Abstract
The combination of marine origin biopolymers for tissue engineering (TE) applications is of high interest, due to their similarities with the proteins and polysaccharides present in the extracellular matrix of different human tissues. This manuscript reports on innovative collagen-chitosan-fucoidan cryogels formed by the simultaneous blending of these three marine polymers in a chemical-free crosslinking approach. The physicochemical characterization of marine biopolymers comprised FTIR, amino acid analysis, circular dichroism and SDS-PAGE, and suggested that the jellyfish collagen used in the cryogels was not denatured (preserved the triple helical structure) and had similarities with type II collagen. The chitosan presented a high deacetylation degree (90.1%) that can strongly influence the polymer physicochemical properties and biomaterial formation. By its turn, rheology, and SEM studies confirmed that these novel cryogels present interesting properties for TE purposes, such as effective blending of biopolymers without visible material segregation, mechanical stability (strong viscoelastic character), as well as adequate porosity to support cell proliferation and exchange of nutrients and waste products. Additionally,in vitrocellular assessments of all cryogel formulations revealed a non-cytotoxic behavior. The MTS test, live/dead assay and cell morphology assessment (phalloidin DAPI) showed that cryogels can provide a proper microenvironment for cell culturing, supporting cell viability and promoting cell proliferation. Overall, the obtained results suggest that the novel collagen-chitosan-fucoidan cryogels herein presented are promising scaffolds envisaging tissue engineering purposes, as both acellular biomaterials or cell-laden cryogels.

Abstract

Abstract
Marine origin polymers represent a sustainable and natural alternative to mammal counterparts regarding the biomedical application due to their similarities with proteins and polysaccharides present in extracellular matrix (ECM) in humans and can reduce the risks associated with zoonosis and overcoming social- and religious-related constraints. In particular, collagen-based biomaterials have been widely explored in tissue engineering scaffolding applications, where cryogels are of particular interest as low temperature avoids protein denaturation. However, little is known about the influence of the parameters regarding their behavior, i.e., how they can influence each other toward improving their physical and chemical properties. Factorial design of experiments (DoE) and response surface methodology (RSM) emerge as tools to overcome these difficulties, which are statistical tools to find the most influential parameter and optimize processes. In this work, we hypothesized that a design of experiments (DoE) model would be able to support the optimization of the collagen-chitosan-fucoidan cryogel manufacturing. Therefore, the parameters temperature (A), collagen concentration (B), and fucoidan concentration (C) were carefully considered to be applied to the Box-Behnken design (three factors and three levels). Data obtained on rheological oscillatory measurements, as well as on the evaluation of antioxidant concentration and adenosine triphosphate (ATP) concentration, showed that fucoidan concentration could significantly influence collagen-chitosan-fucoidan cryogel formation, creating a stable internal polymeric network promoted by ionic crosslinking bonds. Additionally, the effect of temperature significantly contributed to rheological oscillatory properties. Overall, the condition that allowed us to have better results, from an optimization point of view according to the DoE, were the gels produced at -80 degrees C and composed of 5% of collagen, 3% of chitosan, and 10% fucoidan. Therefore, the proposed DoE model was considered suitable for predicting the best parameter combinations needed to develop these cryogels.

Abstract
Cartilage repair after a trauma or a degenerative disease like osteoarthritis (OA) continues to be a big challenge in current medicine due to the limited self-regenerative capacity of the articular cartilage tissues. To overcome the current limitations, tissue engineering and regenerative medicine (TERM) and adjacent areas have focused their efforts on new therapeutical procedures and materials capable of restoring normal tissue functionalities through polymeric scaffolding and stem cell engineering approaches. For this, the sustainable exploration of marine origin materials has emerged in the last years as a natural alternative to mammal sources, benefiting from their biological properties (e.g., biocompatibility, biodegradability, no toxicity, among others) for the develop-ment of several types of scaffolds. In this study, marine collagen(jCOL)-chitosan(sCHT)-fucoidan(aFUC)/ chondroitin sulfate(aCS) were cryo-processed (-20 degrees C,-80 degrees C, and-196 degrees C) and a chemical-free cross -linking approach was explored to establish cohesive and stable cryogel materials. The cryogels were intensively characterized to assess their oscillatory behavior, thermal structural stability, thixotropic properties (around 45 % for the best formulations), injectability, and surface structural organization. Additionally, the cryogels demonstrate an interesting microenvironment in in vitro studies using human adipose-derived stem cells (hASCs), supporting their viability and proliferation. In both physic-chemical and in vitro studies, the systems that contain fucoidan in their formulations, i.e., C1 (jCOL, sCHT, aFUC) and C3 (jCOL, sCHT, aFUC, aCS), submitted at-80 degrees C, are those that demonstrated most promising results for future application in articular cartilage tissues.

