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Autoimmune diseases are life-threatening disorders that cause increasing disability over time. Systemic lupus erythematosus (SLE) and other autoimmune diseases arise when immune stimuli override mechanisms of self-tolerance. Accumulating evidence has demonstrated that protein glycosylation is substantially altered in autoimmune disease development, but the mechanisms by which glycans trigger these autoreactive immune responses are still largely unclear. In this study, we found that presence of microbial-associated mannose structures at the surface of the kidney triggers the recognition of DC-SIGN–expressing γδ T cells, inducing a pathogenic interleukin-17a (IL-17a)–mediated autoimmune response. Mice lacking Mgat5 , which have a higher abundance of mannose structures in the kidney, displayed increased γδ T cell infiltration into the kidney that was associated with spontaneous development of lupus in older mice. N -acetylglucosamine supplementation, which promoted biosynthesis of tolerogenic branched N-glycans in the kidney, was found to inhibit γδ T cell infiltration and control disease development. Together, this work reveals a mannose–γδ T cell–IL-17a axis in SLE immunopathogenesis and highlights glycometabolic reprogramming as a therapeutic strategy for autoimmune disease treatment.

Lung cancer is still the main cause of cancer-related deaths worldwide. Its treatment generally includes surgical resection, immunotherapy, radiotherapy, and chemo-targeted therapies such as the application of tyrosine kinase inhibitors. Gefitinib (GEF) is one of them, but its poor solubility in gastric fluids weakens its bioavailability and therapeutic activity. In addition, like all other chemotherapy treatments, GEF administration can cause damage to healthy tissues. Therefore, the development of novel GEF delivery systems to increase its bioavailability and distribution in tumor site is highly demanded. Herein, an innovative strategy for GEF delivery, by functionalizing PLGA nanoparticles with p28 (p28-NPs), a cell-penetrating peptide derived from the bacterial protein azurin, was developed. Our data indicated that p28 potentiates the selective interaction of these nanosystems with A549 lung cancer cells (active targeting). Further p28-NPs delivering GEF (p28-NPs-GEF) were able to selectively reduce the metabolic activity of A549 cells, while no impact was observed in non-tumor cells (16HBE14o-). In vivo studies using A549 subcutaneous xenograft showed that p28-NPs-GEF reduced A549 primary tumor burden and lung metastases formation. Overall, the design of a p28-functionalized delivery nanosystem to effectively penetrate the membranes of cancer cells while deliver GEF could provide a new strategy to improve lung cancer therapy.

Healing of intestinal chronic wounds remains a major challenge as current therapies are ineffective in promoting proper regeneration of the damaged intestinal wall. An innovative concept, based on a bioinspired multifunctional alginate-melanin hybrid 3D scaffold, to target both inflammatory and regenerative processes, is proposed herein. Hydrogel-entrapped melanin nanoparticles demonstrated free-radical scavenging activity, supported by the neutralization of free-radicals in solution (90%), and the in vitro capture of reactive oxygen species (ROS) produced by stimulated macrophages in an inflammatory-mimicking environment. Notably, scaffolds could be reused (at least 3 times), while maintaining these properties. The extracellular matrix (ECM)-inspired biomaterial, containing protease-sensitive and integrin-binding domains, exhibited remarkable ability for cell colonisation. Human intestinal fibroblasts and epithelial cells (Caco-2) co-seeded on lyophilized scaffolds were able to invade/colonize the construct and produce endogenous ECM, key for neo-tissue formation and re-epithelialization. Scaffolds presented tuneable mechanical properties and could be used both in hydrated and freeze-dried states, maintaining their performance upon rehydration, which are attractive features for clinical application. Collectively, our results highlight the potential of biofunctionalized alginate-melanin hybrid 3D scaffolds as multi-therapeutic patches for modulating inflammation and tissue regeneration in chronic intestinal wounds, which address a major but still unmet clinical need. The proposed multi-therapeutic strategy may potentially be extended to the treatment of other chronic wounds.

Therapeutic and diagnostic payloads are usually associated with properties that compromise their efficacy, such as poor aqueous solubility, short half-life, low bioavailability, nonspecific accumulation and diverse side effects. Nanotechnological solutions have emerged to circumvent some of these drawbacks, augmenting therapeutic and/or diagnostic outcomes. Nanotechnology has benefited from the rise in polymer science research for the development of novel nanosystems for therapeutic and diagnostic purposes. Polymers are a widely used class of biomaterials, with a considerable number of regulatory approvals for application in clinics. In addition to their versatility in production and functionalization, several synthetic and natural polymers demonstrate biocompatible properties that dictate their successful biological performance. This article highlights the physicochemical characteristics of a variety of natural and synthetic biocompatible polymers, as well as their role in the manufacture of nanotechnology-based systems, state-of-art applications in disease treatment and diagnosis, and current challenges in finding a way to clinics.

