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.

Gelatin-based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering.

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.

Local delivery of the Neuregulin1 receptor ecto-domain (ecto-ErbB4) has a positive effect on regenerated nerve fiber maturation.

The Neuregulin/ErbB system plays an important role in the peripheral nervous system, under both normal and pathological conditions. We previously demonstrated that expression of soluble ecto-ErbB4, the released extracellular fragment of the ErbB4 receptor, stimulated glial cell migration in vitro. In this study we examined the possibility of manipulating this system in vivo in order to improve injured peripheral nerve regeneration. Transected rat median nerves of adult female Wistar rats were repaired with a 10-mm-long graft made by muscle-in-vein combined nerve guide previously transduced with either the adeno-associated viral (AAV) vector AAV2-LacZ or AAV2-ecto-ErbB4. Autologous nerve grafts were used as control. Both stereological and functional analyses were performed to assess nerve regeneration. Data show that delivery of soluble ecto-ErbB4 by gene transfer in the muscle-in-vein combined nerve guide has a positive effect on fiber maturation, suggesting that it could represent a potential tool for improving peripheral nerve regeneration.

Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model.

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to develop and test hybrid chitosan membranes to use in peripheral nerve reconstruction, either alone or enriched with N1E-115 neural cells. Hybrid chitosan membranes were tested in vitro, to assess their ability in supporting N1E-115 cell survival and differentiation, and in vivo to assess biocompatibility as well as to evaluate their effects on nerve fiber regeneration and functional recovery after a standardized rat sciatic nerve crush injury. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and ankle kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. All chitosan membranes showed good biocompatibility and proved to be a suitable substrate for plating the N1E-115 cellular system. By contrast, in vivo nerve regeneration assessment after crush injury showed that the freeze-dried chitosan type III, without N1E-115 cell addition, was the only type of membrane that significantly improved posttraumatic axonal regrowth and functional recovery. It can be thus suggested that local enwrapping with this type of chitosan membrane may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery.

Functional, morphological and biomolecular assessment of posttraumatic neuro-muscular recovery in the rat forelimb model.

Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization (using both straight and Y-shaped guides). The outcome of nerve repair at different postoperative times was assessed by functional, morphological and biomolecular analysis. Results showed that both end-to-side and tubulization repair of rat median and ulnar nerves led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function as assessed by grasping test. Biomolecular analysis by means of reverse transcription polymerase chain reaction (RT-PCR) demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3. Finally, our experience also suggests that the rat forelimb experimental model is particularly appropriate for the study of microsurgical reconstruction of major mixed nerve trunks. Furthermore, since the forelimb model is less compromising for the animal, it should be preferred to the hindlimb model for many research purposes.

Nerve regeneration along bioengineered scaffolds.

Tissue engineering has recently seen great advancements in many medical fields, including peripheral nerve reconstruction. In the rat median nerve model, we investigated nerve repair by means of bioengineered tissue scaffolds (muscle-vein-combined tubes) focusing on changes in the neuregulin-1/ErbB-receptor system which represents one of the main regulatory systems of axo-glial interaction in peripheral nerves. Repaired nerves were withdrawn at 5, 15, and 30 days postoperative and processed for morphological and retro-transcriptase polymerase chain reaction (RT-PCR) analysis. Results revealed an early and progressive increase in the expression of NRG1alpha isoform only, while the appearance of the beta isoform of NRG1, which is normally present in peripheral nerves, was delayed. In regards to ErbB2 and ErbB3 receptors, their expression increased progressively inside the muscle-vein-combined scaffolds, though with different kinetics. Taken together, these results suggest that variations in neuregulin-1/ErbB system activation play a key role in peripheral nerve regeneration along bioengineered muscle-vein-combined scaffolds. Since similar variations are also detectable in denervated skeletal muscles, it can be hypothesized that the existence of a NRG1's autocrine/paracrine trophic loop shared by both glial and muscle fibers could be responsible for the effectiveness of muscle-vein-combined conduits for repairing nerve defects.

Osteopontin is an autocrine mediator of hepatocyte growth factor-induced invasive growth.

In epithelial cells, hepatocyte growth factor (HGF) activates a genetic program involving cell-cell dissociation ("scattering"), growth and invasiveness. The full program is not elicited by other growth factors like epidermal growth factor, and is aberrantly activated during cancer progression to the invasive-metastatic phenotype. To identify genes involved in the onset of invasive growth, we explored by cDNA microarrays the in vitro transcriptional response to HGF of mouse embryo liver cells. We identified osteopontin (OPN), a secreted matrix protein, as a major HGF transcriptional target. The wave of OPN induction is maximal at 6 h, in concomitance with the initiation of scattering, and is specific, because no other matrix protein among those explored by the microarray is affected. Interestingly, HGF, but not epidermal growth factor, promotes cell adhesion to OPN via the CD44 receptor. Scattering is significantly impaired by antibodies against OPN and CD44; conversely, constitutive OPN overexpression dramatically increases the motile and invasive responses to HGF, leading to disruption of the ordered morphogenetic program triggered by this ligand.

