In the present work, novel freestanding multilayered films based on chitosan (CHI), alginate (ALG), and functionalized few-layer graphene are developed through layer-by-layer assembly. First, functionalized few-layer graphene aqueous suspensions are prepared from graphite by a stabilizer-assisted liquid phase exfoliation process, using a pyrene derivative as stabilizer. Afterward, the films are produced and their physical, morphological, thermal, and mechanical properties are evaluated. Furthermore, their degradation and swelling profiles, as well as their biological behavior, are assessed. The incorporation of functionalized few-layer graphene results in films with a nanolayered structure, lower roughness than the control CHI/ALG films, and hydrophilic behavior. The mechanical characterization reveals an increase of the Young's modulus, ultimate tensile strength, and elongation at break due to the incorporation of the graphene derivative. A decrease in the electrical resistivity of the multilayered films is also observed. The biological assays reveal improved cytocompatibility toward L929 cells when functionalized few-layer graphene is incorporated in the CHI/ALG matrix. Therefore, these new graphene-reinforced multilayered films exhibit interesting properties and great potential for biomedical applications, particularly in wound healing and cardiac and bone tissue engineering.

This work is the first reporting the use of layer-by-layer to produce adhesive free-standing (FS) films fully produced using natural-based macromolecules: chitosan (CHI), alginate (ALG) and sulfated levan (L-S). The deposition conditions of the natural polymers were studied through zeta potential measurements and quartz crystal microbalance with dissipation monitoring analysis. The properties of the FS films were evaluated and compared with the control ones composed of only CHI and ALG in order to assess the influence of levan polysaccharide introduced in the multilayers. Tensile tests, dynamic mechanical analysis and single lap shear strength tests were performed to evaluate the mechanical properties of the prepared FS films. The presence of L-S conferred both higher tensile strength and shear strength to the developed FS membranes. The results showed an adhesion strength 4 times higher than the control (CHI/ALG) FS films demonstrating the adhesive character of the FS films containing L-S. Morphological and topography studies were carried out revealing that the crosslinking reaction granted the L-S based FS film with a higher roughness and surface homogeneity. Preliminary biological assays were performed by cultivating myoblasts cells on the surface of the produced FS films. Both crosslinked and uncrosslinked FS films containing L-S were cytocompatible and myoconductive. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Developing complex supramolecular biomaterials through highly dynamic and reversible noncovalent interactions has attracted great attention from the scientific community aiming key biomedical and biotechnological applications, including tissue engineering, regenerative medicine, or drug delivery. In this study, the authors report the fabrication of hybrid supramolecular multilayered biomaterials, comprising high‐molecular‐weight biopolymers and oppositely charged low‐molecular‐weight peptide amphiphiles (PAs), through combination of self‐assembly and electrostatically driven layer‐by‐layer (LbL) assembly approach. Alginate, an anionic polysaccharide, is used to trigger the self‐assembling capability of positively charged PA and formation of 1D nanofiber networks. The LbL technology is further used to fabricate supramolecular multilayered biomaterials by repeating the alternate deposition of both molecules. The fabrication process is monitored by quartz crystal microbalance, revealing that both materials can be successfully combined to conceive stable supramolecular systems. The morphological properties of the systems are studied by advanced microscopy techniques, revealing the nanostructured dimensions and 1D nanofibrous network of the assembly formed by the two molecules. Enhanced C2C12 cell adhesion, proliferation, and differentiation are observed on nanostructures having PA as outermost layer. Such supramolecular biomaterials demonstrate to be innovative matrices for cell culture and hold great potential to be used in the near future as promising biomimetic supramolecular nanoplatforms for practical applications.

Novel nanostructured free-standing films based on chitosan, alginate and functionalized flake and ribbon-shaped graphene were developed using the layer-by-layer process.

Natural-based and biomimetic multilayer membranes, whose surface resembles the nanotopography of the native extracellular matrix, are able to induce cellular alignment along with the pattern direction. João F. Mano and co-workers use a simple layer-by-layer technology to produce such platforms, that can be a promising system to control cell behavior and to regenerate tissues, in article number 1601462.