Colorectal cancer (CRC) is the third most common and the second deadliest type of cancer worldwide, urging the development of more comprehensive models and of more efficient treatments. Although the combination of nanotechnology with chemo- and immuno-therapy has represented a promising treatment approach, its translation to the clinic has been hampered by the absence of cellular models that can provide reliable and predictive knowledge about the in vivo efficiency of the formulation. Herein, a 3D model based on CRC multicellular tumor spheroids (MCTS) model was developed by combining epithelial colon cancer cells (HCT116), human intestinal fibroblasts and monocytes. The developed MCTS 3D model mimicked several tumor features with cells undergoing spatial organization and producing extracellular matrix, forming a mass of tissue with a necrotic core. Furthermore, monocytes were differentiated into macrophages with an anti-inflammatory, pro-tumor M2-like phenotype. For a combined chemoimmunotherapy effect, spermine-modified acetalated dextran nanoparticles (NPs) loaded with the chemotherapeutic Nutlin-3a (Nut3a) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were produced and tested in 2D cultures and in the MCTS 3D model. NPs were successfully taken-up by the cells in 2D, but in a significant less extent in the 3D model. However, these NPs were able to induce an anti-proliferative effect both in the 2D and in the 3D models. Moreover, Nut3a was able to partially shift the polarization of the macrophages present in the MCTS 3D model towards an anti-tumor M1-like phenotype. Overall, the developed MCTS 3D model showed to recapitulate key features of tumors, while representing a valuable model to assess the effect of combinatorial nano-therapeutic strategies in CRC. In addition, the developed NPs could represent a promising approach for CRC treatment.

The success of diabetes treatment with bioartificial pancreas is still hindered by a high rate of graft failure caused by poorly functioning cells. The incretin effect of glucagon like peptide-1 (GLP-1) make this peptide an attractive therapeutic agent to improve the success of transplantation of encapsulated beta cells. Our objective was to develop a novel model based on the co-encapsulation of beta cells with nanoparticles containing GLP-1 on alginate hydrogels, allowing its delivery and action in the specific target, beta cells. To this end, poly(lactide-co-glycolide) (PLGA) nanoparticles containing GLP-1 were prepared to be encapsulated together with INS-1E beta cells. Then, cells were encapsulated in alginate alone (Alg 0) and co-encapsulated with PLGA nanoparticles containing GLP-1 (Alg GLP-1). Hydrogels were maintained in culture during 7 days for evaluation of GLP-1’s influence in cellular performance, namely the crucial feature of insulin secretion. PLGA nanoparticles had an average size of 169.3 nm, a polydispersity index of 0.05 and an average zeta-potential of −24.3 mV. The average association efficiency of GLP-1 was 65.4% and the in vitro release test showed 71% of GLP-1 delivery after 7 days. Comparing the cellular conditions (Alg 0 and Alg GLP-1), the presence of nanoparticles led to increased cellular metabolic activity and 8-fold higher insulin levels. Our results reveal a promising successful role of GLP-1 nanoparticles in ensuring the viability of encapsulated beta cells in alginate hydrogel, by promoting its proper delivery and action. Further studies are needed to extensively characterize this new model of bioartifical pancreas.

Blood-contacting devices are increasingly important for the management of cardiovascular diseases. Poly(ethylene glycol) (PEG) hydrogels represent one of the most explored hydrogels to date. However, they are mechanically weak, which prevents their use in load-bearing biomedical applications (e.g., vascular grafts, cardiac valves). Graphene and its derivatives, which have outstanding mechanical properties, a very high specific surface area, and good compatibility with many polymer matrices, are promising candidates to solve this challenge. In this work, we propose the use of graphene-based materials as nanofillers for mechanical reinforcement of PEG hydrogels, and we obtain composites that are stiffer and stronger than, and as anti-adhesive as, neat PEG hydrogels. Results show that single-layer and few-layer graphene oxide can strengthen PEG hydrogels, increasing their stiffness up to 6-fold and their strength 14-fold upon incorporation of 4% w/v (40 mg/mL) graphene oxide. The composites are cytocompatible and remain anti-adhesive towards endothelial cells, human platelets and Staphylococcus aureus, similar to neat hydrogels. To the best of our knowledge, this is the first work to report such an increase of the tensile properties of PEG hydrogels using graphene-based materials as fillers. This work paves the way for the exploitation of PEG hydrogels as a backbone material for load-bearing applications.

Hydrogels can mimic several features of the cell native microenvironment and have been widely used as synthetic extracellular matrices (ECMs) in tissue engineering and regenerative medicine (TERM). However, some applications have specifications that hydrogels cannot efficiently fulfill on their own. Incorporating reinforcing structures like fibrous scaffolds or particles into hydrogels, as hybrid systems, is a promising strategy to improve their functionality. We describe recent advances in the fabrication and application of these hybrid systems, where structural properties and stimuli responsiveness of hydrogels are enhanced while their ECM-like features are preserved. Furthermore, we discuss how these systems can contribute to the development of more complex tissue engineered structures in the rapidly evolving field of TERM.

