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Schematic representation of PDAC-on-chip reassembly the pancreatic acino-ductal unit composed of healthy and pathological human pancreatic ductal epithelial cells (HPDE and KRAS-HPDE, respectively) surrounded by pancreatic stellate cells (PSCs).

1.AbstractPancreatic ductal adenocarcinoma (PDAC) is the most frequent type of pancreatic cancer, one of the leading causes of cancer-related deaths worldwide. PDAC is marked by a dense, fibrous tumor microenvironment, in which stromal fibroblasts surround the cancerous ductal epithelial cells. The crosstalk between pancreatic cells and the surrounding fibroblasts leads to inflammation and stiffening of the surrounding tissue, which is believed to hinder anticancer drugs’ uptake and effectiveness.In vitro, fully human models of the pancreatic cancer microenvironment are needed to foster the development of new, more effective therapies; but it is still challenging to make these models anatomically and functionally relevant. Here, we use tomographic volumetric bioprinting, a novel method to fabricate cell-laden viable constructs within less than a minute, to produce anatomically relevant fibroblast-laden gelatine methacrylate-based pancreatic models, including a duct and an acinus. We then seeded human pancreatic ductal epithelial (HPDE) cells, wild type or cancerous, into these models and evaluated the progression of the constructs for several days after printing. We show that the 3D pancreatic duct models remain viable for up to 14 days after bioprinting. We used immunofluorescence to evaluate the surrounding fibroblasts’ activation by quantifying the relative expressions of alpha smooth muscle actin (αSMA) vs. actin in the fibroblasts. αSMA is known to be overexpressed in inflamed tumor-associated fibroblasts. We show that αSMA expression is significantly higher in fibroblasts co-cultured with cancerous than with wild-type HPDE cells, that this expression increases with time, and that it is higher in fibroblasts that lay closer to HPDE cells than in those deeper into the model. These models could potentially be used in drug development or to shed light on the early progression of the disease.

Pancreatic ductal adenocarcinoma (PDAC) mainly develops in the head of the pancreas, within the acino-ductal unit composed of acinar and ductal cells surrounded by pancreatic stellate cells (PSCs). PSCs strongly influence the tumor microenvironment by triggering an intense stromal deposition, which plays a key role in tumor progression and limits drug perfusion. We have developed a microfluidic

Scaffold pore size plays a fundamental role in the regeneration of new tissue since it has been shown to direct cell activity in situ. It is well known that cellular response changes in relation with pores diameter. Consequently, researchers developed efficient approaches to realize scaffolds with controllable macro-, micro-, and nanoporous architecture. In this context, new strategies aiming at the manufacturing of scaffolds with multiscale pore networks have emerged, in the attempt to mimic the complex hierarchical structures found in living systems. In this review, we aim at providing an overview of the fabrication methods currently adopted to realize scaffolds with controlled, multisized pores highlighting their specific influence on cellular activity.

The multifunctional properties of antimicrobial peptides (AMPs) make them attractive candidates for the treatment of various diseases. AMPs are considered as alternatives to antibiotics due to the increasing number of multidrug-resistant (MDR) bacteria. However, bare AMPs have limited therapeutic potentials due to a low residence time in the blood circulatory system and susceptibility to proteases and an alkaline wound environment. These limitations are the major hurdles for AMPs to succeed as commercial drugs. In contrast, AMP-based materials, for instance, NPs, hydrogels, electrospun fibres, dressings and implants, could overcome these challenges and provide therapeutic efficacies to the conjugated AMPs superior to those of bare AMPs in different disease models. In this review, we discuss the preparation of different compositions of AMP-based materials and their therapeutic potential for the treatment of microbial infections in the brain, eyes, mouth, skin, lungs, and gastrointestinal and urinary tracts. Apart from antimicrobial potential, the applications of AMP-based materials in the regeneration of skin/bone, prevention of implant-associated infections, detection/imaging of bacteria, cancer therapy and gene delivery are discussed in this review. Lastly, we discuss different challenges that hinder the commercialization of AMP-based materials. Overall, this review provides a comprehensive account of the current progress and prospects of AMP-based materials for clinical applications.