Platforms containing multiple arrays for high-throughput screening are demanded in the development of biomaterial libraries. Here, an array platform for the combinatorial analysis of cellular interactions and 3D porous biomaterials is described. Using a novel method based on computer-aided manufacturing, wettable regions are printed on superhydrophobic surfaces, generating isolated spots. This freestanding benchtop array is used as a tool to deposit naturally derived polymers, chitosan and hyaluronic acid, with bioactive glass nanoparticles (BGNPs) to obtain a scaffold matrix. The effect of fibronectin adsorption on the scaffolds is also tested. The biomimetic nanocomposite scaffolds are shown to be osteoconductive, non-cytotoxic, promote cell adhesion, and regulate osteogenic commitment. The method proves to be suitable for screening of biomaterials in 3D cell cultures as it can recreate a multitude of combinations on a single platform and identify the optimal composition that drives to desired cell responses. The platforms are fully compatible with commercially routine cell culture labware and established characterization methods, allowing for a standard control and easy adaptability to the cell culture environment. This study shows the value of 3D structured array platforms to decode the combinatorial interactions at play in cell microenvironments.

Cell-based regenerative medicine strategies hold a great potential to revolutionize the treatment of a large number of injuries with limited regenerative potential. However, the effectiveness of the simple injection of a cell suspension in a target site/tissue of action is often limited by the dispersion of cells toward other tissues, hindering their therapeutic action. Nevertheless, the development of a custom-made cell carrier that can perfectly fit a patient’s defect and be ready on demand is still a challenging task. The present study proposes the development of an off-the-shelf injectable cell delivery system combining a photocross-linkable hyaluronic acid (HA) matrix enriched with platelet lysate (PL) and human adipose tissue-derived stem cells (hASCs), which can be stored using standard cryopreservation methods and used when required. The obtained results indicate that the mechanical and viscoelastic properties of the system are improved in the presence of cells and no significant differences were identified between cell-laden hydrogels produced before or after cryopreservation. In PL-enriched hydrogels, cells tend to better recover from cryopreservation maintaining the values of cell viability and DNA content. Moreover, viable cells laden in our system and expressing stemness markers were detected after 21 days in culture. Altogether, the results obtained in this work demonstrate the potential of the developed strategy as an injectable cell delivery system for ready-to-use applications or as a cryopreserved product to be available on demand for cell-based therapies.

Inspired by native extracellular matrix (ECM) together with the multilevel architecture observed in nature, a material which topography recapitulates topographic features of the ECM and the internal architecture mimics the biological materials organization is engineered. The nanopatterned design along the XY plane is combined with a nanostructured organization along the Z axis on freestanding membranes prepared by layer-by-layer deposition of chitosan and chondroitin sulfate. Cellular behavior is monitored using two different mammalian cell lines, fibroblasts (L929) and myoblasts (C2C12), in order to perceive the response to topography. Viability, proliferation, and morphology of L929 are sensitively controlled by topography; also differentiation of C2C12 into myotubes is influenced by the presence of nanogrooves. This kind of nanopatterned structure has also been associated with strong cellular alignment. To the best of the knowledge, it is the first time that such a straightforward and inexpensive strategy is proposed to produce nanopatterned freestanding multilayer membranes. Controlling cellular alignment plays a critical role in many human tissues, such as muscles, nerves, or blood vessels, so these membranes can be potentially useful in specific tissue regeneration strategies.

This review is focused on the use of membranes for the specific application of bone regeneration. The first section focuses on the relevance of membranes in this context and what are the specifications that they should possess to improve the regeneration of bone. Afterward, several techniques to engineer bone membranes by using bulk-like methods are discussed, where different parameters to induce bone formation are disclosed in a way to have desirable structural and functional properties. Subsequently, the production of nanostructured membranes using a bottom-up approach is discussed by highlighting the main advances in the field of bone regeneration. Primordial importance is given to the promotion of osteoconductive and osteoinductive capability during the membrane design. Whenever possible, the films prepared using different techniques are compared in terms of handability, bone guiding ability, osteoinductivity, adequate mechanical properties, or biodegradability. A last chapter contemplates membranes only composed by cells, disclosing their potential to regenerate bone.