Modular tissue engineering approaches open up exciting perspectives for the biofabrication of vascularized tissues from the bottom-up, using micro-sized units such as spheroids as building blocks. While several techniques for 3D spheroid formation from multiple cell types have been reported, strategies to elicit the extra-spheroid assembly of complex vascularized tissues are still scarce. Here we describe an injectable approach to generate vascularized dermal tissue, as an example application, from spheroids combining fibroblasts and endothelial progenitors (OEC) in a xeno-free (XF) setting. Short-term cultured spheroids (1 day) were selected over mature spheroids (7 days), as they showed significantly higher angiogenic sprouting potential. Embedding spheroids in fibrin was crucial for triggering cell migration into the external milieu, while providing a 3D framework for in-gel extra-spheroid morphogenesis . Migrating fibroblasts proliferated and produced endogenous ECM forming a dense tissue, while OEC self-assembled into stable capillaries with lumen and basal lamina . Massive in vitro interconnection between sprouts from neighbouring spheroids rapidly settled an intricate vascular plexus. Upon injection into the chorioallantoic membrane of chick embryos, fibrin-entrapped pre-vascularized XF spheroids developed into a macrotissue with evident host vessel infiltration . After only 4 days, perfused chimeric capillaries with human cells were present in proximal areas, showing fast and functional inosculation between host and donor vessels. This method for generating dense vascularized tissue from injectable building blocks is clinically relevant and potentially useful for a range of applications.

Correction for ‘Biofunctionalized pectin hydrogels as 3D cellular microenvironments’ by Sara C. Neves et al., J. Mater. Chem. B, 2015, 3, 2096–2108.

The field of stomach-directed therapeutics and diagnosis is still hampered by the lack of reliable in vitro models that closely mimic the gastric mucosa where gastric cancer cells are generally confined. Here we propose a rapid, complex, and innovative 3D in vitro model of the gastric mucosa, by extending a conventional gastric monolayer model to an inner stratum of the mucosa – the lamina propria. The developed model comprises normal stomach fibroblasts embedded in a 3D RGD-modified alginate hydrogel prepared on the basolateral side of a Transwell® insert, mimicking the extracellular matrix and cellular component of the lamina propria, onto which a moderately differentiated adenocarcinoma stomach cell line (MKN74) was seeded, reproducing the physiological conditions of the gastric barrier. The integrity and functionality of the in vitro model was evaluated through permeability studies of FITC-dextran and 200 nm fluorescent polystyrene nanoparticles at gastric conditions. Nanoparticle transport was pH-dependent and strongly impacted by the biomimetic lamina propria, highlighting that a gastric extracellular matrix (ECM)-like microenvironment should be integrated in an in vitro permeability model to be adopted as a reliable evaluation tool of innovative therapeutics and diagnosis of gastric diseases. Statement of Significance Current in vitro models of the gastric mucosa are limited to simplistic 2D cell culture systems, which ignore the dimensionality of the stomach wall and make it difficult to reliably test new therapeutic approaches to gastric pathologies. By combining stomach fibroblasts embedded within a 3D RGD-modified alginate hydrogel and epithelial gastric cancer cells in a Transwell® system, we established a new biomimetic model of the stomach mucosa. Epithelial cells recreate the gastric epithelium, while the cell-laden 3D hydrogel recapitulates both the cellular composition and dimensionality of the extracellular matrix of gastric lamina propria. This cellularized 3D model stands as a promising evaluation platform to assist the development of new strategies for the treatment and diagnosis of gastric diseases.

Efficient cell delivery strategies are urgently needed to improve the outcome of cell-based pro-angiogenic therapies. This study describes the design of an injectable cell delivery platform, based on biomaterial-guided morphogenesis principles. Soft high-mannuronic acid alginate microgels, oxidized and functionalized with integrin-binding peptides, provided adequate biochemical/biomechanical cues for the co-assembly of mesenchymal stem cells and outgrowth endothelial cells (OEC) into pre-vascularized microtissues. In vitro priming conditions regulated OEC tubulogenesis, which only occurred under normoxia (+O2) in the presence of angiogenic factors (+GF) and, importantly, did not revert in an ischemic-like environment. Primed (+O2+GF) microgel-entrapped cells secreted a large variety of angiogenesis-related proteins and produced endogenous extracellular-matrix, rich in fibronectin and collagen type I, fostering cell-cell/cell-matrix interactions and establishing a stable angiogenic niche. Extending the pre-culture time resulted in higher cell outward migration and in vivo angiogenic potential. Microgels partially disintegrated upon implantation in chick embryos, promoting interaction between pre-vascularized microtissues and the host. Preserved human vascular structures were still detected in vivo, and human cells showed the ability to migrate and integrate with the chick vasculature. Our results suggest that an integrated approach combining pro-angiogenic cells, cell-instructive microgels and adequate in vitro priming may provide the basis for successful therapeutic angiogenesis.

