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Fabrication of vascularized tissue constructs plays an integral role in creating clinically relevant tissues. Scaffold materials should be sufficiently vascularized to mimic functional and complex native tissues. Herein, we report the development of bioactive and biomimetic self-assembled peptide (SAP) hydrogels that allow the rapid formation of a vascular structurein vitro. The KLDLKLDLKLDL (KLD peptide) SAP was functionalized with laminin derived peptides IKVAV (V1) and YIGSR (V2) through direct coupling to mimic the natural extracellular matrix (ECM) and human umbilical endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) cultured in 0.5% and 1% SAP hydrogels organized into vascularized structures. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) images proved the molecular integration of the nanofibrous structure in SAP hydrogels. The stability of SAP hydrogels was confirmed by rheological and degradation measurements. Bioactive peptide scaffolds enhanced significantly HUVEC/hMSC proliferation depicted by MTT analysis compared to KLD. Furthermore, the real time quantitative polymerase chain reaction (rt-PCR) was performed to analyse vascular gene expressions such as platelet/endothelial cell adhesion molecule-1 (PECAM-1), von Willebrand factor (vWF), and vascular endothelial cadherin (VE-cadherin). The results indicated that the KLD-V2 hydrogel significantly induced vasculogenesis in hMSC/HUVEC co-culture compared to KLD-V1, Biogelx and KLD because YIGSR in KLD-V2 promoted cell population and ECM secretion by the interaction with cells and increased vasculogenesis. Overall, the designed SAP hydrogel represents an effective scaffold for vascularization of tissue constructs with useful tissue engineering applications. © The Royal Society of Chemistry 2021., Türkiye Bilimsel ve Teknolojik Araştirma Kurumu, TÜBITAK: 117S429, The authors acknowledge funding from TÜBİTAK (The Scientific and Technological Research Council of Turkey) through the Research Project 117S429.

In this paper, the possibility to steer and confine live human cells by means of acoustic waves, such as flexural plate waves (FPWs), generated by piezoelectric actuators applied to non-piezoelectric substrates, has been explored. A device with two lead zirconate titanate (PZT) actuators with an interdigital transducer (IDT) screen-printed on an alumina (Al2O3) substrate has been fabricated and tested. The experimental results show that, by exciting the actuators at their resonant frequencies, FPW modes are generated in the substrate. By exploiting the device, arrangements of cells on lines at frequency-dependent distances have been obtained. To maintain the alignment after switching off the actuator, cells were entrapped in a fibrin clot that was cultured for several days, enabling the formation of cellular patterns.

Scaffolds based on aligned and non-aligned poly (L-lactic acid) (PLLA)/polycaprolactone (PCL) fibers obtained by electrospinning, associated to electrosprayed hydroxyapatite (HA) for tissue engineering applications were developed and their performance was compared in terms of their morphology and biological and mechanical behaviors. The morphological results assessed by scanning electron microscopy showed a mesh of PLLA/PCL fibers (random and perfectly aligned) associated with aggregates of nanophased HA. Fourier transform infrared spectrometry confirmed the homogeneity in the blends and the presence of nanoHA in the scaffold. As a result of fiber alignment a 15-fold increase in Young&rsquo, s Modulus and an 8-fold increase in tensile strength were observed when compared to non-aligned fibers. In PLLA/PCL/HA scaffolds, the introduction of nanoHA caused a remarkable improvement of the mechanical strength of this material acting as a reinforcement, enhancing the response of these constructs to tensile stress. In vitro testing was evaluated using osteoblast (MC3T3-E1) cells. The results showed that both fibrous scaffolds were able to support osteoblast cell adhesion and proliferation and that fiber alignment induced increased cellular metabolic activity. In addition, the adhesion and proliferation of Staphylococcus aureus were evaluated and a lower number of colony forming units (CFUs) was obtained in the scaffolds with aligned fibers.

Microfluidic screening platforms offer new possibilities for performing in vitro cell-based assays with higher throughput and in a setting that has the potential to closely mimic the physiological microenvironment. Integrating functional biomaterials into such platforms is a promising approach to obtain a deeper insight into the interactions occurring at the cell-material interface. The success of such an approach is, however, largely dependent on the ability to miniaturize the biomaterials as well as on the choice of the assay used to study the cell-material interactions. In this work, we developed a microfluidic device, the main component of which is made of a widely used biocompatible polymer, polylactic acid (PLA). This device enabled cell culture under different fluidic regimes, including perfusion and diffusion. Through a combination of photolithography, two-photon polymerization and hot embossing, it was possible to microstructure the surface of the cell culture chamber of the device with highly defined geometrical features. Furthermore, using pyramids with different heights and wall microtopographies as an example, adhesion, morphology and distribution of human MG63 osteosarcoma cells were studied. The results showed that both the height of the topographical features and the microstructural properties of their walls affected cell spreading and distribution. This proof-of-concept study shows that the platform developed here is a useful tool for studying interactions between cells and clinically relevant biomaterials under controlled fluidic regimes.

