Alessandro Faroni

Peripheral nerve regeneration: Experimental strategies and future perspectives.

Peripheral nerve injuries represent a substantial clinical problem with insufficient or unsatisfactory treatment options. This review summarises all the events occurring after nerve damage at the level of the cell body, the site of injury and the target organ. Various experimental strategies to improve neuronal survival, axonal regeneration and target reinnervation are described including pharmacological approaches and cell-based therapies. Given the complexity of nerve regeneration, further studies are needed to address the biology of nerve injury, to improve the interaction with implantable scaffolds, and to implement cell-based therapies in nerve tissue engineering.

Long term peripheral nerve regeneration using a novel PCL nerve conduit

The gold standard in surgical management of a peripheral nerve gap is currently autologous nerve grafting. This confers patient morbidity and increases surgical time therefore innovative experimental strategies towards engineering a synthetic nerve conduit are welcome. We have developed a novel synthetic conduit made of poly ε-caprolactone (PCL) that has demonstrated promising peripheral nerve regeneration in short-term studies. This material has been engineered to permit translation into clinical practice and here we demonstrate that histological outcomes in a long-term in vivo experiment are comparable with that of autologous nerve grafting. A 1cm nerve gap in a rat sciatic nerve injury model was repaired with a PCL nerve conduit or an autologous nerve graft. At 18 weeks post surgical repair, there was a similar volume of regenerating axons within the nerve autograft and PCL conduit repair groups, and similar numbers of myelinated axons in the distal stump of both groups. Furthermore, there was evidence of comparable re-innervation of end organ muscle and skin with the only significant difference the lower wet weight of the muscle from the PCL conduit nerve repair group. This study stimulates further work on the potential use of this synthetic biodegradable PCL nerve conduit in a clinical setting.

Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death.

Schwann cells (SCs) are fundamental for development, myelination and regeneration in the peripheral nervous system. Slow growth rate and difficulties in harvesting limit SC applications in regenerative medicine. Several molecules, including receptors for neurosteroids and neurotransmitters, have been suggested to be implicated in regulating physiology and regenerative potential of SCs. Adipose-derived stem cells (ASCs) can be differentiated into SC-like phenotype (dASC) sharing morphological and functional properties with SC, thus representing a valid SC alternative. We have previously shown that dASC express γ-aminobutyric-acid receptors, which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitters in ASC. In this study, we investigated the expression of purinergic receptors in dASC. Using reverse transriptase (RT)-PCR, western blot analyses and immunocytochemistry, we have demonstrated that ASCs express P2X3, P2X4 and P2X7 purinoceptors. Differentiation of ASCs towards glial phenotype was accompanied by upregulation of P2X4 and P2X7 receptors. Using Ca2+-imaging techniques, we have shown that stimulation of purinoceptors with adenosine 5′-triphosphate (ATP) triggers intracellular Ca2+ signals, indicating functional activity of these receptors. Whole-cell voltage clamp recordings showed that ATP and BzATP induced ion currents that can be fully inhibited with specific P2X7 antagonists. Finally, using cytotoxicity assays we have shown that the increase of intracellular Ca2+ leads to dASC death, an effect that can be prevented using a specific P2X7 antagonist. Altogether, these results show, for the first time, the presence of functional P2X7 receptors in dASC and their link with critical physiological processes such as cell death and survival. The presence of these novel pharmacological targets in dASC might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dASC for nerve repair.

Adipose-derived stem cells and nerve regeneration: promises and pitfalls.

In order to improve the outcome of nerve regeneration following peripheral trauma injuries, the development of bioengineered nerve grafts has attracted great attention in the field of tissue engineering. Adult stem cells constitute the ideal alternative to Schwann cells (SCs) as transplantable cells in bioartificial nerve grafts. Among the various sources of stem cells with potential applications for regenerative medicine, the adipose tissue has been proven to be one of the most promising. Adipose-derived stem cells (ASCs) are easily obtained, rapidly expanded, show low immunogenicity, and can be differentiated into SCs in vitro. This chapter will focus on recent advances in the use of differentiated and undifferentiated ASCs for peripheral nerve regeneration, with a critical attention for the clinical exploitability of ASC in nerve repair strategies.