Skin chronic wounds are associated with a state of persistent inflammation and often with infection, originating a specific microenvironment characterized by increased temperature, alkaline pH, elevated enzymatic activity, and high levels of reactive oxygen species (ROS). These alterations can be explored as intrinsic triggers in the design of stimuli-responsive biomaterials for the release of bioactive molecules at the wound microenvironment. Stimuli-responsive biomaterials may not only prolong the bioactivity of the therapeutic agents but also synchronize it with the healing stages, tuning the wound treatment. In addition, the high activity of enzymes, such as lysozyme in infected chronic wounds, as well as the shift to a more alkaline pH may be used as biomarkers for early detection of infected and/or non-healing wounds. Overall, although a few shortcomings still need to be addressed before clinical translation, the bioengineered smart formulations highlighted in this review stand out as a new generation of therapies to manage skin chronic wounds. This work was supported by Portuguese funds through FCT-MCTES (Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the project “AntINFECT“ (POCI-01-0145-FEDER-031781, PTDC/NAN-MAT/31781/2017). FCT-MCTES is further acknowledged for funding LAQV-REQUIMTE Research Unit (UIDB/50006/2020). P.A. thanks FCT-MCTES for the doctoral grant SFRH/BD/145471/2019 financially supported by national and European Union funds (ESF – European Social Fund through “Programa Operacional Regional Norte”).

13 pages, 7 figures, A novel blue-emitting fluorescent alginate derivative has been successfully synthesised in a simple tworeaction step procedure, using an aqueous conjugation strategy that involved carbodiimide coupling followed by an alkyne–azide ‘‘click’’ reaction. The modified alginate maintained the characteristic ability to form mechanically stable hydrogels by ionic crosslinking. The fluorescent properties of the developed biomaterial were investigated both in solution and hydrogel states, revealing that grafting of the coumarin fluorophore to alginate greatly enhanced its fluorescent properties. Importantly, hydrogels maintained around 80% of their initial fluorescence upon long periods of incubation under physiologic conditions. The fluorescent alginate hydrogels showed to be biocompatible in vitro, supporting the viability, metabolic activity and proliferation of mammary epithelial cells and, more importantly, their morphogenesis into spheroids and polarized acini-like structures. These hydrogels were further applied in the establishment of cell-in-gel microarrays for high-throughput screening of cell behaviour in threedimensional (3D) matrices, being essential for spotting optimization and analysis. Collectively, our results highlight the potential of coumarin-grafted blue-emitting fluorescent alginate as a valuable tool for biomedical applications where hydrogel tracing is required, This work was financed by FEDER – Fundo Europeu de Desenvolvimento Regional funds through COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT – Fundaçao para a Ciencia e a Tecnologia/Ministerio da Ciencia, Tecnologia e Ensino Superior, in the framework of the project 3DEMT (PTDC/BBB-ECT/2518/2014). S. J. B. and C. C. B. thank FCT for SFRH/BPD/80571/2011 fellowship and IF research position IF/00296/2015, respectively. M. F. A. and J. V. gratefully acknowledge Agencia para o Desenvolvimento e Coesao and Ministerio de Hacienda, Direccion General de Fondos Europeos for Interreg V-A Spain-Portugal (POCTEP) 2014–2020 and FEDER (0245_IBEROS_1_E) for the Postdoctoral grant. J. V. and J. A. V. also acknowledge Xunta de Galicia (Grupos de Potencial Crecimiento, IN607B 2018/19)

Introduction: The success of a bioartificial pancreas crucially depends on ameliorating encapsulated beta cells survival and function. By mimicking the cellular in vivo niche, the aim of this study was to develop a novel model for beta cells encapsulation capable of establishing an appropriate microenvironment that supports interactions between cells and extracellular matrix (ECM) components. Methods: ECM components (Arg-Gly-Asp, abbreviated as RGD) were chemically incorporated in alginate hydrogels (alginate-RGD). After encapsulation, INS-1E beta cells outcome was analyzed in vitro and after their implantation in an animal model of diabetes. Results: Our alginate-RGD model demonstrated to be a good in vitro niche for supporting beta cells viability, proliferation, and activity, namely by improving the key feature of insulin secretion. RGD peptides promoted cell–matrix interactions, enhanced endogenous ECM components expression, and favored the assembly of individual cells into multicellular spheroids, an essential configuration for proper beta cell functioning. In vivo, our pivotal model for diabetes treatment exhibited an improved glycemic profile of type 2 diabetic rats, where insulin secreted from encapsulated cells was more efficiently used. Conclusions: We were able to successfully introduce a novel valuable function in an old ally in biomedical applications, the alginate. The proposed alginate-RGD model stands out as a promising approach to improve beta cells survival and function, increasing the success of this therapeutic strategy, which might greatly improve the quality of life of an increasing number of diabetic patients worldwide. The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by FCT/MEC through National Funds and co-financed by FEDER through the PT2020 Partnership Agreement under the 4293 Unit I&D, FCT Strategic Project PEst-C/SAU/UI3282/2011-2013 and UID/NEU/04539/2013, FCT in the framework of project UID/BIM/04293/2013, FCT in the framework of project IF/00939/2013/CP1179/CT0001, FCT for Joana Crisóstomo (grant number SFRH/BD/72964/2010), FCT for Sílvia J Bidarra (grant number SFRH/BPD/80571/2011), and FCT and POPH/ESF (EC) for Cristina C Barrias research position FCT Investigator (IF2013).