A gene trap vector system for identifying transcriptionally responsive genes.

We present a method for fast and efficient trapping of genes whose transcription is regulated by exogenous stimuli. We constructed a promoterless retroviral vector transducing a green fluorescent protein-nitroreductase (GFNR) fusion protein downstream from a splice acceptor site. Flow cytometric analysis of the infected population allows identification and sorting of cells in which the trap is integrated downstream from an active promoter. Conversely, the nitroreductase (NTR) moiety allows pharmacological selection against constitutive GFNR expression. Using hepatocyte growth factor (HGF) stimulation of liver cells combined with either positive or negative selection, we recovered cell populations carrying traps in induced or suppressed genes, respectively. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA level. Positive and negative selection can be calibrated to recover traps in genes showing different levels of basal expression or transcriptional regulation. The flexibility and efficiency of the GFNR-based trap screening procedure make it suitable for wide surveys of transcriptionally regulated genes.

A natural hepatocyte growth factor/scatter factor autocrine loop in myoblast cells and the effect of the constitutive Met kinase activation on myogenic differentiation.

As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way. To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65(tpr-met)) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65(tpr-met) highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand-receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65(tpr-met) kinase. These data indicate that a met kinase signal released from differentiation-dependent control provides a negative stimulus for the onset of myogenic differentiation.

Ets up-regulates MET transcription.

MET, a potentially harmful oncogene controlling invasive growth, is overexpressed in a significant percentage of human cancers. Since amplification of the MET gene occurs only in a fraction of these cases, we investigated the transcriptional mechanisms responsible for up-regulation of the promoter activity. The transcription driven by the 3.1 kbp DNA fragment containing the minimal promoter was studied by 5' progressive deletion analysis. The patterns of MET promoter activity suggest the presence of weak negative and positive elements in the region between 300 and 840 bp upstream to the transcription start site. The region encompassing the first 300 bp strongly up-regulates the promoter. This region contains four putative binding sites for members of the Ets transcription factor family, known to be involved in invasive growth. Transient co-expression of Ets1 resulted in a strong enhancement of the MET promoter activity. Increased expression of the Met protein was observed in cells stably transfected with ETS1. Double stranded oligonucleotides with Ets consensus sequence were used as a 'decoy' to inhibit binding to DNA native sites. They dramatically reduced the amount of Met protein in a human carcinoma cell line overexpressing the oncogene. Interestingly, Met activation induces transcription of ETS1 mRNA, showing that Ets proteins act both upstream and downstream to MET. These data indicate that members of the Ets family promote MET transcription and suggest their contribution to the invasive phenotype through overexpression of MET.

Hepatocyte growth factor (HGF) receptor expression is inducible and is part of the delayed-early response to HGF.

The c-MET proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), also known as scatter factor, a potent mitogen and motogen for epithelial cells. The level of the HGF receptor expressed by epithelial cells varies in different growth conditions, being lower in growth arrested confluent monolayers and higher in growing sparse cells. The amount of HGF receptor mRNA increases from 3- to 5-fold after stimulation of confluent monolayers by serum and up to 10-fold after stimulation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA). An increased level of the receptor mRNA was also observed after cell stimulation with nanomolar concentration of HGF itself. The effect was transient, dose, and time-dependent. Transcription of a reporter gene under control of the cloned 297 base pair c-MET promoter was also stimulated by serum, TPA, or HGF. The accumulation of specific mRNA is followed by appearance of the HGF receptor precursor protein, which is further processed to the receptor mature form. After HGF stimulation, HGF receptor expression follows c-FOS and c-JUN induction with a peak approximately 4 h. Pretreatment with the protein synthesis inhibitor puromycin strongly reduced the response to HGF, while cycloheximide alone increased the level of the receptor mRNA. These data show that c-MET behaves as a delayed early-response gene and suggest that the HGF response is autoamplified by inducing the specific receptor.

Structure and inducible regulation of the human MET promoter.

The MET oncogene, encoding the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor, is expressed in epithelial cells and overexpressed in a significant proportion of human epithelial cancers, suggesting the occurrence of transcriptional alteration(s). To identify the MET promoter, we isolated recombinant cDNA clones encompassing the entire 5'-noncoding sequence of MET messenger RNAs. Using probes derived from this region, we cloned the entire genomic region spanning the first MET exon and the flanking regulatory sequences. The first exon, containing the entire untranslated sequence, is present in the MET mRNAs of 7.1, 5.9, and 4.6 kilobases, showing that the expression of the multiple transcripts is regulated by a single promoter. The start site of transcription was determined by primer extension and by rapid amplification of cDNA ends. We show that a 300-base pair fragment, containing sequences upstream from the start site, efficiently drives the expression of a reporter gene in transfected epithelial cells. This promoter fragment also contains the cis-acting elements responsible for phorbol-ester induction.