The Neurosteroid Allopregnanolone Modulates Specific Functions in Central and Peripheral Glial Cells

Since the first observations on the existence of “neurosteroids” in the 1980s, our understanding of the importance of these endogenous steroids in the control of the central and peripheral nervous system (PNS) has increased progressively. Although most of the observations were made in neuronal cells, equally important are the effects that neurosteroids exert on glial cells. Among the different classes of neurosteroids acting on glial cells, the progesterone 5α-3α metabolite, allopregnanolone, displays a particular mechanism of action involving primarily the modulation of classic GABA receptors. In this review, we focus our attention on allopregnanolone because its effects on the physiology of glial cells of the central and PNS are intriguing and could potentially lead to the development of new strategies for neuroprotection and/or regeneration of injured nervous tissues.

Polymer Scaffolds with Preferential Parallel Grooves Enhance Nerve Regeneration.

We have modified the surface topography of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) blended films to improve cell proliferation and to guide the regeneration of peripheral nerves. Films with differing shaped grooves were made using patterned silicon templates, sloped walls (SL), V-shaped (V), and square-shaped (SQ), and compared with nongrooved surfaces with micropits. The solvent cast films were tested in vitro using adult adipose-derived stem cells differentiated to Schwann cell-like cells. Cell attachment, proliferation, and cell orientation were all improved on the grooved surfaces, with SL grooves giving the best results. We present in vivo data on Sprague-Dawley rat sciatic nerve injury with a 10-mm gap, evaluating nerve regeneration at 3 weeks across a polymer nerve conduit modified with intraluminal grooves (SL, V, and SQ) and differing wall thicknesses (70, 100, 120, and 210 μm). The SL-grooved nerve conduit showed a significant improvement over the other topographical-shaped grooves, while increasing the conduit wall thickness saw no positive effect on the biological response of the regenerating nerve. Furthermore, the preferred SL-grooved conduit (C) with 70 μm wall thickness was compared with the current clinical gold standard of autologous nerve graft (Ag) in the rat 10-mm sciatic nerve gap model. At 3 weeks postsurgery, all nerve gaps across both groups were bridged with regenerated nerve fibers. At 16 weeks, features of regenerated axons were comparable between the autograft (Ag) and conduit (C) groups. End organ assessments of muscle weight, electromyography, and skin reinnervation were also similar between the groups. The comparable experimental outcome between conduit and autograft, suggests that the PCL/PLA conduit with inner lumen microstructured grooves could be used as a potential alternative treatment for peripheral nerve repair.

Schwann-like adult stem cells derived from bone marrow and adipose tissue express γ-aminobutyric acid type B receptors

γ‐Aminobutyric acid type B receptors (GABA‐B) are expressed in glial cells of the central and peripheral nervous systems, and recent evidence has shown their importance in modulating physiological parameters of Schwann cell (SC). SC play essential roles in peripheral nerve regeneration, but several drawbacks prevent their use for nerve repair. Adult stem cells from adipose tissue (ASC) or bone marrow (BM‐MSC) can be differentiated into an SC‐like phenotype and used as SC replacements. The aim of this study was to investigate GABA‐B receptor functional expression in differentiated stem cells by assessing the similarity to SC. By means of RT‐PCR and Western blot methodologies, BM‐MSC and ASC were found to express both GABA‐B1 and GABA‐B2 receptor subunits. The expression levels of GABA‐B1b and GABA‐B2 receptors were influenced by SC‐like differentiation, as shown by Western blot studies. GABA‐B receptor stimulation with baclofen reduced the proliferation rate of SC and differentiated ASC (dASC) but not that of dBM‐MSC. In conclusion, both of the subunits that assemble into a functional GABA‐B receptor are present in differentiated stem cells. Furthermore, GABA‐B receptors in dASC are functionally active, regulating a key process such as proliferation. The presence of functional GABA‐B receptors on differentiated stem cells opens new opportunities for a possible pharmacological modulation of their physiology and phenotype.