The bioartificial pancreas presents a promising methodology to treat diabetes. However, this approach is still associated with a high rate of graft failure caused by poorly functioning cells. The incretin effect of glucagon like peptide-1 (GLP-1) make this peptide an attractive therapeutic agent to improve the success of transplantation of encapsulated beta cells. In present work, we developed a novel model based on the co-encapsulation of beta cells with nanoparticles containing GLP-1 on alginate hydrogels, allowing its delivery and action in the specific target, the beta cells.

The development of three-dimensional (3D) fibrous networks as platforms for tissue engineering applications has been attracting considerable attention. Opportunely arranged microscaled fibers offer an appealing biomimetic 3D architecture, with an open porous structure and a high surface-to-volume ratio. The present work describes the development of modified-alginate hydrogel microfibers for cell entrapment, using a purpose-designed flow circuit. For microfibers biofabrication, cells were suspended in gel-precursor alginate solution and injected in a closed-loop circuit with circulating cross-linking solution. The flow promoted stretching and solidification of continuous cell-loaded micro-scaled fibers that were collected in a strainer, assembling into a microfibrous patch. The process was optimized to allow obtaining a self-standing cohesive structure. After characterization of the microfibrous patch, the behavior of embedded human mesenchymal stem cells (hMSCs) was evaluated. Microfibers of oxidized alginate modified with integrin-binding ligands provided a suitable 3D cellular microenvironment, supporting hMSCs survival and stimulating the production of endogenous extracellular matrix proteins, such as fibronectin and collagen Type I. Collectively, these features make the proposed microfibrous structures stand out as promising 3D scaffolds for regenerative medicine.

Successful strategies to promote neovascularization of ischemic tissues are still scarce, being a central priority in regenerative medicine. Microparticles harboring primitive vascular beds are appealing cell delivery candidates for minimally-invasive therapeutic vascularization. However, dynamic cellular alterations associated with in vitro vascular morphogenesis are still elusive. Here, bioengineered microgels guided the assembly of entrapped outgrowth endothelial cells (OEC) and mesenchymal stem cells (MSC) into cohesive vascularized microtissues. During in vitro maturation, OEC formed capillary-like networks enveloped in newly-formed extracellular matrix. Gene expression profiling showed that OEC acquired a mesenchymal-like phenotype, suggesting the occurrence of partial endothelial-to-mesenchymal transition (EndMT), while MSC remained transcriptionally stable. The secretome of entrapped cells became more pro-angiogenic, with no significant alterations of the inflammatory profile. Importantly, matured microgels showed improved cell survival/retention after transplantation in mice, with preservation of capillary-like networks and de novo formation of human vascular structures. These findings support that in vitro priming and morphogenesis of vessel-forming cells improves their vasculogenic/angiogenic potential, which is of therapeutic relevance, shedding some light on the associated mechanisms.

Spheroids can be used as building-blocks for bottom-up generation of artificial vascular beds, but current biofabrication strategies are often time-consuming and complex. Also, pre-optimization of single spheroid properties is often neglected. Here, we report a simple setup for rapid biomanufacturing of spheroid-based patch-like vascular beds. Prior to patch assembly, spheroids combining mesenchymal stem/stromal cells (MSCs) and outgrowth endothelial cells (OECs) at different ratios (10:1; 5:1; 1:1; 1:5) were formed in non-adhesive microwells and monitored along 7 d. Optimal OEC retention and organization was observed at 1:1 MSC/OEC ratio. Dynamic remodelling of spheroids led to changes in both cellular and extracellular matrix components (ECMs) over time. Some OEC formed internal clusters, while others organized into a peripheral monolayer, stabilized by ECM and pericyte-like cells, with concomitant increase in surface stiffness. Along spheroid culture, OEC switched from an active to a quiescent state, and their endothelial sprouting potential was significantly abrogated, suggesting that immature spheroids may be more therapeutically relevant. Non-adhesive moulds were subsequently used for triggering rapid, one-step, spheroid formation/fusion into square-shaped patches, with spheroids uniformly interspaced via a thin cell layer. The high surface area, endothelial sprouting potential, and scalability of the developed spheroid-based patches make them stand out as artificial vascular beds for modular engineering of large tissue constructs.

Human mesenchymal stem/stromal cells (hMSCs) have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiringin vitroexpansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS) as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way.

