I. González is funded by Instituto de Investigación Sanitaria Gregorio Marañón, Intramural Programme for the Promotion of R&D&I 2021, Sub-programme "Predoctoral training contract".S

Covalently Labeled Fluorescent Exosomes for In Vitro and In Vivo Applications.

The vertiginous increase in the use of extracellular vesicles and especially exosomes for therapeutic applications highlights the necessity of advanced techniques for gaining a deeper knowledge of their pharmacological properties. Herein, we report a novel chemical approach for the robust attachment of commercial fluorescent dyes to the exosome surface with covalent binding. The applicability of the methodology was tested on milk and cancer cell-derived exosomes (from U87 and B16F10 cancer cells). We demonstrated that fluorescent labeling did not modify the original physicochemical properties of exosomes. We tested this nanoprobe in cell cultures and healthy mice to validate its use for in vitro and in vivo applications. We confirmed that these fluorescently labeled exosomes could be successfully visualized with optical imaging.This study was supported by the Comunidad de Madrid, projects: “Y2018/NMT-4949 (NanoLiver-CM)” and “S2017/BMD-3867 (RENIM-CM)”; it was also co-funded by the European Structural and Investment Fund. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation, and it is a Severo Ochoa Center of Excellence (SEV-2015-0505). JV was supported by grants from Instituto de Salud Carlos III (PI18/01833), co-funded by European Regional Development Fund (ERDF) and from Comunidad de Madrid, project “S2017/BMD2737 (ExoHep-CM)”, co-funded by European Structural and Investment Fund. A. Santos-Coquillat is grateful for the financial support from Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III Sara Borrell Fellowship grant CD19/00136.S

Goat milk exosomes as natural nanoparticles for detecting inflammatory processes by optical imaging

Exosomes are cell-derived nanovesicles with a proven intercellular signaling role in inflammation processes and immune response. Due to their natural origin and liposome-like structure, these nanometer-scale vesicles have emerged as novel platforms for therapy and diagnosis. In this work, goat milk exosomes are isolated and fully characterized in terms of their physicochemical properties, proteomics, and biochemical profile in healthy mice, and used to detect inflammatory processes by optical imaging. For the in vitro and in vivo experiments, the exosomes are covalently labeled with the commercial fluorophores sulfo-Cyanine 5 and BODIPY-FL to create nanoprobes. In vitro studies using confocal imaging, flow cytometry, and colorimetric assays confirm the internalization of the nanoprobes as well their lack of cytotoxicity in macrophage populations RAW 264.7. Optical imaging in the mouse peritoneal region confirms the in vivo ability of one of the nanoprobes to localize inflammatory processes. In vivo imaging shows exosome uptake in the inflamed peritoneal region, and flow-cytometric analysis of peritonitis exudates confirms the uptake by macrophage and neutrophil populations. These results support the promising use of goat milk exosomes as natural probes in the detection of inflammatory processes.This study has been funded by Instituto de Salud Carlos III, through the project "PI20/01632", co-funded by European Regional Development Fund (ERDF), "A way to make Europe" and by Comunidad de Madrid, project "Y2018/NMT-4949 (NanoLiver-CM)" and "S2017/BMD-3867 (RENIM-CM)", co-funded by European Structural and Investment Fund. This work has been also supported by "Diagnosis and treatment follow-up of severe Staphylococcal Infections with Anti-Staphylococcal antibodies and Immune-PET - Grant Fundación BBVA a Equipos de Investigación Científica 2018". A. Santos-Coquillat is grateful for financial support to Consejería de Educación e Investigación, co-financed by European Social Fund (ESF) grant PEJD-2018-POST/BMD-9592. A. Santos Coquillat is also funded by Instituto de Salud Carlos III, co-funded by European Social Fund "Investing in your future" (grant CD19/00136). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation. The CNIO Proteomics Unit is funded by the H2020 project EPIC-XS (ref. 823839). Biomedical Imaging has been conducted at the Advanced Imaging Unit of the CNIC (Centro Nacional de Investigaciones Cardiovasculares Carlos III), Madrid, Spain

Encapsulated mesenchymal stromal cells as cyclic providers of immunomodulatory secretomes: a living on-demand delivery system

The stimulation of mesenchymal stromal cells (MSCs) with inflammatory molecules is often used to boost their therapeutic effect. Prolonged exposure to inflammatory molecules has been explored to improve their action because MSCs therapies seem to be improved transiently with such stimuli. However, the possibility of cyclically stimulating MSCs to recover their optimized therapeutic potential is still to be elucidated, although the efficacy of cell-based therapies may be dependent on the ability to readapt to the relapse pathological conditions. Here, the response of MSCs, encapsulated in alginate hydrogels and cultured for 22 d, is explored using three different regimes: single, continuous, and intermittent stimulation with IFNγ. Exposure to IFNγ leads to a decrease in the secretion of IL-10, which is cyclically countered by IFNγ weaning. Conditioned media collected at different stages of pulsatile stimulation show an immunomodulatory potential toward macrophages, which directly correlates with IL-10 concentration in media. To understand whether the correlation between cyclic stimulation of MSCs and other biological actions can be observed, the effect on endothelial cells is studied, showcasing an overall modest influence on tube formation. Overall, the results describe the response of encapsulated MSCs to unusual pulsatile simulation regimens, exploring encapsulated MSCs as a living on-demand release system of tailored secretomes with recoverable immunomodulatory action.publishe

