Publications

Very recently, the controlled release of dopamine (DA), a neurotransmitter whose deficiency is associated with Parkinson's disease, has been postulated as a good alternative to the oral administration of levodopa (L-Dopa), a dopamine precursor, to combat the effects of said disease. However, this is still a very little explored field and there are very few carriers that are capable of releasing DA, a small and water-soluble molecule, in an efficient and controlled manner. In this work, we report a carrier based on a conductive hydrogel capable of loading DA and releasing it progressively and efficiently (100 % release) in a period of five days by applying small electrical stimuli (-0.4 V) daily for a short time (1 min). The hydrogel (CMC/PEDOT), which is electrically active, has been prepared from sodium carboxymethylcellulose and poly(3,4-ethylenedioxythiophene) microparticles, using citric acid as a cross-linking agent. Furthermore, the results have shown that when relatively hydrophobic small molecules, such as chloramphenicol, are loaded, the electrostimulated release is significantly less efficient, demonstrating the usefulness of CMC/PEDOT as a carrier for neurotransmitters.

JTD Keywords: Amines, Carboxymethyl cellulose, Carboxymethylcellulose, Conducting hydrogels, Conducting polymers, Controlled release, Crosslinking, Dopamine, Drug-delivery system, Electrostimulation, Hydrogels, Joining, Levodopa, Loading, Molecules, Neurophysiology, Neurotransmitter release, Neurotransmitters release, Oral administration, Parkinson's disease, Parkinsons-disease, Poly(3,4-ethylenedioxythiophene), Release, Sodium, Transport, Water-soluble molecule

There is a growing interest in developing natural hydrogel-based scaffolds to culture cells in a three-dimensional (3D) millieu that better mimics the in vivo cells' microenvironment. A promising approach is to use hydrogels from animal tissues, such as decellularized extracellular matrices; however, they usually exhibit suboptimal mechanical properties compared to native tissue and their composition with hundreds of different protein complicates to elucidate which stimulus triggers cell's responses. As simpler scaffolds, type I collagen hydrogels are used to study cell behavior in mechanobiology even though they are also softer than native tissues. In this work, type I collagen is mixed with bacterial nanocellulose fibers (BCf) to develop reinforced scaffolds with mechanical properties suitable for 3D cell culture. BCf were produced from blended pellicles biosynthesized from Komagataeibacter xylinus. Then, BCf were mixed with concentrated collagen from rat-tail tendons to form composite hydrogels. Confocal laser scanning microscopy and scanning electron microscopy images confirmed the homogeneous macro- and microdistribution of both natural polymers. Porosity analysis confirmed that BCf do not disrupt the scaffold structure. Tensile strength and rheology measurements demonstrated the reinforcement action of BCf (43% increased stiffness) compared to the collagen hydrogel while maintaining the same viscoelastic response. Additionally, this reinforcement of collagen hydrogels with BCf offers the possibility to mix cells before gelation and then proceed to the culture of the 3D cell scaffolds. We obtained scaffolds with human bone marrow-derived mesenchymal stromal cells or human fibroblasts within the composite hydrogels, allowing a homogeneous 3D viable culture for at least 7 days. A smaller surface shrinkage in the reinforced hydrogels compared to type I collagen hydrogels confirmed the strengthening of the composite hydrogels. These collagen hydrogels reinforced with BCf might emerge as a promising platform for 3D in vitro organ modeling, tissue-engineering applications, and suitable to conduct fundamental mechanobiology studies.

JTD Keywords: 3d cell culture, bacterial cellulose, collagen, composite hydrogels, 3d cell culture, Bacterial cellulose, Cellulose/collagen composite, Collagen, Composite hydrogels, Contraction, Cross-linking, Cytocompatibility, Fibroblasts, Matrix, Mechanical-properties, Reinforcement, Stiffness, Tissue engineering

The mutual interaction between reactive species generated by non-thermal plasma and biopolymers in solution causes oxidative modifications that can have an impact in biomedical applications.