Advances in the understanding of neonatal Fc receptor (FcRn) biology and function have demonstrated that this receptor, primarily identified for the transfer of passive immunity from mother infant, is involved in several biological and immunological processes. In fact, FcRn is responsible for the long half-life of IgG and albumin in the serum, by creating an intracellular protein reservoir, which is protected from lysosomal degradation and, importantly, trafficked across the cell. Such discovery has led researchers to hypothesize the role for this unique receptor in the controlled delivery of therapeutic agents. A great amount of FcRn-based strategies are already under extensive investigation, in which FcRn reveals to have profound impact on the biodistribution and half-life extension of therapeutic agents. This review summarizes the main findings on FcRn biology, function and distribution throughout different tissues, together with the main advances on the FcRn-based therapeutic opportunities and model systems, which indicate that this receptor is a potential target for therapeutic regimen modification.

Human mesenchymal stem/stromal cells (hMSC) are promising therapeutic agents for regenerative medicine. However, therapeutic doses necessary for clinical application require in vitro expansion, ideally under Xeno-Free (XF) conditions to avoid the use of foetal bovine serum (FBS). We previously reported that hMSCs could be expanded using a pharmaceutical-grade human plasma-derived supplement for cell culture (SCC, Plastem®) combined with bFGF and TGFβ1, on fibronectin (Fn)-coated surfaces. hMSCs expansion may also be affected by the chemistry of the culture surface, which modulates protein adsorption at the cell-material interface and, consequently, cell behavior. This work aimed to evaluate the effect of surface chemistry on hMSCs behavior in SCC-based XF media. For that, self-assembled monolayers (SAMs) with hydrophobic (-CH3) and hydrophilic (neutral -OH, positively charged -NH3+ and negatively charged -COO-) groups were used as model surfaces. Under XF conditions, Fn coating showed to be necessary to improve hMSC adhesion (4 h) onto all surfaces, except for OH-SAMs, probably due to a low protein adsorption capacity characteristic of this surface. In terms of cell metabolic activity (5 days) on Fn-coated surfaces, an increase over time under XF conditions was observed in all SAMs except in CH3-SAMs, which can be attributed to strong and irreversible protein adsorption characteristic of hydrophobic surfaces. This trend was also observed under FBS conditions. Nevertheless, none of the surfaces improved hMSC metabolic activity, as compared with tissue-cultured surfaces. Overall, this work describes the role of surface chemistry in XF hMSC expansion.

Three-dimensional (3D) cell culture systems have gained increasing interest among the scientific community, as they are more biologically relevant than traditional two-dimensional (2D) monolayer cultures. Alginate hydrogels can be formed under cytocompatibility conditions, being among the most widely used cell-entrapment 3D matrices. They recapitulate key structural features of the natural extracellular matrix and can be bio-functionalized with bioactive moieties, such as peptides, to specifically modulate cell behavior. Moreover, alginate viscoelastic properties can be tuned to match those of different types of native tissues. Ionic alginate hydrogels are transparent, allowing routine monitoring of entrapped cells, and crosslinking can be reverted using chelating agents for easy cell recovery. In this chapter, we describe some key steps to establish and characterize 3D cultures of mesenchymal stem cells using alginate hydrogels.

Intestinal cell models have been widely studied and used to evaluate absorption and metabolism of drugs in the small intestine, constituting valuable tools as a first approach to evaluate the behavior of new drugs. However, such cell models might not be able to fully predict the absorption mechanisms and metabolic pathways of the tested compounds. In recent years, induced pluripotent stem cells (iPSCs) differentiated into enterocyte-like cells have been proposed as more biorelevant intestinal models. In this review, we describe mechanisms underlying the differentiation of iPSCs into enterocyte-like cells, appraise the usefulness of these cells in tridimensional intestinal models, and discuss their suitability to be used in the future for drug screening.

Although fewer in number, M-cells are considered antigen sampling cells, acting as a gateway for antigens from the gut lumen and presenting an impressive aptitude for particle transcytosis. These features make M-cells attractive targets for oral drug delivery studies, but this has been poorly explored. New and reproducible tissue-like in vitro models for studying intestinal sampling and permeability mechanisms are needed. The combination of different cell players in such models offers improved microenvironments with higher physiologic relevance. Here, a tissue-engineered model was established, by co-culturing Caco-2 absorptive cells, HT29-MTX mucus-producing cells and Raji B lymphocytes. After 3 weeks of cell co-culture, the presence of M-like cells was evidenced by the loss of brush-border organization, detected by the lack of microvilli. The triple-culture model showed to be efficient for insulin transport, a process that was influenced by the tightness of junctions between epithelial cells and the presence of mucus and M-like cells. Ultimately, the proposed tissue-engineered model provides a more complete and reliable tool to perform drug permeability tests, as compared to traditional models, and may also find applicability as an in vitro system to study transdifferentiation mechanisms of M cells.