Antimicrobial peptides (AMPs), such as LL37 peptides, may be immobilized on the surface of medical devices to render them with antimicrobial and angiogenic properties. However, little is known about LL37 properties after immobilization. Here, we have studied the antimicrobial and pro-angiogenic activity of soluble and immobilized LL37 peptides (conjugated to gold nanoparticles). Both soluble and immobilized LL37 peptides have potent antimicrobial activity against Gram-positive and Gram-negative bacteria in the presence of 10% human serum (HS). However, the immobilized LL37 peptides showed less cytotoxicity to endothelial cells (ECs) at a concentration that was able to kill bacteria. Interestingly, although both soluble and immobilized LL37 peptides showed pro-angiogenic activities in Matrigel and chorioallantoic membrane (CAM) assays, they induce different signalling pathways in ECs. The results described here highlight the importance of investigating the properties of immobilized AMPs that might act as a new entity.

This project aims at reproducing the morphology and the composition of the pancreatic acino-ductal unit. More specifically, this work involves the use of additive manufacturing technologies to fabricate a 3D exocrine glandular tissue model that mimics in vitro the physiological structure experienced by cells in vivo.

The design and fabrication of a system to both mimic the multicellular composition of the lung and its vascular network, as well as the composition and structure of extracellular matrix (ECM) was developed.

Conductive polymers (CPs) have recently been applied in the development of scaffolds for tissue engineering applications in attempt to induce additional cues able to enhance tissue growth. Polyaniline (PANI) is one of the most widely studied CPs, but it requires to be blended with other polymers in order to be processed through conventional technologies. Here, we propose the fabrication of nanofibers based on a polycaprolactone (PCL)-PANI blend obtained using electrospinning technology. An extracellular matrix-like fibrous substrate was obtained showing a good stability in the physiological environment (37 °C in PBS solution up 7 days). However, since the high hydrophobicity of the PCL-PANI mats (133.5 ± 2.2°) could negatively affect the biological response, a treatment with atmospheric plasma was applied on the nanofibrous mats, obtaining a hydrophilic surface (67.1 ± 2°). In vitro tests were performed to confirm the viability and the physiological-like morphology of human foreskin fibroblast (HFF-1) cells cultured on the plasma treated PCL-PANI nanofibrous scaffolds.

Promising UV-curable starch-based coatings were fabricated by utilizing methacrylated starch. The aqueous methacrylated starch solution was cast on a glass substrate, and UV-cured after drying. The efficiency of UV-curing process was monitored with gel percentage measurements. The thermal and mechanical properties of the fabricated UV-cured coatings were investigated through differential scanning calorimetry and tensile test and compared with the starch-based uncured casted coatings. A complete characterization of the surface properties was performed by means of pencil hardness, adhesion, solvent resistance, and surface tension measurements. The cross-linking by UV-curing significantly enhanced the mechanical and surface properties of the coating. The effect of UV-curing on the biodegradability of the coating was evaluated by following the enzymatic degradation by &alpha, amylase by determining the amount of glucose and maltose released from the coatings. UV-cured methacrylated starch based coating with promising material and surface properties and retained biodegradation potential was demonstrated.

This project aims at reproducing the morphology and the composition of the pancreatic acino-ductal unit. More specifically, this work involves the use of a 4D printing system that combines melt electrospinning technology with an automatized layer-by-layer surface functionalization which provides biological cues to the surface of the melt electrospun structure. This 3D exocrine glandular tissue model mimics in vitro the physiological structure experienced by cells in vivo and serves as a powerful tool to investigate pathological processes such as the Pancreatic Ductal Adenocarcinoma (PDAC), https://youtu.be/jn_5ZNTQ-AU

The high drug loading capacity, cytocompatibility and easy functionalization of ordered mesoporous carbons (OMCs) make them attractive nanocarriers to treat several pathologies. OMCs&rsquo, efficiency could be further increased by embedding them into a hydrogel phase for an in loco prolonged drug release. In this work, OMCs were embedded into injectable thermosensitive hydrogels. In detail, rod-like (diameter ca. 250 nm, length ca. 700 nm) and spherical (diameter approximately 120 nm) OMCs were synthesized by nanocasting selected templates and loaded with ibuprofen through a melt infiltration method to achieve complete filling of their pores (100% loading yield). In parallel, an amphiphilic Poloxamer&reg, 407-based poly(ether urethane) was synthesized (Mn&macr, 72 kDa) and solubilized at 15 and 20% w/v concentration in saline solution to design thermosensitive hydrogels. OMC incorporation into the hydrogels (10 mg/mL concentration) did not negatively affect their gelation potential. Hybrid systems successfully released ibuprofen at a slower rate compared to control gels (gels embedding ibuprofen as such), but with no significant differences between rod-like and spherical OMC-loaded gels. OMCs can thus work as effective drug reservoirs that progressively release their payload over time and also upon encapsulation in a hydrogel phase, thus opening the way to their application to treat many different pathological states (e.g., as topical medications).

