RGD-pectin microfiber patches for guiding muscle tissue regeneration.

Opportunely arranged microscaled fibers offer an attractive 3D architecture for tissue regeneration as they may enhance and stimulate specific tissue regrowth. Among different scaffolding options, encapsulating cells in degradable hydrogel microfibers appears as particularly attractive strategy. Hydrogel patches, in fact, offer a highly hydrated environment, allow easy incorporation of biologically active molecules, and can easily adapt to implantation site. In addition, microfiber architecture is intrinsically porous and can improve mass transport, vascularization, and cell survival after grafting. Anionic polysaccharides, as pectin or the more popular alginate, represent a particularly promising choice for the fabrication of cell-laden patches, due to their extremely mild gelation in the presence of divalent ions and widely accepted biocompatibility. In this study, to combine the favorable properties of hydrogel and fibrous architecture, a simple coaxial flow wet-spinning system was used to prepare cell-laden, 3D fibrous patches using RGD-modified pectin. Rapid fabrication of coherent self-standing patches, with diameter in the range of 100-200 µm and high cell density, was possible by accurate choice of pectin and calcium ions concentrations. Cells were homogeneously dispersed throughout the microfibers and remained highly viable for up to 2 weeks, when the initial stage of myotubes formation was observed. Modified-pectin microfibers appear as promising scaffold to support muscle tissue regeneration, due to their inherent porosity, the favorable cell-material interaction, and the possibility to guide cell alignment toward a functional tissue.

Influence of Culture Substrates on Morphology and Function of Pulmonary Alveolar Cells In Vitro.

Cell's microenvironment has been shown to exert influence on cell behavior. In particular, matrix-cell interactions strongly impact cell morphology and function. The purpose of this study was to analyze the influence of different culture substrate materials on phenotype and functional properties of lung epithelial adenocarcinoma (A549) cells. A549 cells were seeded onto two different biocompatible, commercially available substrates: a polyester coverslip (Thermanox™ Coverslips), that was used as cell culture plate control, and a polydimethylsiloxane membrane (PDMS, Elastosil® Film) investigated in this study as alternative material for A549 cells culture. The two substrates influenced cell morphology and the actin cytoskeleton organization. Further, the Yes-associated protein (YAP) and its transcriptional coactivator PDZ-binding motif (TAZ) were translocated to the nucleus in A549 cells cultured on polyester substrate, yet it remained mostly cytosolic in cells on PDMS substrate. By SEM analysis, we observed that cells grown on Elastosil® Film maintained an alveolar Type II cell morphology. Immunofluorescence staining for surfactant-C revealing a high expression of surfactant-C in cells cultured on Elastosil® Film, but not in cells cultured on Thermanox™ Coverslips. A549 cells grown onto Elastosil® Film exhibited morphology and functionality that suggest retainment of alveolar epithelial Type II phenotype, while A549 cells grown onto conventional plastic substrates acquired an alveolar Type I phenotype.

Characterization of the Microflow Through 3D Synthetic Niche Microenvironments Hosted in a Millifluidic Bioreactor.

Background: Development of new medicines is a lengthy process with high risk of failure since drug efficacy measured in vitro is difficult to confirm in vivo. Intended to add a new tool aiding drug discovery, the MOAB-NICHOID device was developed: a miniaturized optically accessible bioreactor (MOAB) housing the 3D engineered scaffold NICHOID. The aim of our study was to characterize the microflow through the 3D nichoid microenvironment hosted in the MOAB-NICHOID device. Methods: We used computational fluid dynamics (CFD) simulations to compute the flow field inside a very fine grid resembling the scaffold microenvironment. Results: The microflow inside the multi-array of nichoid blocks is fed and locally influenced by the mainstream flow developed in the perfusion chamber of the device. Here we have revealed a low velocity, complex flow field with secondary, backward, or local recirculation micro-flows induced by the intricate architecture of the nichoid scaffold. Conclusion: Knowledge of the microenvironment inside the 3D nichoids allows planning of cell experiments, to regulate the transport of cells towards the scaffold substrate during seeding or the spatial delivery of nutrients and oxygen which affects cell growth and viability.

Cross-Linking Optimization for Electrospun Gelatin: Challenge of Preserving Fiber Topography.

