Green approaches for extraction, chemical modification and processing of marine polysaccharides for biomedical applications.

Over the past few decades, natural-origin polysaccharides have received increasing attention across different fields of application, including biomedicine and biotechnology, because of their specific physicochemical and biological properties that have afforded the fabrication of a plethora of multifunctional devices for healthcare applications. More recently, marine raw materials from fisheries and aquaculture have emerged as a highly sustainable approach to convert marine biomass into added-value polysaccharides for human benefit. Nowadays, significant efforts have been made to combine such circular bio-based approach with cost-effective and environmentally-friendly technologies that enable the isolation of marine-origin polysaccharides up to the final construction of a biomedical device, thus developing an entirely sustainable pipeline. In this regard, the present review intends to provide an up-to-date outlook on the current green extraction methodologies of marine-origin polysaccharides and their molecular engineering toolbox for designing a multitude of biomaterial platforms for healthcare. Furthermore, we discuss how to foster circular bio-based approaches to pursue the further development of added-value biomedical devices, while preserving the marine ecosystem.

Liquefied Microcapsules Compartmentalizing Macrophages and Umbilical Cord-Derived Cells for Bone Tissue Engineering.

Extraordinary capabilities underlie the potential use of immune cells, particularly macrophages, in bone tissue engineering. Indeed, the depletion of macrophages during bone repair often culminates in disease scenarios. Inspired by the native dynamics between immune and skeletal systems, this work proposes a straightforward in vitro method to bioengineer biomimetic bone niches using biological waste. For that, liquefied and semipermeable reservoirs generated by electrohydrodynamic atomization and layer-by-layer techniques are developed to coculture umbilical cord-derived human cells, namely monocyte-derived macrophages, mesenchymal-derived stromal cells (MSCs), and human umbilical vein endothelial cells (HUVECs). Poly(ε-caprolactone) microparticles are also added to the liquefied core to act as cell carriers. The fabricated microcapsules grant the successful development of viable microtissues, ensuring the high diffusion of bioactive factors. Interestingly, macrophages within the bioengineered microcapsules increase the release of osteocalcin, osteoprotegerin, and vascular endothelial growth factor. The cytokines profile variation indicates macrophages' polarization into a prohealing phenotype. Altogether, the incorporation of macrophages within the fabricated microcapsules allows to recreate an appropriate bone microenvironment for developing new bone mineralized microtissues. The proposed bioencapsulation protocol is a powerful self-regulated system, which might find great applicability in bone tissue engineering based on bottom-up approaches or disease modeling.

Bioengineered Hierarchical Bonelike Compartmentalized Microconstructs Using Nanogrooved Microdiscs.

Fabrication of vascularized large-scale constructs for regenerative medicine remains elusive since most strategies rely solely on cell self-organization or overly control cell positioning, failing to address nutrient diffusion limitations. We propose a modular and hierarchical tissue-engineering strategy to produce bonelike tissues carrying signals to promote prevascularization. In these 3D systems, disc-shaped microcarriers featuring nanogrooved topographical cues guide cell behavior by harnessing mechanotransduction mechanisms. A sequential seeding strategy of adipose-derived stromal cells and endothelial cells is implemented within compartmentalized, liquefied-core macrocapsules in a self-organizing and dynamic system. Importantly, our system autonomously promotes osteogenesis and construct's mineralization while promoting a favorable environment for prevascular-like endothelial organization. Given its modular and self-organizing nature, our strategy may be applied for the fabrication of larger constructs with a highly controlled starting point to be used for local regeneration upon implantation or as drug-screening platforms.

Freestanding Magnetic Microtissues for Tissue Engineering Applications.

A long-sought goal in tissue engineering (TE) is the development of tissues able to recapitulate the complex architecture of the native counterpart. Microtissues, by resembling the functional units of living structures, can be used to recreate tissues' architecture. Howbeit, microfabrication methodologies fail to reproduce cell-based tissues with uniform shape. At the macroscale, complex tissues are already produced by magnetic-TE using solely magnetized cells as building materials. The enhanced extracellular matrix (ECM) deposition guaranties the conservation of tissues' architecture, leading to a successful cellular engraftment. Following the same rational, now the combination of a versatile microfabrication-platform is proposed with magnetic-TE to generate robust micro-tissues with complex architecture for TE purposes. Small tissue units with circle, square, and fiber-like shapes are designed with high fidelity acting as building blocks for engineering complex tissues. Notably, freestanding microtissues maintain their geometry after 7 days post-culturing, overcoming the challenges of microtissues fabrication. Lastly, the ability of microtissues in invading distinct tissue models while releasing trophic factors is substantiated in methacryloyl laminarin (LAM) and platelet lysates (PLMA) hydrogels. By simply using cells as building units and such microfabrication-platform, the fabrication of complex multiscale and multifunctional tissues with clinical relevance is envisaged, including for therapies or disease models.