Absorption evaluation plays an increasingly important role at the early stage of drug discovery due to its potential to scan the ADME (absorption, distribution, metabolism and excretion) properties of new drug candidates. Therefore, a new three-dimensional (3D) in vitro model replicating the intestinal functioning is herein proposed aiming to dissect the stromal-epithelial interactions and evaluate the permeation of a model drug, insulin. Inspired on the intestinal mucosal architecture, the present model comprises intestinal myofibroblasts (CCD18-Co cells) embedded in Matrigel, onto which epithelial enterocytes (Caco-2 cells) and mucus-producing cells (HT29-MTX cells) were seeded. CCD18-Co myofibroblasts showed to have a central role in the remodeling of the surrounding matrix confirmed by the production of fibronectin. Subsequently, this matrix revealed to be essential to the maintenance of the model architecture by supporting the overlying epithelial cells. In terms of functionality, this model allowed the efficient prediction of insulin permeability in which the presence of mucus, the less tight character between Caco-2 and HT29-MTX epithelial cells and the 3D assembly were critical factors. Concluding, this model constitutes a robust tool in the drug development field with potential to bridge the traditional 2D cell culture models and in vivo animal models.

Hydrogels have been widely investigated as provisional matrices for tissue engineering and regenerative medicine, namely, as analogs of the natural extracellular matrix (ECM). In this context, different types of biofunctionalization strategies have been proposed to enhance the biological performance of hydrogels, particularly when they lack intrinsic bioactivity. Polymer-peptide hybrid hydrogels have emerged as promising systems, combining the ECM-like structural/mechanical properties of polymeric 3D networks with the biological activity of peptides. In this chapter, we present an overview of strategies involving the chemical functionalization of hydrogels with different types of bioactive peptides, for the design of cell instructive/responsive artificial 3D matrices.

Provascularization strategies are critical to the success of many tissue engineering applications and may also be of great value in the clinical management of ischemic vascular diseases. The use of biofunctional biomaterials to promote vascularization is increasing, supported by advances in the understanding of vascular cell biology and key elements of neovascularization processes. In particular, the design of increasingly sophisticated systems has been supported by greater knowledge on the dynamic interaction of multiple growth factors, acting on different time windows and spatial locations, and on the cross talk between vascular cells and the extracellular matrix (ECM). Polymer–peptide hybrids stand out as a promising class of biomaterials, combining the ECM-like structural features of hydrogels with the biological activity, specificity, and design flexibility of peptide moieties, being commonly used as reservoirs of angiogenic compounds and/or vascular cells. In this chapter, we present an overview and critical perspective on strategies involving the biofunctionalization of hydrogels with different types of angiogenic peptides for therapeutic vascularization.

Three-dimensional (3D) cell culture systems have gained increasing interest among the scientific community, as they are more biologically relevant than traditional two-dimensional (2D) monolayer cultures. Alginate hydrogels can be formed under cytocompatibility conditions, being among the most widely used cell-entrapment 3D matrices. They recapitulate key structural features of the natural extracellular matrix and can be bio-functionalized with bioactive moieties, such as peptides, to specifically modulate cell behavior. Moreover, alginate viscoelastic properties can be tuned to match those of different types of native tissues. Ionic alginate hydrogels are transparent, allowing routine monitoring of entrapped cells, and crosslinking can be reverted using chelating agents for easy cell recovery. In this chapter, we describe some key steps to establish and characterize 3D cultures of mesenchymal stem cells using alginate hydrogels.

This work was supported by the project Norte-01-0145-FEDER- 000012 – Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and supported by the project P2020-PTDC/BBB ECT/2145/2014 funded by POCI via FEDER and by Foundation for Science and Technology (FCT) via OE. R. P., A. S. and C. B. thank FCT for the doctoral grant SFRH/ BD/91151/2012, the post-doctoral grant SFRH/BPD/90047/2012 and the FCT Investigator research position IF/00296/2015 (FCT and POPH/ESF), respectively. The authors thank D. S. and M. A. from CEMUP (Centro de Materiais da Universidade do Porto) for the CryoSEM and 1H NMR analyses, respectively. Bioprinting is attractive to create cellularized constructs for skin repair. However, the vast majority of bioinks present limitations in the printing of chemically defined 3D constructs with controllable biophysical and biochemical properties. To address this challenge, a single-component hydrogel bioink with a controlled density of cell-adhesive ligands, tuneable mechanical properties and adjustable rheological behaviour is developed for extrusion bioprinting and applied for the biofabrication of 3D dermal constructs. A methacrylate modified pectin bioink is designed to allow the tethering of integrin-binding motifs and the formation of hydrogels by UV photopolymerization and ionic gelation. The rheological behaviour of a low polymer concentration (1.5 wt%) solution is adjusted by ionic crosslinking, yielding a printable bioink that holds the predesigned shape upon deposition for postprinting photocrosslinking. Printed constructs provide a suitablemicroenvironment that supports the deposition of endogenous extracellular matrix, rich in collagen and fibronectin, by entrapped dermal fibroblasts. This approach enables the design of chemically defined and cell-responsive bioinks for tissue engineering applications and particularly for the generation of biomimetic skin constructs. info:eu-repo/semantics/publishedVersion