This paper reports on the preparation of cellular materials by different techniques. Bioactive and resorbable scaffolds based on biphasic calcium phosphate were produced by 3D-printing using extrudable pastes through fine nozzles, according to a pre-defined spatial arrangement considered suitable for bone regeneration and tissue engineering applications. Milled powders of cathode ray glass tubes and egg shell wastes were mixed and compacted to produce recycled glass foams via viscous flow sintering and thermal decomposition of the egg shell component that played the role of foaming agent. © 2016 Portuguese Society of Materials (SPM)

Biointerface design is widely used to functionalize biomaterials with controllable physicochemical properties. Functionalized biointerface provides a versatile platform to connect biological entities and nonbiogenic materials. Existing nanofabrication approaches to create such a nanostructured biointerface involve in low stability of the functionalized nanolayer and simple functionalities that limit its applicability. Here, a stable nanolayered synthetic polypeptide (poly[LA-co-(Glc-alt-Lys)] and modified with arginine-glycine-aspartic acid, PRGD)/basic fibroblast growth factor (bFGF) biointerface is created via structural matching, charge interaction, and hydrogen bonding. The cooperative effect of the PRGD/bFGF biointerface shows multiple functionalities in promoting stem cell adhesion by 33% increase in cell adhesion to poly(d,l-lactic acid) substrate as compared to experiments on bare substrate as a control. Moreover, the biointerface enhances proliferation by 40% in cell density, potential differentiation by 62%, and gene expression by 40 and 80% respectively as compared to the control samples. The fabricated biointerface may have applications in nerve regeneration, tissue repair, and stem cell-based therapy.

The external auditory canal (EAC) is an osseocartilaginous structure extending from the auricle to the eardrum, which can be affected by congenital, inflammatory, and neoplastic diseases, thus reconstructive materials are needed. Current biomaterial-based approaches for the surgical reconstruction of EAC posterior wall still suffer from resorption (biological) and extrusion (synthetic). In this study, 3D fiber deposited scaffolds based on poly(ethylene oxide terephthalate)/poly(butylene terephthalate) were designed and fabricated to replace the EAC wall. Fiber diameter and scaffold porosity were optimized, leading to 200 ± 33 µm and 55% ± 5%, respectively. The mechanical properties were evaluated, resulting in a Young's modulus of 25.1 ± 7.0 MPa. Finally, the EAC scaffolds were tested in vitro with osteo-differentiated human mesenchymal stromal cells (hMSCs) with different seeding methods to produce homogeneously colonized replacements of interest for otologic surgery. This study demonstrated the fabrication feasibility of EAC wall scaffolds aimed to match several important requirements for biomaterial application to the ear under the Tissue Engineering paradigm, including shape, porosity, surface area, mechanical properties and favorable in vitro interaction with osteoinduced hMSCs. This study demonstrated the fabrication feasibility of outer ear canal wall scaffolds via additive manufacturing. Aimed to match several important requirements for biomaterial application to ear replacements under the Tissue Engineering paradigm, including shape, porosity and pore size, surface area, mechanical properties and favorable in vitro interaction with osteo-differentiated mesenchymal stromal cells.

Collagen based devices are frequently associated with foreign body response. Although several pre(e.g. species, state of animal, tissue) and post-(e.g. cross-linking, scaffold architecture) extraction method factors have a profound effect on foreign body response, little is known about which and how during the extraction process factors mediate foreign body response. In this study, we assessed the influence of acetic acid and hydrochloric acid and the utilisation or not of pepsin or salt precipitation during collagen extraction on the yield, purity, free amines, denaturation temperature, resistance to collagenase degradation and macrophage response. Acetic acid/pepsin extracted collagen exhibited the highest yield, purity and free amine content and the lowest denaturation temperature. No differences in resistance to collagenase digestion were detected between the groups. Although all treatments exhibited similar macrophage morphology comprised of round cells (M1 phenotype), elongated cells (M2 phenotype) and cell aggregates (foreign body response), significantly more elongated cells were observed on HC films. Although no differences in metabolic activity were observed between the groups, the DNA concentration was significantly lower for the hydrochloric acid treatments. Further, cytokine analysis revealed that hydrochloric acid treatments induced significantly higher IL-1 beta and TNF-alpha release with respect to acetic acid treatments. Salt precipitation did not influence the parameters assessed. Collectively, these data suggest that during the collagen extraction process variables should also be monitored as, evidently, they affect the physicochemical and biological properties of collagen preparations.