Opportunely arranged micro/nano-scaled fibers represent an extremely attractive architecture for tissue engineering, as they offer an intrinsically porous structure, a high available surface, and an ideal microtopography for guiding cell migration. When fibers are made with naturally occurring polymers, matrices that closely mimic the architecture of the native extra-cellular matrix and offer specific chemical cues can be obtained. Along this track, electrospinning of collagen or gelatin is a typical and effective combination to easily prepare fibrous scaffolds with excellent properties in terms of biocompatibility and biomimicry, but an appropriate cross-linking strategy is required. Many common protocols involve the use of swelling solvents and can result in significant impairment of fibrous morphology and porosity. As a consequence, the efforts for processing gelatin into a fiber network can be vain, as a film-like morphology will be eventually presented to cells. However, this appears to be a frequently overlooked aspect. Here, the effect on fiber morphology of common cross-linking protocols was analyzed, and different strategies to improve the final morphology were evaluated (including alternative solvents, cross-linker concentration, mechanical constraint, and evaporation conditions). Finally, an optimized, fiber-preserving protocol based on carbodiimide (EDC) chemistry was defined.

Electrophoretic processing of chitosan based composite scaffolds with Nb-doped bioactive glass for bone tissue regeneration.

Bioactive glasses (BGs), due to their ability to influence osteogenic cell functions, have become attractive materials to improve loaded and unloaded bone regeneration. BG systems can be easily doped with several metallic ions (e.g., Ag, Sr, Cu, Nb) in order to confer antibacterial properties. In particular, Nb, when compared with other metal ions, has been reported to be less cytotoxic and possess the ability to enhance mineralization process in human osteoblast populations. In this study, we co-deposited, through one-pot electrophoretic deposition (EPD), chitosan (CS), gelatin (GE) and a modified BG containing Nb to obtain substrates with antibacterial activity for unloaded bone regeneration. Self-standing composite scaffolds, with a defined porosity (15-90 µm) and homogeneous dispersion of BGs were obtained. TGA analysis revealed a BG loading of about 10% in the obtained scaffolds. The apatite formation ability of the scaffolds was evaluated in vitro in simulated body fluid (SBF). SEM observations, XRD and FT-IR spectra showed a slow (21-28 days) yet effective nucleation of CaP species on BGs. In particular, FT-IR peak around 603 cm-1 and XRD peak at 2θ = 32°, denoted the formation of a mineral phase after SBF immersion. In vitro biological investigation revealed that the release of Nb from composite scaffolds had no cytotoxic effects. Interestingly, BG-doped Nb scaffolds displayed antibacterial properties, reducing S. lutea and E. coli growth of ≈60% and ≈50%, respectively. Altogether, the obtained results disclose the produced composite scaffolds as promising materials with inherent antibacterial activity for bone tissue engineering applications.

Bottom-up engineering of cell-laden hydrogel microfibrous patch for guided tissue regeneration.

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.

Cross-Linking Strategies for Electrospun Gelatin Scaffolds.

Electrospinning is an exceptional technology to fabricate sub-micrometric fiber scaffolds for regenerative medicine applications and to mimic the morphology and the chemistry of the natural extracellular matrix (ECM). Although most synthetic and natural polymers can be electrospun, gelatin frequently represents a material of choice due to the presence of cell-interactive motifs, its wide availability, low cost, easy processability, and biodegradability. However, cross-linking is required to stabilize the structure of the electrospun matrices and avoid gelatin dissolution at body temperature. Different physical and chemical cross-linking protocols have been described to improve electrospun gelatin stability and to preserve the morphological fibrous arrangement of the electrospun gelatin scaffolds. Here, we review the main current strategies. For each method, the cross-linking mechanism and its efficiency, the influence of electrospinning parameters, and the resulting fiber morphology are considered. The main drawbacks as well as the open challenges are also discussed.

3D Encapsulation Made Easy: A Coaxial-Flow Circuit for the Fabrication of Hydrogel Microfibers Patches.