Spheroid culture has gained increasing popularity, arising as a promising tool for regenerative medicine applications. Importantly, spheroids may present advantages over single-cell suspensions in cell-based therapies (CT). Unfortunately, most growth media used for spheroid culture contain animal origin-components, such as fetal bovine serum (FBS). The presence of FBS compromises the safety of CT and presents economic and ethical constraints. SCC (supplement for cell culture) is a novel xeno-free (XF) industrial cell culture supplement, derived from well-controlled pooled human plasma and processed under good manufacturing practice rules. Here, we developed a XF SCC-based formulation for 2D-culture of outgrowth endothelial cells (OEC), and then used it for generating co-culture spheroids of OEC and mesenchymal stem cells (MSC). XF MSC-OEC spheroids were characterized in detail and compared to spheroids cultured in FBS-supplemented medium. XF spheroids presented comparable integrity, size and morphology as the reference culture. The use of both media resulted in spheroids with similar structure, abundant extracellular matrix deposition and specific patterns of OEC distribution and organization. Notably, XF spheroids presented significantly enhanced angiogenic potential, both in vitro (fibrin sprouting assay) and in vivo (CAM assay). These findings are particularly promising in the context of potential therapeutic applications. This work was supported by: i) European Regional Development Fund (ERDF) through COMPETE 2020 -Operacional Programme for Competitiveness and Internationalisation (POCI), in the framework of project NORTE-01- 0145-FEDER-000012, funded by North Portugal Regional Operational Program (Norte2020) under PORTUGAL2020 Partnership Agreement; ii) European Union 7th Framework Programme, under the Marie Curie Initial Training Programme Network IB2 (MC ITN-EID nº317052[ABP1]) (Ewa Bauman scholarship); iii) Project 3DEMT funded by POCI via FEDER (POCI-01-0145-FEDER-016627) and by FCT via OE (PTDC/BBBECT/251872014). CC Barrias acknowledges FCT for her research position FCT Investigator (IF/00296/2015). The authors acknowledge Eduardo A. Silva (UC Davis, US) for providing OEC; and i3S Scientific platforms “b.IMAGE”, “In vivo CAM assays” and “Histology and Electron Microscopy” for service providence.

In situ-forming hydrogels of pectin, a polysaccharide present in the cell wall of higher plants, were prepared using an internal ionotropic gelation strategy based on calcium carbonate/D-glucono-delta-lactone, and explored for the first time as cell delivery vehicles. Since no ultrapure pectins are commercially available yet, a simple and efficient purification method was established, effectively reducing the levels of proteins, polyphenols and endotoxins of the raw pectin. The purified pectin was then functionalized by carbodiimide chemistry with a cell-adhesive peptide (RGD). Its gelation was analyzed by rheometry and optimized. Human mesenchymal stem cells embedded within unmodified and RGD-pectin hydrogels of different viscoelasticities (1.5 and 2.5 wt%) remained viable and metabolically active for up to 14 days. On unmodified pectin hydrogels, cells remained isolated and round-shaped. In contrast, within RGD-pectin hydrogels they elongated, spread, established cell-to-cell contacts, produced extracellular matrix, and migrated outwards the hydrogels. After 7 days of subcutaneous implantation in mice, acellular pectin hydrogels were considerably degraded, particularly the 1.5 wt% hydrogels. Altogether, these findings show the great potential of pectin-based hydrogels, which combine an interesting set of easily tunable properties, including the in vivo degradation profile, for tissue engineering and regenerative medicine.

Bioprinting technologies are powerful additive biofabrication techniques to produce cellular constructs for skin tissue engineering owing to their unique ability to precisely pattern living and non-living materials in pre-defined spatial locations. This unique feature, combined with the computer controlled printing and medical imaging techniques, enable researchers and clinicians to generate patient specific constructs partly replicating the intricate compositional and architectural organization of the skin. Bioprinting has been used to automatically dispense hydrogels with skin cells located in prescribed sites that promote skin formation in vitro and in vivo. Current skin bioprinting approaches mostly rely on the sequential printing of fibroblasts and keratinocytes embedded within a homogeneous hydrogel. Although such approaches have already been translated to pre-clinical scenarios, they still present limitations in terms of fully replicating the cellular and extracellular matrix (ECM) heterogeneity in native skin. The success of bioprinting for skin repair strongly depends on the design of printable bioinks capable of supporting the function of printed cells and stimulating the production of new ECM components. To better mimic the human skin, novel developments in dedicated bioprinting technologies, in the design of bioinks, as well as in the printing of vascularised constructs are necessary. This paper presents an overview regarding the use of bioprinting for skin tissue engineering applications. The operating principles of bioprinting technologies are outlined along with requirements of printed skin constructs. Finally, pre-clinical results are summarized and future perspectives for the field are highlighted.