In this review, we discuss recent developments in the field of nanoparticles and their use in tissue regeneration approaches. Owing to their unique chemical properties and flexibility in design, nanoparticles can be used as drug delivery systems, to create novel features within materials or as bioimaging agents, or indeed these properties can be combined to create smart multifunctional structures. This review aims to provide an overview of this research field where the focus will be on nanoparticle-based strategies to stimulate bone regeneration; however, the same principles can be applied for other tissue and organ regeneration strategies. In the first section, nanoparticle-based methods for the delivery of drugs, growth factors and genetic material to promote tissue regeneration are discussed. The second section deals with the addition of nanoparticles to materials to create nanocomposites. Such materials can improve several material properties, including mechanical stability, biocompatibility and biological activity. The third section will deal with the emergence of a relatively new field of research using nanoparticles in advanced cell imaging and stem cell tracking approaches. As the development of nanoparticles continues, incorporation of this technology in the field of regenerative medicine will ultimately lead to new tools that can diagnose, track and stimulate the growth of new tissues and organs.

International audience; Cyclic monomers bearing either vinyl or exomethylene groups have the ability to be polymerized through a radical pathway via a ring-opening mechanism (addition fragmentation process), leading to the introduction of functionalities in the polymer backbone. Radical ring-opening polymerization (rROP) combines the advantages of both ring-opening polymerization and radical polymerization, that is the preparation of polymers bearing heteroatoms in the backbone but with the ease and robustness of a radical process. This current review presents a comprehensive description of rROP by detailing: (0 the various monomers that polymerize through rROP; (ii) the main parameters that govern the rROP mechanism; (iii) the copolymerization by conventional or controlled/living radical polymerization between rROP monomers and traditional vinyl monomers to obtain copolymers with advanced properties; (iv) the different applications (low shrinkage materials and pieparation of (bio)degradable materials) of rROP monomer-containing materials, and (v) the main alternatives to rROP to induce degradability to materials obtained by a radical polymerization.

In this work, gelatin was crosslinked with oxidized alginate in the presence of borax yielding a hybrid biohydrogel system with the ability to self-repair upon mechanical damage. A judicious balance between concentration, stoichiometric ratio of the two biopolymers and gelatin source was found necessary to achieve optimal self-healing properties of these biohydrogels. The pH was found to have a major influence on the reconstruction of the damaged hydrogel interface, confirming that the dynamic Schiff base linkages between the amine groups of gelatin and the aldehyde groups of oxidized alginate play a fundamental role on the healing process of the hybrid gels. © 2017 the Partner Organisations., This work was supported the University of Regensburg, the Deutsche Forschungsgemeinschaft (DFG, DI 1748/3-1) and the SINCHEM Joint Doctorate Programme selected under the Erasmus Mundus Action 1 Programme (FPA 2013-0037). We are thankful to the Laboratoire Charles Coulomb (Universitéde Montpellier & CNRS, Montpellier, France) for granting access to the rheometer and to M. Jean-Marc Fromental for assistance with the rheological measurements. D. D. D. thanks the DFG for the Heisenberg Professorship Award.

Nanocomposite gels are a fascinating class of polymeric materials with an integrative assembly of organic molecules and organic/inorganic nanoparticles, offering a unique hybrid network with synergistic properties. The mechanical properties of such networks are similar to those of natural tissues, which make them ideal biomaterial candidates for tissue engineering applications. Existing nanocomposite gel systems, however, lack many desirable gel properties, and their suitability for surface coatings is often limited. To address this issue, this article aims at generating multifunctional nanocomposite gels that are injectable with an appropriate time window, functional with bicyclononynes (BCN), biocompatible and slowly degradable, and possess high mechanical strength. Further, the in situ network-forming property of the proposed system allows the fabrication of ultrathin nanocomposite coatings in the submicrometer range with tunable wettability and roughness. Multifunctional nanocomposite gels were fabricated under cytocompatible conditions (pH 7.4 and T = 37 °C) using laponite clays, isocyanate (NCO)-terminated sP(EO-stat-PO) macromers, and clickable BCN. Several characterization techniques were employed to elucidate the structure-property relationships of the gels. Even though the NCO-sP(EO-stat-PO) macromers could form a hydrogel network in situ on contact with water, the incorporation of laponite led to significant improvement of the mechanical properties. BCN motifs with carbamate links were used for a metal-free click ligation with azide-functional molecules, and the subsequent gradual release of the tethered molecules through the hydrolysis of carbamate bonds was shown. The biocompatibility of the hydrogels was examined through murine macrophages, showing that the material composition strongly affects cell behavior.