To fully exploit the potential of hydrogel micro-fibers in the design of regenerative medicinal materials, we designed a simple, easy to replicate system for cell embedding in degradable fibrous scaffolds, and validated its effectiveness using alginate-based materials. For scaffold fabrication, cells are suspended in a hydrogel-precursor and injected in a closed-loop circuit, where a pump circulates the ionic cross-linking solution. The flow of the cross-linking solution stretches and solidifies a continuous micro-scaled, cell-loaded hydrogel fiber that whips, bends, and spontaneously assembles in a self-standing, spaghetti-like patch. After investigation and tuning of process- and solution-related parameters, homogeneous microfibers with controlled diameters and consistent scaffolds were obtained from different alginate concentrations and blends with biologically favorable macromolecules (i.e., gelatin or hyaluronic acid). Despite its simplicity, this coaxial-flow encapsulation system allows for the rapid and effortless fabrication of thick, well-defined scaffolds, with viable cells being homogeneously distributed within the fibers. The reduced fiber diameter and the inherent macro-porous structure that is created from the random winding of fibers can sustain mass transport, and support encapsulated cell survival. As different materials and formulations can be processed to easily create homogeneously cell-populated structures, this system appears as a valuable platform, not only for regenerative medicine, but also, more in general, for 3D cell culturing in vitro.

An injectable, degradable hydrogel plug for tracheal occlusion in congenital diaphragmatic hernia (CDH).

Congenital Diaphragmatic Hernia (CDH) is a birth defect characterized by failed closure of diaphragm, herniation of viscera in the thoracic cavity and impaired lung development. In most severe cases, fetoscopic endoluminal tracheal occlusion (FETO) is performed through a catheter-mounted balloon to contain pulmonary hypoplasia. To support the success rate of FETO and overcome some of the associated drawbacks, this work aimed at finding an injectable, degradable substitute for the balloon not requiring prenatal removal. Two different types of gels were evaluated as tracheal plugs: calcium-alginate and hyaluronan/methylcellulose blends (HA-MC). The effect of composition on hydrogel properties was evaluated, to identify formulations meeting design requirements. Anatomical tracheal casts were fabricated and filled with a simulated lung fluid to assess injectability, cohesiveness, sealing pressure and persistence of plugs. In vitro cytotoxicity and adhesion of mouse fibroblasts on hydrogels were also investigated. For both hydrogels, adjusting formulation enabled to obtain gelation time within the design range. Furthermore, when injected in the tracheal model, good cohesion and capability to adapt to the anatomical shape were also observed. Effective sealing up to 80 cm H20 was reached for selected compositions and was maintained for over 4 weeks in the in vitro model. Swelling and weight loss were also strongly influenced by composition and varied from few days to several weeks. Despite the absence of in vitro cytotoxicity, a favourably low cell-adhesion was observed for both hydrogels. However, due to their shear-thinning behaviour, HA-MC blends appeared as a particularly suitable candidate for single-surgery tracheal occlusion.

Hierarchical microchannel architecture in chitosan/bioactive glass scaffolds via electrophoretic deposition positive-replica.

One of the main challenges in the design of scaffolds for cortical bone regeneration is mimicking the highly oriented, hierarchical structure of the native tissue in an efficient, simple, and consistent way. As a possible solution to this challenge, positive replica based on electrophoretic deposition (EPD) was here evaluated as a technique to produce organic/inorganic scaffolds with oriented micro-porosities mimicking Haversian canals diameter and spacing. Two different sizes of 45S5 bioactive glass (BG) powders were chosen as inclusions and loaded in a chitosan matrix via EPD on micro-patterned cathodes. Self-standing chitosan scaffolds, with a homogeneous dispersion of BG particles and regularly-oriented micro-channels (ϕ = 380 ± 50 µm, inter-channel spacing = 600 ± 40 µm), were obtained. In vitro analysis in simulated body fluid (SBF) revealed the ability to induce a deposition of a homogenous layer of hydroxyapatite (HA), with Ca/P nucleation reactions appearing kinetically favored by smaller BG particles. Cell interaction with hybrid scaffolds was evaluated in vitro with bone osteosarcoma cells (SAOS-2). The osteoconductive potential of EPD structures was assessed by evaluating cells proliferation, viability and scaffold colonization. Results indicate that EPD is a simple yet extremely effective technique to prepare composite micro-patterned structures and can represent a platform for the development of a new generation of bone scaffolds. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.