Abstract
In the recent decade, marine origin products have been growingly studied as building blocks complying with the constant demand of the biomedical sector regarding the development of new devices for Tissue Engineering and Regenerative Medicine (TERM). In this work, several combinations of marine collagen-chitosan-fucoidan hydrogel were formed using a newly developed eco-friendly compressive and absorption methodology to produce hydrogels (CAMPH), which consists of compacting the biopolymers solution while removing the excess of water. The hydrogel formulations were prepared by blending solutions of 5% collagen from jellyfish and/or 3% collagen from blue shark skin, with solutions of 3% chitosan from squid pens and solutions of 10% fucoidan from brown algae, at different ratios. The biopolymer physico-chemical characterization comprised Amino Acid analysis, ATR-FTIR, CD, SDS-PAGE, ICP, XRD, and the results suggested the shark/jellyfish collagen(s) conserved the triple helical structure and had similarities with type I and type II collagen, respectively. The studied collagens also contain a denaturation temperature of around 30-32 degrees C and a molecular weight between 120 and 125 kDa. Additionally, the hydrogel properties were determined by rheology, water uptake ability, degradation rate, and SEM, and the results showed that all formulations had interesting mechanical (strong viscoelastic character) and structural stability properties, with a significant positive highlight in the formulation of H-3 (blending all biopolymers, i.e., 5% collagen from jellyfish, 3% collagen from skin shark, 3% chitosan and 10% of fucoidan) in the degradation test, that shows a mass loss around 18% over the 30 days, while the H-1 and H-2, present a mass loss of around 35% and 44%, respectively. Additionally, the in vitro cellular assessments using chondrocyte cells (ATDC5) in encapsulated state revealed, for all hydrogel formulations, a non-cytotoxic behavior. Furthermore, Live/Dead assay and Phalloidin/DAPI staining, to assess the cytoskeletal organization, proved that the hydrogels can provide a suitable microenvironment for cell adhesion, viability, and proliferation, after being encapsulated. Overall, the results show that all marine collagen (jellyfish/shark)-chitosan-fucoidan hydrogel formulations provide a good structural architecture and microenvironment, highlighting the H-3 biomaterial due to containing more polymers in their composition, making it suitable for biomedical articular cartilage therapies.

Abstract
Miniaturized three-electrode electrochemical sensors (MES) are widely used in the advancement of innovative technologies for remote sensing applications. MESs consist of conductive electrodes that are applied onto an inert solid substrate using various techniques, such as photolithography, electroplating, and screen printing. Typical MES systems comprise working (WE) and counter (CE) electrodes based on gold (Au), paired with a reference electrode (RE) based on silver (Ag). This configuration is commonly selected due to Au's high conductivity, low resistance, and compatibility with robust organothiol chemistries, especially for the WE. Moreover, Ag is often preferred for REs owing to its low toxicity, stability, and high conductivity. Nevertheless, in uncontrolled environments outside of cleanrooms, both Au and Ag surfaces are prone to atmospheric contamination, resulting in significant sensor variability and compromised analytical performance. Therefore, it is crucial to integrate a pre-processing stage into the sensor manufacturing process to guarantee the quality and cleanliness of MES electrode surfaces for sensor functionalization and precise electrochemical measurements. Considering the potential negative effects of methods tailored for a specific electrode material on another material, this study extensively investigates 18 different treatment methods for MESs incorporating Au CEs and WEs, along with Ag REs. Employing a multi-parametric analysis, this study aims to identify the most effective treatment for a variety of electrode materials, thereby improving analytical accuracy and reproducibility for subsequent MES (bio)sensor applications. Miniaturized three-electrode electrochemical sensors (MES) are essential for advancing remote sensing technologies. However, the inherent morpho-chemical heterogeneity of built-in electrodes challenges MES analytical performance. This study investigates treatments for the different electrode materials, providing new methods to enhance quality control, analytical accuracy, and reproducibility in MES biosensing applications. image