Chitosan based biomaterials: soft tissue engineering applications

In recent years, considerable attention has been given to chitosan (CS)-based biomaterials and their applications in the field of soft tissue engineering (TE). CS is a glycosaminoglycan derived from chitin, the primary structural polymer in crustacean exoskeletons. CS is biocompatible, biodegradable, easily formed into various structures (i.e. sponges, nanofibers and films) under mild processing conditions and can be chemically modified through graft copolymerization and crosslinking. However, the rapid degradation of CS and its low mechanical strength are concerns that may limit its use in clinical applications. In the first part of the thesis, different non cytotoxic crosslinkers were used aiming at improving the structural properties of CS. Genipin (GP), γ-glycidoxypropyltrimethoxysilane (GPTMS), dibasic sodium phosphate (DSP) were selected as biocompatible CS crosslinkers as reported in literature. After a preliminary physico-chemical and mechanical characterization, the proper crosslinking compounds were selected for the development of different typologies of CS scaffolds for both human and veterinary applications. CS- based scaffolds were developed as nerve guidance channels (NGCs) and internal fillers fabrication to promote peripheral nerve regeneration in humans. Two CS based hollow NGCs were prepared and tested in vitro and in vivo (coded as CS flat membrane and bi-layer CS membrane) and a CS based nanostructured internal filler was optimized and characterized in vitro. i. CS flat membranes were prepared by solvent casting. According to the results obtained in the first part of the thesis, DSP alone (CS/DSP) or in association with the GPTMS (CS/GPTMS_DSP) were used as crosslinkers. CS crosslinked membranes showed permeation to nutrients and did not exert any cytotoxic effect on RT4-D6P2T. The higher mechanical stability of CS/GPTMS_DSP under wet state allowed to confirm the RT4-D6P2T attachment and proliferation as well as the neurite outgrowth of dorsal root ganglia (DRG) on CS substrates. Before in vivo implantation in rats, CS/GPTMS_DSP and CS/DSP membranes were easily rolled up to form a NGC. Then, membranes were used to bridge median nerve defects in rats. After 12 week post-operative CS/GPTMS_DSP tubes were found to be detached from the distal suturing site and functional recovery did not occurred. On the other hand, crushed nerve encircled with CS/DSP membranes, allowed nerve fibre regeneration and functional recovery, showing similar results to autografts. ii. Bi-layer CS membranes were developed using a two-step coating technique. CS/DSP and CS/GPTMS_DSP flat membranes were combined to produce scaffold structures with good biocompatibility in the inner layer (CS/DSP) and with the desired mechanical strength imparted by the outer (CS/GPTMS_DSP, GPTMS 25% wt./wt.). Gradual water uptake and permeation to small molecules was observed compared to single layers. From in vivo tests, median nerves treated with bi-layer tubes displayed regenerated and aligned fibres at the injury site. iii. CS crosslinked electrospun nanofibres were fabricated by electrospinning solutions containing CS, polyethylene oxide (PEO), and dimethylsulphoxide (DMSO). PEO and DMSO were introduced to allow the spinnability of CS solutions at high polymer concentration with controllable fiber size and increase fiber yields by relaxing CS chain entanglement. Optimization of the process and solution parameters allowed to obtain CS nanofibres with size of 128±17 nm. To increase CS stability in aqueous media, DSP was used as crosslinker After DSP crosslinking fibre size decreased to 109±17 nm while an increase in the mechanical strength (E, from 63±10 MPa to 113±8 MPa) was observed compared to uncrosslinked nanofibrous matrices. In the third part of the thesis, CS porous membranes with improved antimicrobial properties were prepared for veterinary application. The developed scaffolds were fabricated by freeze-drying to promote the wound healing process and to reduce the bacterial proliferation in chelonian shell injury site. Different ratios of silver nanoparticles (AgNPs, 5%, 10% and 15% wt. /wt.) and gentamicin sulphate (GS, 3.5 mg/ml) were loaded into the CS/GPTMS_DSP membranes to impart the proper antibacterial properties and to favor drug release avoiding the risk of systemic toxicity. After a preliminary in vitro characterization, CS/GPTMS_DSP loaded with AgNPs at a concentration of 10% wt./wt (CS/GPTMS_DSP_AgNP10) was selected as ideal candidate for this application field. GS release profile from CS/GPTMS_DSP_GS evidenced high burst release of the antibiotics in the first day (about 70%). Finally, GS and AgNPs (10 % wt./wt.) effect on bacterial inhibition was evaluated and confirmed against Gram+ and Gram-. The results reported in this thesis work demonstrate that CS is a promising candidate for applications in human and veterinary soft TE. Mechanical and physico-chemical properties of CS scaffolds can be tuned by using different crosslinking methods. By the in vitro characterization, GPTMS and DSP were selected as ideal compounds to the development of scaffolds for peripheral nerve regeneration (in human) and wound healing (in animals). Four different morphologies (3 for peripheral nerve regeneration and 1 for wound healing application) were obtained by varying the fabrication methods and the final composition. All membranes were found to satisfy the requirements for the application of interest. CS based membranes developed for peripheral nerve regeneration were found to be biocompatible, and successful functional recovery was observed in case of CS/DSP and bi-layer membranes. Porous membranes with improved antimicrobial properties were prepared to enhance wound healing in chelonians and were found to be effective against a broad spectrum of bacteria following the release of two different investigated antimicrobial agents (AgNPs and GS)