Deletion of GABA-B Receptor in Schwann Cells Regulates Remak Bundles and Small Nociceptive C-fibers

The mechanisms regulating the differentiation into non‐myelinating Schwann cells is not completely understood. Recent evidence indicates that GABA‐B receptors may regulate myelination and nociception in the peripheral nervous system. GABA‐B receptor total knock‐out mice exhibit morphological and molecular changes in peripheral myelin. The number of small myelinated fibers is higher and associated with altered pain sensitivity. Herein, we analyzed whether these changes may be produced by a specific deletion of GABA‐B receptors in Schwann cells. The conditional mice (P0‐GABA‐B1fl/fl) show a morphological phenotype characterized by a peculiar increase in the number of small unmyelinated fibers and Remak bundles, including nociceptive C‐fibers. The P0‐GABA‐B1fl/fl mice are hyperalgesic and allodynic. In these mice, the morphological and behavioral changes are associated with a downregulation of neuregulin 1 expression in nerves. Our findings suggest that the altered pain sensitivity derives from a Schwann cell‐specific loss of GABA‐B receptor functions, pointing to a role for GABA‐B receptors in the regulation of Schwann cell maturation towards the non‐myelinating phenotype. GLIA 2014;62:548–565

Expression of Functional γ-Aminobutyric Acid Type A Receptors in Schwann-Like Adult Stem Cells

Gamma-Aminobutyric acid (GABA) receptors are present in peripheral and central glia and modulate important physiological parameters of glial cells. Schwann cells (SC), the peripheral nervous system glial cells, play essential roles in nerve regeneration, but they are unsuitable for bioengineering of nerve repair. Increasing interest has been focused on adult stem cells derived from bone marrow (BM-MSC) or adipose tissue (ASC), which can be differentiated into SC-like phenotype and used as SC replacements. SC-like adult stem cells express GABA-B receptors that can modulate their proliferation. The aim of this study was to investigate GABA-A receptors functional expression in differentiated stem cells. BM-MSC and ASC were found to express GABA-A α2 and β3, but not β1 mRNA transcripts. Protein expression levels of GABA-A α2 and β3 receptors were upregulated following SC-like differentiation as shown by Western blot studies. GABA-A receptor stimulation with muscimol increased the proliferation rate of SC, differentiated BM-MSC and differentiated ASC. In conclusion, GABA-A α2 and β3 receptor subunits are present in BM-MSC and ASC and upregulated following glial differentiation. GABA-A subunits in differentiated stem cells and SC assemble in functional receptors modulating cell proliferation. Functional GABA-A and GABA-B receptors represent a possible pharmacological target to modulate SC-like stem cells physiology.

Human Schwann‐like cells derived from adipose‐derived mesenchymal stem cells rapidly de‐differentiate in the absence of stimulating medium

Finding a viable cell‐based therapy to address peripheral nerve injury holds promise for enhancing the currently suboptimal microsurgical approaches to peripheral nerve repair. Autologous nerve grafting is the current gold standard for surgical repair of nerve gaps; however, this causes donor nerve morbidity in the patient, and the results remain unsatisfactory. Transplanting autologous Schwann cells (SCs) results in similar morbidity, as well as limited cell numbers and restricted potential for expansion in vitro. Adipose‐derived stem cells (ASCs), ‘differentiated’ towards an SC‐like phenotype in vitro (dASCs), have been presented as an alternative to SC therapies. The differentiation protocol stimulates ASCs to mimic the SC phenotype; however, the efficacy of dASCs in nerve repair is not yet convincing, and the practicality of the SC‐like phenotype is unproven. Here, we examined the stability of dASCs by withdrawing differentiation medium for 72 h after the full 18‐day differentiation protocol, and measuring changes in morphology, gene expression, and protein levels. Withdrawal of differentiation medium from dASCs resulted in a rapid reversion to stem cell‐like characteristics. Quantitative real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay analyses demonstrated a significant reduction in gene and protein expression of growth factors that were expressed at high levels following ‘differentiation’. Therefore, we question the relevance of differentiation to an SC‐like phenotype, as withdrawal of differentiation medium, a model of transplantation into an injured nerve, results in rapid reversion of the dASC phenotype to stem cell‐like characteristics. Further investigation into the differentiation process and the response of dASCs to an injured environment must be undertaken prior to the use of dASCs in peripheral nerve repair therapies.