The development of new bio-based inks is a stringent request for the expansion of additive manufacturing towards the development of 3D-printed biocompatible hydrogels. Herein, methacrylated carboxymethyl cellulose (M-CMC) is investigated as a bio-based photocurable ink for digital light processing (DLP) 3D printing. CMC is chemically modified using methacrylic anhydride. Successful methacrylation is confirmed by 1H NMR and FTIR spectroscopy. Aqueous formulations based on M-CMC/lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator and M-CMC/Dulbecco&rsquo, s Modified Eagle Medium (DMEM)/LAP show high photoreactivity upon UV irradiation as confirmed by photorheology and FTIR. The same formulations can be easily 3D-printed through a DLP apparatus to produce 3D shaped hydrogels with excellent swelling ability and mechanical properties. Envisaging the application of the hydrogels in the biomedical field, cytotoxicity is also evaluated. The light-induced printing of cellulose-based hydrogels represents a significant step forward in the production of new DLP inks suitable for biomedical applications.

Light processable hydrogels were successfully fabricated by utilizing maize starch as raw material. To render light processability, starch was gelatinized and methacrylated by simple reaction with methacrylic anhydride. The methacrylated starch was then evaluated for its photocuring reactivity and 3D printability by digital light processing (DLP). Hydrogels with good mechanical properties and biocompatibility were obtained by direct curing from aqueous solution containing lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photo-initiator. The properties of the hydrogels were tunable by simply changing the concentration of starch in water. Photo-rheology showed that the formulations with 10 or 15 wt% starch started curing immediately and reached G&rsquo, plateau after only 60 s, while it took 90 s for the 5 wt% formulation. The properties of the photocured hydrogels were further characterized by rheology, compressive tests, and swelling experiments. Increasing the starch content from 10 to 15 wt% increased the compressive stiffness from 13 to 20 kPa. This covers the stiffness of different body tissues giving promise for the use of the hydrogels in tissue engineering applications. Good cell viability with human fibroblast cells was confirmed for all three starch hydrogel formulations indicating no negative effects from the methacrylation or photo-crosslinking reaction. Finally, the light processability of methacrylated starch by digital light processing (DLP) 3D printing directly from aqueous solution was successfully demonstrated. Altogether the results are promising for future application of the hydrogels in tissue engineering and as cell carriers

Piezoelectric ceramic nanomaterials have recently attracted attention in the biomedical field thanks to their interesting electrical properties in response to mechanical stimulation (and vice versa) combined with a good biocompatibility and the ability to promote the regeneration of electrically sensitive tissues. In tissue engineering approaches, in order to obtain smart scaffolds these materials must be combined with other biomaterials for processing through conventional as well as non-conventional technologies. In this work, a novel composite electrospun membrane was produced by combining extracellular matrix-like gelatin nanofibers with barium titanate nanoparticles (BTNPs). The electrospinning process was optimized to achieve a high BTNP load, reducing the formation of aggregates which could alter the morphology and stability of the membrane. A complete morphological, mechanical and chemical–physical characterization of the composite membranes was performed, confirming the integration of the BTNPs into the polymer fibers. Furthermore, the biocompatibility of the developed membranes was assessed using a sarcoma osteogenic cell line (SaOS-2).

The coatings application onto medical devices has experienced a continuous growth in the last few years. Medical device coating market is expected to grow at a CAGR of 5.16% to reach USD 10 million by 2023 due to the increasing geriatric population and the growing demand for continuous innovation. Layer-by-Layer (LbL) assembly represents a versatile method to modify the surface properties, in order to control cell interaction and thus enhance biological functions. Furthermore, LbL is environmentally friendly, able to coat all types of surfaces with the creation of homogenous film and to include and control the release of biomolecules/drugs. This feature review provides a critical overview on recent progresses in functionalizing materials by LbL assembly for bone regeneration and disorder treatment. An overview of emerging and visionary opportunities on LbL technologies and further combination with other existing methods used in biomedical field, is also discussed to evidence the new challenges and potential developments in bone regenerative medicine.