The effects of opioids on the peripheral terminals of rat and guinea pig sensory neurons

Studies in vivo and in vitro suggest that opioids can modulate nociceptive signals by interacting with receptors on peripheral neurons. We investigated the peripheral actions of mu (μ), delta (δ), and kappa (κ) opioid agonists using an electrophysiological model of inflammatory-type nociception. Dorsal horn convergent neurons were recorded extracellularly in the halothane anesthetized intact adult rat. Subcutaneous injection of formalin into the hindpaw receptive field of these neurons results in two distinct phases of cell firing. Neither morphine, exogenous ligand for the μ receptor, nor the δ agonist Tyr-D-Ser-(tbu)-Gly-Phe-Leu-Thr (DSTBULET) influenced the formalin response when administered peripherally into the paw. The κ-selective ligand U50488H produced a dose-dependent, naloxone-reversible inhibition of both phases of formalin-induced activity which does not result from leakage of the drug into the systemic circulation. Intrathecal administration of μ and δ, but not κ, opioids has previously been shown to inhibit the biphasic formalin response in the adult rat. Our data suggest that different types of opioid receptors may be important in the periphery and spinal cord. There is some indication that opioid receptor populations are different in adult and neonatal rat spinal cord. For example there are functional μ and κ, but not δ, opioid receptors in an in vitro model of nociceptive activity in the neonatal rat spinal cord. We looked at these apparent developmental differences in binding assays in which opioid receptors in the two tissues were characterized by measurements of ligand binding to crude membrane fractions. Results from binding studies agreed well with fictional studies, in that δ opioid binding sites were not detected on neonatal rat spinal cord membranes. Levels of κ binding were higher in the neonate than in the adult. Novel continuous clonal cell lines with some characteristics of nociceptive dorsal root ganglion (DRG) neurons were tested as a potential model system for the action of opioids on primary afferent nerve fibers. Two of the cell lines expressed δ, but not μ or κ, opioid binding sites. We could not detect effects of δ opioids on potassium currents (as measured by 86Rubidium efflux) or on the release of substance P-like immunoreactivity (SP-LI). We concluded that these cell lines were not good models for studying opioid action on sensory neurons. Measurement of SP-LI release from guinea pig cardiac right ventricular slices did provide a useful model to study peripheral actions of opioids. Formalin (0.2%), capsaicin (100 nM-3μM), and a depolarizing concentration of potassium (100 mM K+) increased the outflow of SP-LI from heart slices. Agonists at μ, δ, and κ opioid receptors inhibited K+-stimulated release and these effects were reversed by naloxone to differing degrees. High concentrations of μ and κ ligands, in their own right, increased the outflow of SP-LI, and these results are compared to previous reports of opioid excitation. Formalin-evoked SP-LI release from heart slices was subject to modulation by opioids. These results agreed well with in vivo results, in that SP-LI release evoked by formalin was not inhibited by μ or δ opioid agonists, but was sensitive to blockade by the κ ligand U50488H. We have demonstrated effects of opioids on the peripheral terminals of sensory neurons in two different models. Peripheral κ, but not μ or δ, receptors were important in modulating formalin-induced effects both in vivo and in vitro. Central κ receptors, at least in adult rats, have been shown not to influence the formalin response in vivo to the same extent as μ and δ receptors. In vitro μ, δ, and κ opioids modulated responses to K+ depolarization of sensory neurons at the peripheral terminals. These results provide strong evidence that peripheral opioid receptors can modulate nociceptive signals

Investigating small molecule therapeutics to improve regeneration and functional recovery following peripheral nerve damage

Peripheral nerve injury (PNI) can be debilitating and results in loss of function, coupled with slow neuron regeneration. Microsurgical treatments remain the gold standard therapy, with no drug therapies currently available. Effective pharmacological treatments could potentially maintain neuronal viability, encourage axonal growth, improve axonal specificity to targets and reduce neuropathic pain. Some drugs and targets have been identified but challenges remain with clinical translation. Advancements in understanding the molecular and cellular events occurring following PNI identifies signalling pathways that could be targeted with drug therapies. The failure in drug therapies reaching PNI clinical trials may be due to the lack of effective in vitro and in vivo pre-clinical models. This study developed and applied models to be used as effective screening tools to address this need. Many compounds demonstrated positive effects on neurite growth when screened in a 3D-engineered co-culture model. NSAIDs (ibuprofen and sulindac sulfide) demonstrated beneficial effects and were studied further in two injury models demonstrating increased axonal growth and improved function. Local controlled-release drug delivery systems have become more attractive because of the drawbacks in conventional drug treatments. This study investigated drug release from various biomaterials in order to obtain an optimal material for implantation and sustained drug delivery. Suitable biomaterials were implanted in vivo to deliver ibuprofen or sulindac sulfide. Both drugs demonstrated beneficial effects on axonal regeneration and functional recovery. Embedding drugs into biocompatible and bio-degradable materials provides effective delivery systems for future translation. Studying NSAIDs revealed a previously unreported relationship between PPAR-γ affinity and regeneration. A NSAID derivative demonstrated the greatest effects on neurite growth in vitro at lower doses than other compounds tested. In summary, this work has identified therapeutic targets to aid the development of novel compounds, as well as, drug repurposing, and effective tools for the pre-clinical screening of these drugs