A direct-writing electrospinning system for designing complex architectures in tissue engineering.

Recently, direct-writing electrospinning has been pursued to reach a higher accuracy and complexity in fiber scaffold fabrication compared to other extrusion techniques more frequently encountered in tissue engineering. However, to date, direct-writing electrospinning lacks a wide application to process materials such as nature-derived polymers, of huge importance in tissue engineering given their chemical properties similar to that of native tissues. In this work, a setup to perform direct-writing electrospinning was developed and demonstrated versatility and efficiency in obtaining submicrometric fibers and guiding their deposition along various types of paths and patterns, resulting in a user-friendly method to create structures closely resembling tissue architecture.

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.

In Vitro Models for the Development of Peripheral Nerve Conduits, Part I: Design of a Fibrin Gel-Based Non-Contact Test.

Current clinical strategies to repair peripheral nerve injuries draw on different approaches depending on the extent of lost tissue. Nerve guidance conduits (NGCs) are considered to be a promising, off-the-shelf alternative to autografts when modest gaps need to be repaired. Unfortunately, to date, the implantation of an NGC prevents the sacrifice of a healthy nerve at the price of suboptimal clinical performance. Despite the significant number of materials and fabrication strategies proposed, an ideal combination has not been yet identified. Validation and comparison of NGCs ultimately requires in vivo animal testing due to the lack of alternative models, but in the spirit of the 3R principles, a reliable in vitro model for preliminary screening is highly desirable. Nevertheless, more traditional in vitro tests, and direct cell seeding on the material in particular, are not representative of the actual regeneration scenario. Thus, we have designed a very simple set-up in the attempt to appreciate the relevant features of NGCs through in vitro testing, and we have verified its applicability using electrospun NGCs. To this aim, neural cells were encapsulated in a loose fibrin gel and enclosed within the NGC membrane. Different thicknesses and porosity values of two popular polymers (namely gelatin and polycaprolactone) were compared. Results indicate that, with specific implementation, the system might represent a useful tool to characterize crucial NGC design aspects.

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.

Biopolymer-based strategies in the design of smart medical devices and artificial organs.

Advances in regenerative medicine and in modern biomedical therapies are fast evolving and set goals causing an upheaval in the field of materials science. This review discusses recent developments involving the use of biopolymers as smart materials, in terms of material properties and stimulus-responsive behavior, in the presence of environmental physico-chemical changes. An overview on the transformations that can be triggered in natural-based polymeric systems (sol-gel transition, polymer relaxation, cross-linking, and swelling) is presented, with specific focus on the benefits these materials can provide in biomedical applications.

Electrospun silk fibroin-gelatin composite tubular matrices as scaffolds for small diameter blood vessel regeneration.

In this work an innovative method to obtain natural and biocompatible small diameter tubular structures is proposed. The biocompatibility and good mechanical properties of electrospun silk fibroin tubular matrices (SFts), extensively studied for tissue engineering applications, have been coupled with the excellent cell interaction properties of gelatin. In fact, an innovative non-cytotoxic gelatin gel, crosslinked in mild conditions via a Michael-type addition reaction, has been used to coat SFt matrices and obtain SFt/gel structures (I.D. = 6 mm). SFts/gel exhibited homogeneous gelatin coating on the electrospun fibrous tubular structure. Circumferential tensile tests performed on SFts/gel showed mechanical properties comparable to those of natural blood vessels in terms of UTS, compliance and viscoelastic behavior. Finally, SFt/gel in vitro cytocompatibility was confirmed by the good viability and spread morphology of L929 fibroblasts up to 7 days. These results demonstrated that SFt/gel is a promising off-the-shelf graft for small diameter blood vessel regeneration.

The effect of scaffold pore size in cartilage tissue engineering.

INTRODUCTION: The effect of scaffold pore size and interconnectivity is undoubtedly a crucial factor for most tissue engineering applications. The aim of this study was to examine the effect of pore size and porosity on cartilage construct development in different scaffolds seeded with articular chondrocytes. METHODS: We fabricated poly-L-lactide-co-trimethylene carbonate scaffolds with different pore sizes, using a solvent-casting/particulate-leaching technique. We seeded primary bovine articular chondrocytes on these scaffolds, cultured the constructs for 2 weeks and examined cell proliferation, viability and cell-specific production of cartilaginous extracellular matrix proteins, including GAG and collagen. RESULTS: Cell density significantly increased up to 50% with scaffold pore size and porosity, likely facilitated by cell spreading on the internal surface of bigger pores, and by increased mass transport of gases and nutrients to cells, and catabolite removal from cells, allowed by lower diffusion barriers in scaffolds with a higher porosity. However, both the cell metabolic activity and the synthesis of cartilaginous matrix proteins significantly decreased by up to 40% with pore size. We propose that the association of smaller pore diameters, causing 3-dimensional cell aggregation, to a lower oxygenation caused by a lower porosity, could have been the condition that increased the cell-specific synthesis of cartilaginous matrix proteins in the scaffold with the smallest pores and the lowest porosity among those tested. CONCLUSIONS: In the initial steps of in vitro cartilage engineering, the combination of small scaffold pores and low porosity is an effective strategy with regard to the promotion of chondrogenesis.

Polymeric materials as artificial muscles: an overview.

PURPOSE: The accurate selection of materials and the fine tuning of their properties represent a fundamental aspect in the realization of new active systems able to produce actuating forces, such as artificial muscles. In this regard, exciting opportunities for the design of new advanced systems are offered by materials belonging to the emerging class of functional polymers: exploiting their actuation response, specific devices can be realized. Along this direction, materials showing either shape-memory effect (SME) or shape-change effect (SCE) have been the subject of extensive studies aimed at designing of actuators as artificial muscles. Here, we concisely review active polymers in terms of properties and main applications in artificial muscle design. STRUCTURE: The main aspects related to material properties in both shape-memory polymers (SMPs) and electroactive polymers (EAPs) are reviewed, based on recent scientific literature. SME in thermally activated SMPs is presented by preliminarily providing a definition that encompasses the new theories regarding their fundamental properties. EAPs are briefly presented, describing the working mechanisms and highlighting the main properties and drawbacks, in view of their application as actuators. For both classes of materials, some key examples of effective application in artificial muscles are offered. OUTLOOK: The potential in polymer architecture design for the fabrication of actively moving systems is described to give a perspective on the main achievements and new research activities.

Understanding telecardiology success and pitfalls by a systematic review.

Cardiology is the clinical area where death causes are more frequent than in any other clinical area, and it could benefit from telemedicine. At present, assessment about telecardiology application are hard to find, and sometimes can be found in review article about telemedicine, but there is a lack of review on telecardiology application literature. So we reviewed studies regarding telemedicine applications on cardiology specialty.Sixty-one articles were selected, searching the PubMed database for all years the database were available. All considered articles were published on peer reviewed telemedicine and biomedical journals, from 1992 to 2004. We defined an evaluation grid for the articles in our research result set. Each article was reviewed and catalogued, identifying: 1) Article identification, 2) Content description, 3) Telemedicine manifesto classification, 4) Telemedicine system paradigm, 5) Involved actors.Most of the analysed literature referred only to feasibility studies, pilot projects and to short-term outcomes, for example only 21 cases reported a project duration greater than one year. The method proposed can be used to analyse literature on others telemedicine specialties.