Articular cartilage (AC) lacks the ability to self-repair and cell-based approaches, primarily based on using chondrocytes and mesenchymal stem cells (MSCs), are emerging as effective technology to restore cartilage functionality, because cells synergic functionality may support the maintenance of chondrogenic phenotype and promote extracellular matrix regeneration. This work aims to develop a more physiologically representative co-culture system to investigate the influence of MSCs on the activity of chondrocytes. A thermo-sensitive chitosan-based hydrogel, ionically crosslinked with β–glycerophosphate, is optimised to obtain sol/gel transition at physiological conditions within 5 minutes, high porosity with pores diameter in vitro mechanical integrity with compressive and equilibrium Young’s moduli of 37 kPa and 17 kPa, respectively. Live/dead staining showed that after 1 and 3 days in culture, the encapsulated MSCs into the hydrogels are viable and characterised by round-like morphology. Furthermore chondrocyte spheroids, seeded on top of gels that contained either MSCs or no cells, show that the encapsulated MSCs stimulate chondrocyte activity within a gel co-culture, both in terms of maintaining the coherence of chondrocyte spheroids, leading to a larger quantity of CD44 (by immunofluorescence) and a higher production of collagen and glycosaminoglycans (by histology) compared with the mono-culture.

Localised controlled release of simvastatin from porous freeze-dried chitosan-gelatin (CH-G) scaffolds was investigated by incorporating simvastatin loaded poly-(dl-lactide-co-glycolide) acid (PLGA) microparticles (MSIMs) into the scaffolds. MSIMs at 10% w/w simvastatin loading were prepared using a single emulsion-solvent evaporation method. The MSIM optimal amount to be incorporated into the scaffolds was selected by analysing the effect of embedding increasing amounts of blank PLGA microparticles (BL-MPs) on the scaffold physical properties and on the in vitro cell viability using a clonal human osteoblastic cell line (hFOB). Increasing the BL-MP content from 0% to 33.3% w/w showed a significant decrease in swelling degree (from 1245±56% to 570±35%). Scaffold pore size and distribution changed significantly as a function of BL-MP loading. Compressive modulus of scaffolds increased with increasing BL-MP amount up to 16.6% w/w (23.0±1.0kPa). No significant difference in cell viability was observed with increasing BL-MP loading. Based on these results, a content of 16.6% w/w MSIM particles was incorporated successfully in CH-G scaffolds, showing a controlled localised release of simvastatin able to influence the hFOB cell proliferation and the osteoblastic differentiation after 11 days.

Recent progresses in tissue engineering are directed towards the development of technologies able to provide personalized scaffolds recreating the defect shape in a patient specific manner. To achieve this ambitious goal, 3D bioprinting can be combined with a suitable bioink, able to create a physiological milieu for cell growth. In this work, a novel chitosan-based hydrogel was developed combining photocrosslinking and thermo-sensitive properties. Commercial chitosan (CS) was first methacrylated and then mixed with β glycerol phosphate salt (β-GP) to impart a thermally induced phase transition. The absence of cytotoxic degradation products and the excellent biocompatibility of the developed hydrogel was confirmed through in vitro tests using different cell lines (NIH/3T3, Saos-2, SH-SY5Y). Cellularized 3D structures were obtained though 3D bioprinting technologies confirming the processability of the developed hydrogels and its unique biological properties.

Hypothesis Wound healing is a complex process that often requires treatment with antibacterial agents to avoid infection, which affects the optimal tissue regeneration process. Ideal scaffolds for wound healing treatment should combine biomimetic features to ensure the tissue growth on properly designed extracellular matrix (ECM)-like scaffolds and antibacterial properties in order to avoid bacterial colonization. Experiments In this work, gelatin cross-linked nanofibers (GL-nanofibres), with diameters ranging from 200 to 300 nm, were prepared via a “green electrospinning technique” to mimic the structure and composition of the extracellular matrix (ECM), and promote the normal skin wound healing process. Nanofibres were doped with two antibacterial agents (gentamicin sulphate or silver nanoparticles) to achieve an antibacterial effect against Gram + and Gram- bacteria. Findings The ECM-mimicking structure of GL-nanofibres was not affected by the presence of the antibacterial agents, which were homogeneously distributed within the mats as shown by SEM and EDS. The antibacterial properties of the developed matrices were confirmed using 4 strains (S. aureus, S. epidermidis, P. aeruginosa and E. coli) while the biocompatibility of the developed substrates and their ability to induce cell growth was assessed using Neonatal Normal Human Dermal Fibroblasts (NHDF-Neo).