Nanocomposite scaffolds ate studied widely due to their resemblance with the natural extracellular matrix of bone, but their use as a bone tissue engineered scaffold is clinically hampered due to low mechanical stiffness, inadequate osteoconduction, and graft associated infections. The purpose of the current study was the development of a mechanically stiff nanocomposite scaffold using biodegradable gellan and xanthan polymers reinforced with bioglass nanoparticles (nB) (Size; 20-120 nm). These nanocomposite scaffolds, were cross-linked with zinc sulfate ions to improve their osteoconduction and antibacterial properties for the regeneration of a functional bone. The compressive strength and modulus of the optimized nanocomposite scaffold (1% w/v polymer reinforced with 4%w/v nB nanoparticles, cross-linked with 1.5 mM zinc sulfate) was 1.91 +/- 0.31 MPa and 2.036 +/- 1.08 MPa, respectively, which was comparable to the trabecular bone and very high compared to nanocomposite scaffolds reported in earlier studies. Further, in vitro simulated body fluid (SBF) study suggested deposition of biomimetic apatite on the surface of zinc cross-linked nanocomposite scaffolds confirming their bioactivity. MG 63 osteoblast-like cells cultured with the nanocomposite scaffold's responded to matrix stiffness with better adhesion, spreading and cellular interconnections compared to the polymeric gellan and xanthan scaffolds. Incorporation of bioglass nanoparticles and zinc cross linker in nanocomposite scaffolds demonstrated 62% increment in expression of alkaline phosphatase activity (ALP) and 150% increment in calcium deposition of MG 63 osteoblast-like cells compared to just gellan and xanthan polymeric scaffolds. Furthermore, zinc cross-linked nanocomposite scaffolds significantly inhibited the growth of Gram-positive Bacillus subtilis (70% reduction) and Gram-negative Escherichia coli (81% reduction) bacteria. This study demonstrated a facile approach to tune the mechanical stiffness, bioactivity, osteoconduction potential and bacteriostatic properties of scaffolds, which marked it as a potential bone tissue engineered scaffold.

International audience; To develop bioactive scaffolds of targeted properties for tissue repair or biomedical applications, hybrid microfiber-nanoparticle (MF-NP) matrices capable of controlled nanoparticle (NP) delivery were prepared through two novel approaches. In a first strategy, the suppleness of the jet-spraying method to produce polymer microfibers (MF) was used to deposit poly(D,L-lactide) (PLA) NP on poly(lactic-co-glycolic acid) (PLGA) MF by direct co-projection. The second approach relied on the post-incubation of PLA NP aqueous dispersion with MF preliminarily prepared by jet-spraying. NP coverage density onto MF and NP release was assessed by scanning electron microscopy and fluorescence measurements using coumarin-6 loaded NP. The first process was shown to allow high coverage density of NP onto MF (300 mu g/mg MF) and strong association, with no NP release observed over time. In the second approach, direct incubation of PLA NP with PLA MF led to lower NP coverage density (40 mu g/mg MF) with very fast release of NP from MF. The pre-coating of MF with poly-L-lysine (PLL) or the one of NP with lysozyme as a model protein drug afforded a higher coverage density and stronger association, coupled with a more sustained release of NP from MF over time. These results show the possibility to control the immobilization density and release of NP through appropriate preparation process and surface modification, and are of prime interest for the development of complex scaffolds with orchestrated bioactivity. (C) 2015. Elsevier B.V. All rights reserved.