JTD Keywords: atmospheric plasma, cellulose, dftb3, gas, oxidation, parameterization, simulations, water, Biopolymers, Hydrogen peroxide, Molecular dynamics simulation, Molecular-dynamics, Nitrites, Reactive oxygen species

The low endogenous regenerative capacity of the heart,added tothe prevalence of cardiovascular diseases, triggered the advent ofcardiac tissue engineering in the last decades. The myocardial nicheplays a critical role in directing the function and fate of cardiomyocytes;therefore, engineering a biomimetic scaffold holds excellent promise.We produced an electroconductive cardiac patch of bacterial nanocellulose(BC) with polypyrrole nanoparticles (Ppy NPs) to mimic the naturalmyocardial microenvironment. BC offers a 3D interconnected fiber structurewith high flexibility, which is ideal for hosting Ppy nanoparticles.BC-Ppy composites were produced by decorating the network of BC fibers(65 & PLUSMN; 12 nm) with conductive Ppy nanoparticles (83 & PLUSMN; 8 nm).Ppy NPs effectively augment the conductivity, surface roughness, andthickness of BC composites despite reducing scaffolds' transparency.BC-Ppy composites were flexible (up to 10 mM Ppy), maintained theirintricate 3D extracellular matrix-like mesh structure in all Ppy concentrationstested, and displayed electrical conductivities in the range of nativecardiac tissue. Furthermore, these materials exhibit tensile strength,surface roughness, and wettability values appropriate for their finaluse as cardiac patches. In vitro experiments withcardiac fibroblasts and H9c2 cells confirmed the exceptional biocompatibilityof BC-Ppy composites. BC-Ppy scaffolds improved cell viability andattachment, promoting a desirable cardiomyoblast morphology. Biochemicalanalyses revealed that H9c2 cells showed different cardiomyocyte phenotypesand distinct levels of maturity depending on the amount of Ppy inthe substrate used. Specifically, the employment of BC-Ppy compositesdrives partial H9c2 differentiation toward a cardiomyocyte-like phenotype.The scaffolds increase the expression of functional cardiac markersin H9c2 cells, indicative of a higher differentiation efficiency,which is not observed with plain BC. Our results highlight the remarkablepotential use of BC-Ppy scaffolds as a cardiac patch in tissue regenerativetherapies.

JTD Keywords: bacterial nanocellulose, cardiac patches, conducting polymers, polypyrrole, Arrhythmias, Bacterial nanocellulose, Biomaterials, Cardiac patches, Cell therapy, Cellulose, Conductingpolymers, H9c2, In-vitro, Polymer, Polypyrrole, Scaffolds, Tissue, Tissue engineering, Viability

Local cancer treatment by in situ injections of thermo-responsive hydrogels (HG) offers several advantages over conventional systemic anti-cancer treatments. In this work, a biodegradable and multicompartmental HG composed of N-isopropylacrylamide, cellulose, citric acid, and ceric ammonium nitrate was developed for the controlled release of hydrophilic (doxorubicin) and hydrophobic (niclosamide) drugs. The formulation presented ideal properties regarding thermo-responsiveness, rheological behavior, drug release profile, biocompatibility, and biological activity in colon and ovarian cancer cells. Cellulose was found to retard drugs release rate, being only 4 % of doxorubicin and 30 % of niclosamide released after 1 week. This low release was sufficient to cause cell death in both cell lines. Moreover, HG demonstrated a proper injectability, in situ prevalence, and safety profile in vivo. Overall, the HG properties, together with its natural and eco-friendly composition, create a safe and efficient platform for the local treatment of non-resectable tumors or tumors requiring pre-surgical adjuvant therapy.