Bioinstructive Layer-by-Layer-Coated Customizable 3D Printed Perfusable Microchannels Embedded in Photocrosslinkable Hydrogels for Vascular Tissue Engineering.

The development of complex and large 3D vascularized tissue constructs remains the major goal of tissue engineering and regenerative medicine (TERM). To date, several strategies have been proposed to build functional and perfusable vascular networks in 3D tissue-engineered constructs to ensure the long-term cell survival and the functionality of the assembled tissues after implantation. However, none of them have been entirely successful in attaining a fully functional vascular network. Herein, we report an alternative approach to bioengineer 3D vascularized constructs by embedding bioinstructive 3D multilayered microchannels, developed by combining 3D printing with the layer-by-layer (LbL) assembly technology, in photopolymerizable hydrogels. Alginate (ALG) was chosen as the ink to produce customizable 3D sacrificial microstructures owing to its biocompatibility and structural similarity to the extracellular matrices of native tissues. ALG structures were further LbL coated with bioinstructive chitosan and arginine-glycine-aspartic acid-coupled ALG multilayers, embedded in shear-thinning photocrosslinkable xanthan gum hydrogels and exposed to a calcium-chelating solution to form perfusable multilayered microchannels, mimicking the biological barriers, such as the basement membrane, in which the endothelial cells were seeded, denoting an enhanced cell adhesion. The 3D constructs hold great promise for engineering a wide array of large-scale 3D vascularized tissue constructs for modular TERM strategies.

Geometrically Controlled Liquefied Capsules for Modular Tissue Engineering Strategies.

A plethora of bioinspired cell-laden hydrogels are being explored as building blocks that once assembled are able to create complex and highly hierarchical structures recapitulating the heterogeneity of living tissues. Yet, the resulting 3D bioengineered systems still present key limitations, mainly related with limited diffusion of essential molecules for cell survival, which dictates the failure of most strategies upon implantation. To maximize the hierarchical complexity of bioengineered systems, while simultaneously fully addressing the exchange efficiency of biomolecules, the high-throughput fabrication of liquefied capsules is proposed using superhydrophobic-superhydrophilic microarrays as platforms to produce the initial structures with high fidelity of geometry and size. The liquefied capsules are composed by i) a permselective multilayered membrane; ii) surface-functionalized poly(ε-caprolactone) microparticles loaded into the liquefied core acting as cell adhesion sites; and iii) cells. It is demonstrated that besides the typical spherical liquefied capsules, it is also possible to obtain multi-shaped blocks with high geometrical precision and efficiency. Importantly, the internal gelation approach used to produce such blocks does not jeopardize cell viability, evidencing the mild conditions of the proposed cell encapsulation technique. The proposed system is intended to be used as hybrid devices implantable using minimally invasive procedures for multiple tissue engineering applications.

Freeform 3D printing using a continuous viscoelastic supporting matrix.

Embedded bio-printing has fostered significant advances toward the fabrication of soft complex tissue-like constructs, by providing a physical support that allows the freeform shape maintenance within the prescribed spatial arrangement, even under gravity force. Current supporting materials still present major drawbacks for up-scaling embedded 3D bio-printing technology towards tissue-like constructs with clinically relevant dimensions. Herein, we report a a cost-effective and widely available supporting material for embedded bio-printing consisting on a continuous pseudo-plastic matrix of xanthan-gum (XG). This natural polisaccharide exhibits peculiar rheological properties that have enabled the rapid generation of complex volumetric 3D constructs with out-of-plane features. The freedom of design within the three orthogonal axes through the independent and controlled bio-printing process opens new opportunities to produce on demand large arbitrary shapes for personalized medicine. Additionally, we have demonstrated the versatile functionality of XG as a photocurable gel reservoir to engineer perfused cell-laden hydrogel constructs, addressing other practical biomedical applications such as in vitro models and organ-on-chip platforms.