Random and aligned gelatin (GL) and chitosan (CS) nano-fibres have been prepared by electrospinning tuning the collector rotation speed. The effect of fibre alignment on cell adhesion and proliferation was assessed in vitro by using different Schwann cell (SC) and neuronal models. Moreover, actin cytoskeleton organization, lamellipodia and filipodia formation, and axon outgrowth were evaluated. GL and CS fibres induced similar adhesion and proliferation rates. GL and CS random fibres promoted higher adhesion and proliferation rates induction in comparison to the aligned ones, although GL and CS fibres alignment resulted in SC and axon-oriented growth. Filipodia formation was higher on aligned fibres, suggesting that these substrates can promote higher cell migration in comparison to random ones. 50B11 (neuronal cell line) differentiation was higher on GL fibres, whereas no differences were observed in dorsal root ganglia explants model. These data suggest that both GL and CS fibres can be promising substrates to be used in peripheral nerve reconstruction. Copyright © 2016 John WileySons, Ltd.

Nerve guidance channels facilitate nerve regeneration and represent an attractive alternative to nerve graft. Actually, nano- and microstructured biomaterials for nerve reconstruction have gained much attention, thanks to recent discoveries about topography effects on cell behavior and morphology. Electrospun fibres have been proposed as filler or structural component for nerve guidance channels, principally due to their similarity with extracellular matrices which facilitate nerve regeneration. Among several tested biomaterials, gelatin has been used to prepare fibres able to support Schwann cell migration and neurite outgrowth. In this work, the effects of gelatin fibre size on axon elongation and Schwann cell migration have been tested using dorsal root ganglia cultures. Moreover, we analyzed how fibres might affect the expression of specific neuronal subtype markers in sensory neuron cultures and how the combined effect of substrate and biological cues affects neurite growth and gene expression. Data show that fibre topography differentially affects both neurite outgrowth and gene expression and suggest that fibre size and topography associated to specific growth factor exposure might be used to select neuron subpopulations and favor the axonal growth of specific neurons. Anat Rec, 301:1668-1677, 2018. © 2018 Wiley Periodicals, Inc.

Current therapeutic strategies to treat neurodegenerative diseases, such as alpha-synucleinopathies, aim at enhancing the amount of drug reaching the brain. Methods proposed, such as intranasal administration, should be able to bypass the blood brain barrier (BBB) and even when directly intracerebrally injected they could require a carrier to enhance local release of drugs. We have investigated the effect of a model synthetic hydrogel to be used as drug carrier on the amount of alpha-synuclein aggregates in cells in culture. The results indicated that alpha-synuclein aggregation was affected by the synthetic polymer, suggesting the need for testing the effect of any used material on the pathological process before its application as drug carrier.

Biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles, containing human parathyroid hormone (PTH (1–34)), prepared by a modified double emulsion-solvent diffusion-evaporation method, were incorporated in porous freeze-dried chitosan-gelatin (CH-G) scaffolds. The PTH-loaded nanoparticles (NPTH) were characterised in terms of morphology, size, protein loading, release kinetics and in vitro assessment of biological activity of released PTH and cytocompatibility studies against clonal human osteoblast (hFOB) cells. Structural integrity of incorporated and released PTH from nanoparticles was found to be intact by using Tris-tricine SDS-PAGE. In vitro PTH release kinetics from PLGA nanoparticles were characterised by a burst release followed by a slow release phase for 3–4 weeks. The released PTH was biologically active as evidenced by the stimulated release of cyclic AMP from hFOB cells as well as increased mineralisation studies. Both in vitro and cell studies demonstrated that the PTH bioactivity was maintained during the fabrication of PLGA nanoparticles and upon release. Finally, a content of 33.3% w/w NPTHs was incorporated in CH-G scaffolds, showing an intermittent release during the first 10 days and, followed by a controlled release over 28 days of observation time. The increased expression of Alkaline Phosphatase levels on hFOB cells further confirmed the activity of intermittently released PTH from scaffolds.

Background The design of efficient nerve conduits able to sustain the axonal outgrowth and its guidance towards appropriate targets is of paramount importance in nerve tissue engineering. Methods In this work, we propose the preparation of highly aligned nanocomposite fibers of gelatin/cerium oxide nanoparticles (nanoceria), prepared by electrospinning. Nanoceria are powerful self-regenerative antioxidant nanomaterials, that behave as strong reactive oxygen species scavengers, and among various beneficial effects, they have been proven to inhibit the cell senescence and to promote the neurite sprouting. Results After a detailed characterization of the developed substrates, they have been tested on neuron-like SH-SY5Y cells, demonstrating strong antioxidant properties and beneficial multi-cue effects in terms of neurite development and alignment. Conclusions Obtained findings suggest efficiency of the proposed substrates in providing combined topographical stimuli and antioxidant effects to cultured cells. General significance Proposed nanocomposite scaffolds represent a promising approach for nerve tissue engineering and regenerative medicine.