Electroactive electrospun fiber mat composites based on poly (vinylidene fluoride) (PVDF) with 5 and 10% of 1-ethyl-3-methylimidazolium bis (trifluoromethanesulfonyl)amide ([C2mim][NTf2]) ionic liquid (IL) were developed for potential applications in the biomedical area. The morphology and polymer crystalline phase content of the fibers were evaluated as a function of the processing conditions. Hydrophobic random and aligned fibers have been obtained with average fiber diameters between ~700 and 500 nm, the smaller diameters corresponding to the aligned fiber mats. The results show that the charge structure of [C2mim][NTf2] induces the crystallization of the PVDF fibers in the piezoelectric B-phase with full crystallization in this phase for an ionic liquid content of 10 wt%. Furthermore, the presence of the ionic liquid also increases the degree of crystallinity of the fibers. Thermal degradation studies show a single degradation process which is strongly influenced by the polymer-IL interactions. Finally, the non-cytotoxicity of the fiber mats indicates their suitability for biomedical applications., This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the project UID/Multi/04551/2013 and 2012 Investigator FCT program. The authors also thank support from the COST Action, MP1206 “Electrospun Nano-fibers for bio inspired composite materials and innovative industrial applications”. JCD, DMC, SR and CR thank the FCT for the SFRH/BD/90215/2012, SFRH/BD/82411/2011, SFRH/BD/111478/2015 and SFRH/BPD/90870/2012 grants, respectively. The authors thank financial support from the Basque Government Industry Department under the ELKARTEK Program. SLM thanks the Diputación de Bizkaia for financial support under the Bizkaia Talent program.

The present work aims at constructing the ideal scaffold matrix of which the physico-chemical properties can be altered according to the targeted tissue regeneration application. Ideally, this scaffold should resemble the natural extracellular matrix (ECM) as close as possible both in terms of chemical composition and mechanical properties. Therefore, hydrogel films were developed consisting of methacrylamide-modified gelatin and starch-pentenoate building blocks because the ECM can be considered as a crosslinked hydrogel network consisting of both polysaccharides and structural, signaling and cell-adhesive proteins. For the gelatin hydrogels, three different substitution degrees were evaluated including 31%, 72% and 95%. A substitution degree of 32% was applied for the starch-pentenoate building block. Pure gelatin hydrogels films as well as interpenetrating networks with gelatin and starch were developed. Subsequently, these films were characterized using gel fraction and swelling experiments, high resolution-magic angle spinning H-1 NMR spectroscopy, rheology, infrared mapping and atomic force microscopy. The results indicate that both the mechanical properties and the swelling extent of the developed hydrogel films can be controlled by varying the chemical composition and the degree of substitution of the methacrylamide-modified gelatin applied. The storage moduli of the developed materials ranged between 14 and 63 kPa. Phase separation was observed for the IPNs for which separated starch domains could be distinguished located in the surrounding gelatin matrix. Furthermore, we evaluated the affinity of aggrecan for gelatin by atomic force microscopy and radiolabeling experiments. We found that aggrecan can be applied as a bioactive coating for gelatin hydrogels by a straightforward physisorption procedure. Thus, we achieved distinct fine-tuning of the physico-chemical properties of these hydrogels which render them promising candidates for tissue engineering approaches. (C) 2016 Elsevier Ltd. All rights reserved.

A key goal of tissue engineering is to use cells, materials, and supporting structures to substitute for damaged tissue and repair its function. For tissue specificity, using appropriate materials for scaffold construction is imperative. An ideal material used for fabricating tissue-engineered scaffolds would provide both structural support and the control of bioactivity. In this regard, chitin-based materials are regarded as appropriate candidates because of their biodegradable properties and ability to be easily fabricated into distinct types of tissue scaffolds. Quaternary ammonium groups that are present within chitin-based materials efficiently interact with the cell. Chitin-based materials can be readily fabricated into many forms of scaffolds, including hydrogels, microcapsules, membranous films, sponges, tubes, and a variety of three-dimensional porous structures (1–4). Chitosan, a specific moiety of chitin, is a mucopolysaccharide which is used in bioengineering. It is be biocompatible and biodegradable due to cellular lysosomal action. As a non-toxic and non-immunogenic material, its degradation products are not thought to cause any reaction in the body. (1–3,5) Chitosan, being an important biomaterial for use in the treatment of skin damage and burns, has also been reported to increase regeneration processes in the dermis and accelerate wound healing (6–10). Chitosan, which is similar to glycosaminoglycans, has been suggested as a very suitable biomaterial to repair connective tissue (11). Recent studies have revealed the frequent interaction of chitosan and its derivatives with different cell types (4, 12). Chitosan hydrogel can be prepared and cross-linked either covalently or ionically. Covalent cross-linking provides chitosan hydrogel with a permanent architecture that can be used to absorb and release bioactive factors or water. Chitin-based materials and their derivatives are receiving increased attention in tissue engineering because of their unique and appealing biological properties (4–6, 8–10, 13, 14). Despite numerous primary cell culture studies investigating the effects of chitosan on cellular proliferation, there is little information available about the effects of chitosan on continuous cell lines. Preliminary experiments in our group suggested that the simple addition of soluble chitosan to cell culture media (as opposed to the generation of membranes in culture vessels) had positive effects on cell growth and behaviour. The present study was designed to investigate the putative effects of a solution form of chitosan on the activity of Swiss 3T3 mouse embryonic fibroblast continuous cell line (15, 16), comparing the effects to those induced by membranous chitosan.

Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction.

Low back pain is one of the most reported medical conditions associated to intervertebral disc (IVD) degeneration. Nucleus pulposus (NP) is often regarded as the structure where intervertebral disc degeneration begins. Gellan gum-based (GG) hydrogels for acellular and cellular tissue engineering strategies have been developed for finding applications as NP substitutes. The innovative strategy is based on the reinforcement of the hydrogel matrix with biocompatible and biodegradable GG microparticles (MPs), which are expected to improve the mechanical properties, while allowing to tailor its degradation rate. In this study, several GG MPs/hydrogels discs formulations were prepared by means of mixing high (HAGG 0.75% (w/v)) and low acyl (LAGG 2% (w/v)) GG aqueous solutions at different ratios, namely 75%:25% (v/v), 50%:50% (v/v), 25%:75% (v/v), respectively. The GG MPs size was measured using a stereo microscope and their dispersion within the hydrogel matrix was evaluated by means of staining the MPs with Toluidine Blue-O. The developed GG MPs/hydrogel discs were physico-chemically characterized by Fourier-transform infrared spectroscopy and 1H-nuclear magnetic resonance spectroscopy. The swelling behaviour and degradation rate were assessed by immersion in a phosphate buffer saline solution for the period of 14 days. The morphology and mechanical behaviour were investigated by scanning electron microscopy and dynamic mechanical analysis, respectively. The mechanical properties of the hydrogels discs were improved by mixing the gels with the MPs. In addition, the possible cytotoxicity of the leachables released by MPs/hydrogel discs was screened in vitro, using a mouse lung fibroblast cell line (L929 cells). In order to investigate the encapsulation efficacy of L929 cells into the GG MPs/hydrogel discs, cells were stained with DAPI blue/Texas Red-Phalloidin and observed by confocal microscopy, after 24, 48 and 72 hours of culturing. A cell viability assay was also performed using Calcein AM staining. The cell culture studies demonstrated that MPs/hydrogel discs are non-cytotoxic over L929 cells. It was also demonstrated that L929 cells can be successfully encapsulated into the GG MPs of different formulations, remaining viable after 72 hours of culturing. This study showed that GG hydrogel matrices reinforced with cell-loaded MPs could be a candidate strategy for NP regeneration., Fundação para a Ciência e a Tecnologia (FCT) throught the POCTI and FEDER, including ProteoLight

This paper describes the biological consequences of presenting electrospun fibrinogen (FBG) to endothelial cells as a spatially organized nanofibrous matrix. Aligned and randomly oriented FBG nanofibers with an average diameter of less than 200 nm were obtained by electrospinning of native FBG solution. Electrophoretic profiling confirmed that the electrospun FBG resembled the native protein structure, and fluorescent tracing of FITC-labeled FBG showed that electrospun fibers withstood immersion in physiological solutions reasonably well for several days. With respect to cellular interactions, the nanofibrous FBG matrix provided better conditions for initial recognition by human umbilical vein endothelial cells compared to pre-adsorbed FBG on a flat surface. Furthermore, the spatial organization of electrospun FBG fibers presented opportunities for guiding the cellular behavior in a way that is not possible when the protein is presented in another form (e. g. adsorbed or soluble). For example, on aligned FBG fibers, cells rapidly oriented themselves along the fibers, and time-lapse recordings revealed pronounced cellular movements restricted to the fiber direction. In great contrast, on randomly deposited fibers, cells acquired a stellate-like morphology and became locally immobilized by the fibers. We also show that the FBG fiber orientation significantly influenced both the cytoskeleton organization in confluent cell layers and the orientation of the extracellular fibronectin matrix secreted by the cells. In conclusion, this study demonstrates that electrospun FBG nanofibers can be a promising tool for guiding endothelial cell behavior for tissue engineering applications.