The lack of knowledge on the degradation of layer-by-layer structures is one of the causes hindering its translation to preclinical assays. The enzymatic degradation of chitosan/hyaluronic acid films in the form of ultrathin films, freestanding membranes, and microcapsules was studied resorting to hyaluronidase. The reduction of the thickness of ultrathin films was dependent on the hyaluronidase concentration, leading to thickness and topography variations. Freestanding membranes exhibited accelerated weight loss up to 120 h in the presence of the enzyme, achieving complete degradation. Microcapsules with around 5 μm loaded simultaneously with FITC-BSA and hyaluronidase showed that the coencapsulation of such enzyme and protein mixture led to a FITC-BSA release four times higher than in the absence of hyaluronidase. The results suggest that the degradation of LbL devices may be tuned via embedded enzymes, namely, in the controlled release of active agents in biomedical applications.

Moldable superhydrophobic surfaces are produced by freely covering a solid surface with specifically designed microcapsules. Owing to the presence of Fe-particles within the former microcapsules, they can be temporarily fixed on different substrates using a permanent magnet. Such systems are prospective candidates for the fabrication of water/oil repellent surfaces, liquid marbles, or microfluidic channels and as templates for microspheres fabrication. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The design of self-standing multilayered structures based on biopolymers has been attracting increasing interest due to their potential in the biomedical field. However, their use has been limited due to their gel-like properties. Herein, we report the combination of covalent and ionic cross-linking, using natural and non-cytotoxic cross-linkers, such as genipin and calcium chloride (CaCl2). Combining both cross-linking types the mechanical properties of the multilayers increased and the water uptake ability decreased. The ionic cross-linking of multilayered chitosan (CHI)-alginate (ALG) films led to freestanding membranes with multiple interesting properties, such as: improved mechanical strength, calcium-induced adhesion and shape memory ability. The use of CaCl2 also offered the possibility of reversibly switching all of these properties by simple immersion in a chelate solution. We attribute the switch-ability of the mechanical properties, shape memory ability and the propensity for induced-adhesion to the ionic cross-linking of the multilayers. These findings suggested the potential of the developed polysaccharide freestanding membranes in a plethora of research fields, including in biomedical and biotechnological fields.

In this work, novel free-standing (FS) films based on chitosan, alginate and graphene oxide (GO) were developed through layer-by-layer assembly. First, GO was synthesized from graphite and multi-walled carbon nanotubes using a modified Hummer's method, yielding oxidized graphene flakes (o-GFs) and oxidized graphene nanoribbons (o-GNRs), respectively, which were then characterized. Then FS films were produced and their morphological, thermal and mechanical properties, as well as the o-GF and o-GNR dispersion along the films were assessed. Their degradation and swelling profiles as well as their biological behavior were evaluated. Graphite and nanotubes were successfully oxidized and exfoliated forming stable suspensions that could be combined with chitosan (CHI) and alginate (ALG) solutions by layer-by-layer processing. The addition of o-GFs and o-GNRs resulted in rougher, hydrophilic FS films with significantly improved mechanical properties relative to CHI/ALG films. The presence of o-GFs or o-GNRs did not affect the thermal stability and the addition of o-GFs resulted in films with enhanced cytocompatibility. The results demonstrate the high potential of the GO reinforced films for biomedical applications, in particular o-GF films, for wound healing, and cardiac and bone engineering applications.

Chitosan (CHT) spheres incorporating bioactive glass nanoparticles (BGNPs) were prepared to obtain a composite system able to induce the deposition of an apatite layer upon immersion in a biological-like environment. Spheres were synthesized with different concentrations of BGNPs obtained from a sol–gel route and genipin (GNP, the crosslinking agent). Biomimetic superhydrophobic surfaces were used to support droplets of chitosan-based solutions that after crosslinking enabled to produce well developed spherical particles with controlled sizes. From SEM and EDS analysis it was observed the successful formation of bone-like apatite on the surface when the spheres were immersed in a simulated body fluid (SBF). Lower GNP concentration promoted more apatite formation. The spheres presented shape memory behaviour triggered by hydration with high values of shape fixity and shape recovery. This effect was used to introduce these spheres in a bone defect showing a good geometrical accommodation in the implanted site. The bioactive spheres allowed the incorporation of a drug model and its effective release. Overall the developed nanocomposite spheres showed great potential for bone tissue engineering in particular as a device to be implanted using minimal invasive procedures.