Chitosan (CS) membranes obtained by electrospinning are potentially ideal substrates for soft tissue engineering as they combine the excellent biological properties of CS with the extracellular matrix (ECM)-like structure of nanofibrous mats. However, the high amount of acid solvents required to spun CS solutions interferes with the biocompatibility of CS fibres. To overcome this limitation, novel CS based solutions were investigated in this work. Low amount of acidic acid (0.5 M) was used and dibasic sodium phosphate (DSP) was introduced as ionic crosslinker to improve nanofibres water stability and to neutralize the acidic pH of electrospun membranes after fibres soaking in biological fluids. Randomly oriented and aligned nanofibres (128 ± 19 nm and 140 ± 41 nm, respectively) were obtained through electrospinning process (voltage of 30 kV, 30 μL/min flow rate and temperature of 39 °C) showing mechanical properties similar to those of soft tissues (Young Modulus lower than 40 MPa in dry condition) and water stability until 7 days. C2C12 myoblast cell line was cultured on CS fibres showing that the aligned architecture of substrate induces cell orientation that can enhance skeletal muscle regeneration.

Polyurethanes (PURs) are a class of block copolymers synthesised from three different reagents: a polyol, a di- or multi-isocyanate, and a chain extender. Biodegradable PURs can be synthesised by properly selecting the building blocks with suitable properties for applications in regenerative medicine. The nature and relative amount of hard and soft segments affect the mechanical properties. PURs can be bulk or surface functionalised with biomimetic peptide sequences or natural polymers, affecting cell response and/or the biodegradation rate. PUR particles, fibres, and hydrogels can be designed with a suitable composition for drug encapsulation and controlled release. This chapter offers an overview of the chemistry of biodegradable and biocompatible PURs and reports their application as scaffolds for tissue engineering and carriers for drug delivery.

Binary blends of poly(L-lactic) acid (PLLA: 201790 Da) and poly(lactide-co-glycolide) (PLGA) (LA:GA = 50:50 mol:mol; 32030 Da) with various compositions were prepared. Physicochemical properties of PLLA/PLGA blends were analyzed. Blends showed a biphasic morphology, with distinct glass transition temperatures, which only slightly approximated compared to pure polymers. Analysis of tensile mechanical properties through the Kerner-Uemura-Takayanagi model showed compatibility for PLLA/PLGA 75/25 blend. Rapid degradation of PLGA phase (2–8 weeks) in PLLA/PLGA 75/25 blend led to porous samples, which appear promising for drug delivery and tissue engineering. A limited inflammatory reaction resulted from subcutaneous implantation of PLLA/PLGA 75/25 in Balb-c mice.

Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF-controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF-releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin-based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

Injectable hydrogels are becoming of increasing interest in the field of tissue engineering thanks to their versatile properties and to the possibility of being injected into tissues or devices during surgery. In peripheral nerve tissue engineering, injectable hydrogels having shear-thinning properties are advantageous as filler of nerve guidance channels (NGCs) to improve the regeneration process. In the present work, gelatin-based hydrogels were developed and specifically designed for the insertion into the lumen of hollow NGCs through a syringe during surgery. Injectable hydrogels were obtained using an agar-gelatin 20:80 weight ratio, (wt/wt) blend crosslinked by the addition of genipin (A/GL_GP). The physicochemical properties of the A/GL_GP hydrogels were analysed, including their injectability, rheological, swelling and dissolution behaviour, and their mechanical properties under compression. The hydrogel developed showed shear-thinning properties and was applied as filler of NGCs. The A/GL_GP hydrogel was tested in vitro using different cell lines, among them Schwann cells which have been used because they have an important role in peripheral nerve regeneration. Viability assays demonstrated the lack of cytotoxicity. In vitro experiments showed that the hydrogel is able to promote cell adhesion and proliferation. Two- and three-dimensional migration assays confirmed the capability of the cells to migrate both on the surface and within the internal framework of the hydrogel. These data show that A/GL_GP hydrogel has characteristics that make it a promising scaffold material for tissue engineering and nerve regeneration. Copyright © 2014 John Wiley & Sons, Ltd.