JTD Keywords: biodegradable, cellulose, controlled-release formulation, drug delivery systems, hydrogel, thermo-responsiveness, Ammonium-nitrate, Biodegradable, Cancer treatment, Cellulose, Controlled-release formulation, Delivery, Drug delivery systems, Hydrogel, Reduce, Thermo-responsiveness

Hydrogels have been recently proposed as suitable materials to generate reactive oxygen and nitrogen species (RONS) upon gas-plasma treatment, and postulated as promising alternatives to conventional cancer therapies. Acting as delivery vehicles that allow a controlled release of RONS to the diseased site, plasma-treated hydrogels can overcome some of the limitations presented by plasma-treated liquids in in vivo therapies. In this work, we optimized the composition of a methylcellulose (MC) hydrogel to confer it with the ability to form a gel at physiological temperatures while remaining in the liquid phase at room temperature to allow gas-plasma treatment with suitable formation of plasma-generated RONS. MC hydrogels demonstrated the capacity for generation, prolonged storage and release of RONS. This release induced cytotoxic effects on the osteosarcoma cancer cell line MG-63, reducing its cell viability in a dose-response manner. These promising results postulate plasma-treated thermosensitive hydrogels as good candidates to provide local anticancer therapies.

JTD Keywords: Case-control studies, Cellulose, Hydrogels, Methylcellulose, Phase-separation, Plasma gases, Reactive oxygen species, Stability, Substituent, Temperature, Thermoreversible gelation

Technologies to cryogenically preserve (a.k.a. cryopreserve) living tissue, cell lines and primary cells have matured greatly for both clinicians and researchers since their first demonstration in the 1950s and are widely used in storage and transport applications. Currently, however, there remains an absence of viable cryopreservation and thawing methods for bioengineered, three-dimensional (3D) cell models, including patients' samples. As a first step towards addressing this gap, we demonstrate a viable protocol for spheroid cryopreservation and survival based on a 3D carboxymethyl cellulose scaffold and precise conditions for freezing and thawing. The protocol is tested using hepatocytes, for which the scaffold provides both the 3D structure for cells to self-arrange into spheroids and to support cells during freezing for optimal post-thaw viability. Cell viability after thawing is improved compared to conventional pellet models where cells settle under gravity to form a pseudo-tissue before freezing. The technique may advance cryobiology and other applications that demand high-integrity transport of pre-assembled 3D models (from cell lines and in future cells from patients) between facilities, for example between medical practice, research and testing facilities.

JTD Keywords: 3d cell culture, biofabrication, biomaterials, carboxymethyl cellulose, cryopreservation, hepatocytes, 3d cell culture, Biofabrication, Biomaterials, Carboxymethyl cellulose, Cell culture techniques, three dimensional, Cell survival, Cryopreservation, Freezing, Hepatocytes, Humans, Prevention, Scaffolds, Spheroids

The limitations of obtaining pancreatic islets from differentsources as animal models or human donors complicate the study oftype 2 diabetes (T2D) in vitro. Immortalized cell lines as the in-sulin-producing INS1Eb-cells appeared as a valid alternative tomodel insulin-related diseases. The formation of 3D structures topromote cell aggregations from single cells is a handy tool togenerate resemblance islet-like pseudoislets. Traditionally usedhydrogel encapsulation methods induce a lack of nutrient and ox-ygen diffusion for pancreatic tissue engineering. Here, we usecryogelation technology to develop a more resemblance scaffoldwith the mechanical and physical properties needed to engineerpancreatic tissue. This study shows that carboxymethyl cellulose(CMC) cryogels prompted cells to generateb-cell clusters. Thehigh porosity achieved with this approach allowed us to createspecific range pseudoislets. However, gelatin-based scaffolds didnot induce this cell organization. Pseudoislets formed within CMC-scaffolds showed cell viability for up to 7 days and responded betterto the glucose over conventional monolayer cultures. Overall, ourresults demonstrate that CMC-scaffolds can be used to control theorganization and function of insulin-producingb-cells, represent-ing a suitable technique to generateb-cell clusters to study pan-creatic islet function.

