Development and characterization of novel agar and gelatin injectable hydrogel as filler for peripheral nerve guidance channels.

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.

Chitosan crosslinked flat scaffolds for peripheral nerve regeneration.

Chitosan (CS) has been widely used in a variety of biomedical applications, including peripheral nerve repair, due to its excellent biocompatibility, biodegradability, readily availability and antibacterial activity. In this study, CS flat membranes, crosslinked with dibasic sodium phosphate (DSP) alone (CS/DSP) or in association with the γ-glycidoxypropyltrimethoxysilane (CS/GPTMS_DSP), were fabricated with a solvent casting technique. The constituent ratio of crosslinking agents and CS were previously selected to obtain a composite material having both adequate mechanical properties and high biocompatibility. In vitro cytotoxicity tests showed that both CS membranes allowed cell survival and proliferation. Moreover, CS/GPTMS_DSP membranes promoted cell adhesion, induced Schwann cell-like morphology and supported neurite outgrowth from dorsal root ganglia explants. Preliminary in vivo tests carried out on both types of nerve scaffolds (CS/DSP and CS/GPTMS_DSP membranes) demonstrated their potential for: (i) protecting, as a membrane, the site of nerve crush or repair by end-to-end surgery and avoiding post-operative nerve adhesion; (ii) bridging, as a conduit, the two nerve stumps after a severe peripheral nerve lesion with substance loss. A 1 cm gap on rat median nerve was repaired using CS/DSP and CS/GPTMS_DSP conduits to further investigate their ability to induce nerve regeneration in vivo. CS/GPTMS_DSP tubes resulted to be more fragile during suturing and, along a 12 week post-operative lapse of time, they detached from the distal nerve stump. On the contrary CS/DSP conduits promoted nerve fiber regeneration and functional recovery, leading to an outcome comparable to median nerve repaired by autograft.

Porous CS based membranes with improved antimicrobial properties for the treatment of infected wound in veterinary applications.

Recently, much attention has been given to the use of innovative solution for the treatment of infected wounds in animals. Current applied treatments are often un-effective leading to infection propagation and animal death. Novel engineered membranes based on chitosan (CS) can be prepared to combine local antimicrobial effect, high flexibility and easy manipulation. In this work, CS crosslinked porous membranes with improved antimicrobial properties were prepared via freeze-drying technique to promote wound healing and to reduce the bacterial proliferation in infected injuries. Silver nanoparticles (AgNPs) and gentamicin sulfate (GS) were incorporated into the CS matrices to impart antibacterial properties on a wild range of strains. CS based porous membranes were tested for their physicochemical, thermal, mechanical as well as swelling and degradation behavior at physiological condition. Additionally, GS release profile was investigated, showing a moderate burst effect in the first days followed by a decreasing release rate which it was maintained for at least 56 days. Moreover, porous membranes loaded with GS or AgNPs showed good bactericidal activity against both of Gram-positive and Gram-negative bacteria. The bacterial strains used in this work were collected in chelonians after carapace injuries to better mimic the environment after trauma.

Chitosan membranes for tissue engineering: comparison of different crosslinkers.

Chitosan (CS), a derivative of the naturally occurring biopolymer chitin, is an attractive material for biomedical applications thanks to its biocompatibility, biodegradability, antibacterial properties and ability to enhance cell adhesion and growth compared to other biopolymers. However, the physical and mechanical stability of CS based materials in aqueous solutions is limited and crosslinking agents are required to increase CS performances in a biological environment. In this work, the effect of three highly-biocompatible crosslinkers as genipin (GP), γ-glycidoxypropyltrimethoxysilane (GPTMS), dibasic sodium phosphate (DSP) and a combination of GPTMS and DSP (GPTMS_DSP) on CS physicochemical, thermal, morphological, mechanical properties, swelling and degradation behavior was investigated. Infrared spectroscopy and thermogravimetric analyses confirmed the chemical reaction between CS and the different crosslinkers. CS wettability was enhanced when CS was DSP ionically crosslinked showing contact angle values of about 65° and exhibiting a higher swelling behavior compared to covalently crosslinked films. Moreover, all the crosslinking methods analyzed improved the stability of CS in aqueous media, showed model molecule permeation in time and increased the mechanical properties when compared with non-crosslinked films. The possibility to tailor the final properties of CS scaffolds through crosslinking is a key strategy in applying CS in different biomedical and tissue engineering applications. The obtained results reveal that the optimization of the crosslinking mechanism provides CS membrane properties required in different biomedical applications.