The regeneration of soft biological tissues requires new substitutes that exhibit mechanical properties similar to the native tissue. Herein, thin saloplastic membranes with tunable physical properties are prepared by complexation of chitosan and alginate solutions containing different concentrations of sodium chloride. Polyelectrolyte complexes (PECs) are transferred to flat Petri dishes for compaction into membrane shapes by sedimentation and solvent evaporation. All membranes are resistant to degradation by lysozyme and are stable in solutions with pH values between 1 and 13. Immersing the different membranes in new doping solutions of increasing salt concentrations triggers the typical saloplastic behavior, with a high water absorption and decrease of the rigidity and ultimate tensile strength. The range of such variations is tuned by the sodium chloride amount used in the synthesis: high salt concentrations increase water uptake and tensile moduli, while decreasing the ultimate strength. Cellular assays demonstrate high proliferation rates and viability of L929 fibroblasts seeded onto the most rigid membranes. The results validate the use of saloplastic membranes as soft tissue substitutes for future biomedical applications.

We investigated the pH-dependent properties of multilayered films made of chitosan (CHI) and alginate (ALG) and focused on their postassembly response to different pH environments using a quartz crystal microbalance with dissipation monitoring (QCM-D), swelling studies, ζ potential measurements, and dynamic mechanical analysis (DMA). In an acidic environment, the multilayers presented lower dissipation values and, consequently, higher moduli when compared with the values obtained for the pH used during the assembly (5.5). When the multilayers were exposed to alkaline environments, the opposite behavior occurred. These results were further corroborated by the ability of this multilayered system to exhibit a reversible swelling-deswelling behavior within the pH range from 3 to 9. The changes in the physicochemical properties of the multilayer system were gradual and different from those of individual solubilized polyelectrolytes. This behavior is related to electrostatic interactions between the ionizable groups combined with hydrogen bonding and hydrophobic interactions. Beyond the pH range of 3-9, the multilayers were stabilized by genipin cross-linking. The multilayered films also became more rigid while the pH responsiveness conferred by the ionizable moieties of the polyelectrolytes was preserved. This work demonstrates the versatility and feasibility of LbL methodology to generate inherently pH stimulus-responsive nanostructured films. Surface functionalization using pH responsiveness endows several biomedical applications with abilities such as drug delivery, diagnostics, microfluidics, biosensing, and biomimetic implantable membranes.

Fundacao para a Ciencia e Tecnologia (grant SFRH/BPD/95446/2013), ‘‘Fundo Social Europeu’’ and ‘‘Programa Operacional de Potencial Humano’’

Free-standing (FS) membranes have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we studied the potential of free-standing membranes made by the layer-by-layer assembly of chitosan and alginate to be used as a simple biomimetic system of the periosteum. The design of a periosteum-like membrane implies the elaboration of a thick membrane suitable for both muscle and bone formation. Our aim was to produce well-defined ∼50 μm thick polysaccharide membranes that could be easily manipulated, were mechanically resistant, and would enable both myogenesis and osteogenesis in vitro and in vivo. The membranes were chemically crosslinked to improve their mechanical properties. Crosslinking chemistry was followed via Fourier transform infrared spectroscopy and the mechanical properties of the membranes were assessed using dynamic mechanical analysis. The loading and release of the potent osteoinductive growth factor bone morphogenetic protein 2 (BMP-2) inside and outside of the FS membrane was followed by fluorescence spectroscopy in a physiological buffer over 1 month. The myogenic and osteogenic potentials of the membranes in vitro were assessed using BMP-2-responsive skeletal myoblasts. Finally, their osteoinductive properties in vivo were studied in a preliminary experiment using a mouse ectopic model. Our results showed that the more crosslinked FS membranes enabled a more efficient myoblast differentiation in myotubes. In addition, we showed that a tunable amount of BMP-2 can be loaded into and subsequently released from the membranes, depending on the crosslinking degree and the initial BMP-2 concentration in solution. Only the more crosslinked membranes were found to be osteoinductive in vivo. These polysaccharide-based membranes have strong potential as a periosteum-mimetic scaffold for bone tissue regeneration.