JTD Keywords: Cellulose, Cryogel, Diabetes

In vitro research for the study of type 2 diabetes (T2D) is frequently limited by the availability of a functional model for islets of Langerhans. To overcome the limitations of obtaining pancreatic islets from different sources, such as animal models or human donors, immortalized cell lines as the insulin-producing INS1E beta-cells have appeared as a valid alternative to model insulin-related diseases. However, immortalized cell lines are mainly used in flat surfaces or monolayer distributions, not resembling the spheroid-like architecture of the pancreatic islets. To generate islet-like structures, the use of scaffolds appeared as a valid tool to promote cell aggregations. Traditionally-used hydrogel encapsulation methods do not accomplish all the requisites for pancreatic tissue engineering, as its poor nutrient and oxygen diffusion induces cell death. Here, we use cryogelation technology to develop a more resemblance scaffold with the mechanical and physical properties needed to engineer pancreatic tissue. This study shows that carboxymethyl cellulose (CMC) cryogels prompted cells to generate beta-cell clusters in comparison to gelatin-based scaffolds, that did not induce this cell organization. Moreover, the high porosity achieved with CMC cryogels allowed us to create specific range pseudoislets. Pseudoislets formed within CMC-scaffolds showed cell viability for up to 7 d and a better response to glucose over conventional monolayer cultures. Overall, our results demonstrate that CMC-scaffolds can be used to control the organization and function of insulin-producing beta-cells, representing a suitable technique to generate beta-cell clusters to study pancreatic islet function.

JTD Keywords: biomaterial, cryogel, pancreatic islets, scaffold, tissue engineering, ?-cell, Architecture, Beta-cell, Beta-cell heterogeneity, Biomaterial, Carboxymethyl cellulose, Cell culture, Cell death, Cell engineering, Cell organization, Cells, Cellulose, Cryogel, Cryogels, Cytoarchitecture, Delivery, Encapsulation methods, Gelation, Gene-expression, Immortalized cells, Insulin, Insulin secretory responses, Islets of langerhans, Mechanical and physical properties, Monolayer culture, Monolayers, Pancreatic islets, Pancreatic tissue, Pancreatic-islets, Proliferation, Scaffold, Scaffolds, Scaffolds (biology), Size, Tissue, Tissue engineering, Β-cell

© 2020 Elsevier B.V. Semi-interpenetrated nanogels (NGs) able to release and sense diclofenac (DIC) have been designed to act as photothermal agents with the possibility to ablate cancer cells using mild-temperatures (<45 °C). Combining mild heat treatments with simultaneous chemotherapy appears as a very promising therapeutic strategy to avoid heat resistance or damaging the surrounding tissues. Particularly, NGs consisted on a poly(N-isopropylacrylamide) (PNIPAM) and dendritic polyglycerol (dPG) mesh containing a semi-interpenetrating network (SIPN) of poly(hydroxymethyl 3,4-ethylenedioxythiophene) (PHMeEDOT). The PHMeEDOT acted as photothermal and conducting agent, while PNIPAM-dPG NG provided thermoresponsivity and acted as stabilizer. We studied how semi-interpenetration modified the physicochemical characteristics of the thermoresponsive SIPN NGs and selected the best condition to generate a multifunctional photothermal agent. The thermoswitchable conductiveness of the multifunctional NGs and the redox activity of DIC could be utilized for its electrochemical detection. Besides, as proof of the therapeutic concept, we investigated the combinatorial effect of photothermal therapy (PTT) and DIC treatment using the HeLa cancer cell line in vitro. Within 15 min NIR irradiation without surpassing 45 °C we were able to kill 95% of the cells, demonstrating the potential of SIPN NGs as drug carriers, sensors and agents for mild PTT.

JTD Keywords: cells, cellulose, conducting polymers, controlled delivery, diclofenac, efficiency, electrochemical oxidation, electrochemical sensors, nanogels, nanoparticles, photothermal therapy, pnipam, poly(3,4-ethylenedioxythiophene), Conducting polymers, Electrochemical sensors, Nanogels, Photothermal therapy