In vitro and in vivo characterization of anodised zirconium as a potential material for biomedical applications

In vitro studies offer the insights for the understanding of the mechanisms at the tissue–implant interface that will provide an effective functioning in vivo. The good biocompatibility of zirconium makes a good candidate for biomedical applications and the attractive in vivo performance is mainly due to the presence of a protective oxide layer. The aim of this study is to evaluate by in vitro and in vivo approach, the influence of surface modification achieved by anodisation at 30 and 60 V on zirconium implants on the first steps of the osseointegration process. In this study cell attachment, proliferation and morphology of mouse myoblast C2C12-GFP and in mouse osteoprogenitor MC3T3-E1 cells was evaluated. Also, together with the immune system response, osteoclast differentiation and morphology with RAW 264.7 murine cell line were analysed. It was found that anodisation treatment at 60 V enhanced cell spreading and the osteoblastic and osteoclastic cells morphology, showing a strong dependence on the surface characteristics. In vivo tests were performed in a rat femur osteotomy model. Dynamical and static histological and histomorphometric analyses were developed 15 and 30 days after surgery. Newly formed bone around Zr60V implants showed a continuous newly compact and homogeneous bone just 15 after surgery, as judged by the enhanced thickness and mineralization rate. The results indicate that anodising treatment at 60 V could be an effective improvement in the osseointegration of zirconium by stimulating adhesion, proliferation, morphology, new bone thickness and bone mineral apposition, making zirconium an emerging candidate material for biomedical applications.Fil: Katunar, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Gomez Sanchez, Andrea Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Santos Coquillat, Ana. Universidad Complutense de Madrid; EspañaFil: Civantos, Ana. Instituto de Ciencia y Tecnología de Polímeros; EspañaFil: Martinez Campos, Enrique. Universidad Complutense de Madrid; EspañaFil: Ballarre, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Vico, Tamara Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Baca, Matías. Hospital Interzonal General de Agudos “Oscar Alende”; ArgentinaFil: Ramos, Viviana. Instituto de Ciencia y Tecnología de Polímeros; EspañaFil: Cere, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentin

Thermosensitive hydrogel platforms with modulated ionic load for optimal cell sheet harvesting

Tissue engineering and regenerative medicine demand affordable and robust technologies for cell manipulation that are capable of culturing cells and collecting them by gentle cell detachment. Nowadays, technologies able to detach and transplant cell monolayers in a controlled and complete manner are particularly relevant. In this work, the capability of vinyl-caprolactam-based hydrogels functionalized with different ionic groups (anionic, cationic or two types of zwitterions) to support mouse endothelial C166-GFP cell growth until confluence and to allow for subsequent cell and cell sheet detachment using thermal stimuli is shown. These hydrogel-type supports, which are robust and easy to ‘handle’ and maneuver, are obtained in a simple, one step radical photopolymerization procedure that requires two types of cross-linkers. For comparison reasons, these formulations were studied together with a neutral hydrogel. Nature and type of charge were shown to greatly influence cell growth. Furthermore, only the neutral and zwitterionic hydrogels exhibited excellent detachment efficiency upon decrease of temperature in spite of the capability of most of the hydrogels to form cell monolayers. The ability of cell sheet detachment was related to the extent of thermosensitivity. The use of hydrogels allowed for transplanting the cells without the need of a superstrate and did not show a thin-thickness requirement, which are usual limitations for the use of poly-N-isopropylacrylamide (pNIPAm) supports, such as commercial grafts. The whole family may actually be considered as a candidate to compete with the expensive and technologically complex thermoresponsive cell platforms based on those pNIPAm grafts.The authors gratefully acknowledge support from the Consejo Superior de Investigaciones Científicas (CSIC). Equally, this work was financially supported by the Ministerio de Economía y Competitividad (MINECO) through MAT2013-42957-R, MAT2016-78437-R.Peer Reviewe

Toward Cell Selective Surfaces: Cell Adhesion and Proliferation on Breath Figures with Antifouling Surface Chemistry

We report the preparation of microporous functional polymer surfaces that have been proven to be selective surfaces toward eukaryotic cells while maintaining antifouling properties against bacteria. The fabrication of functional porous films has been carried out by the breath figures approach that allowed us to create porous interfaces with either poly­(ethylene glycol) methyl ether methacrylate (PEGMA) or 2,3,4,5,6-pentafluorostyrene (5FS). For this purpose, blends of block copolymers in a polystyrene homopolymer matrix have been employed. In contrast to the case of single functional polymer, using blends enables us to vary the chemical distribution of the functional groups inside and outside the formed pores. In particular, fluorinated groups were positioned at the edges while the hydrophilic PEGMA groups were selectively located inside the pores, as demonstrated by TOF-SIMS. More interestingly, studies of cell adhesion, growth, and proliferation on these surfaces confirmed that PEGMA functionalized interfaces are excellent candidates to selectively allow cell growth and proliferation while maintaining antifouling properties