Dynamic microfactories co-encapsulating osteoblastic and adipose-derived stromal cells for the biofabrication of bone units.

Cells with differentiation potential into mesodermal types are the focus of emerging bone tissue engineering (TE) strategies as an alternative autologous source. When the source of cells is extremely limited or not readily accessible, such as in severe injuries, a tissue biopsy may not yield the required number of viable cells. In line, adipose-derived stromal cells (ASCs) quickly became attractive for bone TE, since they can be easily and repeatably harvested using minimally invasive techniques with low morbidity. Inspired by the multiphenotypic cellular environment of bone, we propose the co-encapsulation of ASCs and osteoblasts (OBs) in self-regulated liquefied and multilayered microcapsules. We explore the unique architecture of such hybrid units to provide a dynamic environment using a simple culture in spinner flasks. Results show that microtissues were successfully obtained inside the proposed microcapsules with an appropriate diffusion of essential molecules for cell survival and signaling. Remarkably, microcapsules cultured in the absence of supplemental osteogenic differentiation factors presented osteopontin immunofluorescence, evidencing that the combined effect of the dynamic environment, and the paracrine signaling between ASCs and OBs may prompt the development of bone-like microtissues. Furthermore, microcapsules cultured under dynamic environment presented an enhanced mineralized matrix and a more organized extracellular matrix ultrastructure compared to static cultures used as control. Altogether, data in this study unveil an effective engineered bioencapsulation strategy for the in vitro production of bone-like microtissues in a more realistic and cost-effective manner. Accordingly, we intend to use the proposed system as hybrid devices implantable by minimally invasive procedures for bone TE applications.

Screening of perfused combinatorial 3D microenvironments for cell culture.

Biomaterials combining biochemical and biophysical cues to establish close-to-extracellular matrix (ECM) models have been explored for cell expansion and differentiation purposes. Multivariate arrays are used as material-saving and rapid-to-analyze platforms, which enable selecting hit-spotted formulations targeting specific cellular responses. However, these systems often lack the ability to emulate dynamic mechanical aspects that occur in specific biological milieus and affect physiological phenomena including stem cells differentiation, tumor progression, or matrix modulation. We report a tailor-made strategy to address the combined effect of flow and biochemical composition of three-dimensional (3D) biomaterials on cellular response. We suggest a simple-to-implement device comprising (i) a perforated platform accommodating miniaturized 3D biomaterials and (ii) a bioreactor that enables the incorporation of the biomaterial-containing array into a disposable perfusion chamber. The system was upscaled to parallelizable setups, increasing the number of analyzed platforms per independent experiment. As a proof-of-concept, porous chitosan scaffolds with 1 mm diameter were functionalized with combinations of 5 ECM and cell-cell contact-mediating proteins, relevant for bone and dental regeneration, corresponding to 32 protein combinatorial formulations. Mesenchymal stem cells adhesion and production of an early osteogenic marker were assessed on-chip on static and under-flow dynamic perfusion conditions. Different hit-spotted biomaterial formulations were detected for the different flow regimes using direct image analysis. Cell-binding proteins still poorly explored as biomaterials components - amelogenin and E-cadherin - were here shown as relevant cell response modulators. Their combination with ECM cell-binding proteins - fibronectin, vitronectin, and type 1 collagen - rendered specific biomaterial combinations with high cell adhesion and ALP production under flow. The developed versatile system may be targeted at widespread tissue regeneration applications, and as a disease model/drug screening platform. STATEMENT OF SIGNIFICANCE: A perfusion system that enables cell culture in arrays of three-dimensional biomaterials under dynamic flow is reported. The effect of 31 cell-binding protein combinations in the adhesion and alkaline phosphatase (ALP) production of mesenchymal stem cells was assessed using a single bioreactor chamber. Flow perfusion was not only assessed as a classical enhancer/accelerator of cell growth and early osteogenic differentiation. We hypothesized that flow may affect cell-protein interactions, and that key components driving cell response may differ under static or dynamic regimes. Indeed, hit-spotted formulations that elicited highest cell attachment and ALP production on static cell culture differed from the ones detected for dynamic flow assays. The impacting role of poorly studied proteins as E-cadherin and amelogenin as biomaterial components was highlighted.