Differentiated adipose-derived stem cells act synergistically with RGD-modified surfaces to improve neurite outgrowth in a co-culture model.

Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a ß1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that ß1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd.

The biomechanical and functional relationships of the proximal radioulnar joint, distal radioulnar joint, and interosseous ligament.

This biomechanical study assessed integrated function of the proximal radioulnar joint (PRUJ), interosseous ligament (IOL), and distal radioulnar joint (DRUJ). Tekscan™ pressure sensors were inserted into the DRUJ and PRUJ of 15 cadaveric specimens. MicroStrain(®) sensors were mounted onto the IOL on nine of these specimens. A customized biomechanical jig was used to apply axial loads and take measurements through pronosupination. The PRUJ, IOL, and DRUJ were shown to function as an integrated osseoligamentous system distributing applied load. The PRUJ has transmitted pressure profiles similar to those of the DRUJ. Different IOL components support loading at different stages of pronosupination. The IOL is lax during pronation. Mid-IOL tension peaks in the midrange of forearm rotation; distal-IOL tension peaks in supination. Axial loading consistently increases IOL strain in a non-linear fashion. There are clinical implications of this work: disease or surgical modification of any of these structures may compromise normal biomechanics and function.

Schwann cell interactions with polymer films are affected by groove geometry and film hydrophilicity.

We have developed a biodegradable polymer scaffold made of a polycaprolactone/polylactic acid (PCL/PLA) film. Surface properties such as topography and chemistry have a vital influence on cell-material interactions. Surface modifications of PCL/PLA films were performed using topographical cues and UV-ozone treatment to improve Schwann cell organisation and behaviour. Schwann cell attachment, alignment and proliferation were evaluated on the grooved UV-ozone treated and non-treated films. Solvent casting of the polymer solution on patterned silicon substrates resulted in films with different groove shapes: V (V), sloped (SL) and square (SQ) shapes. Pitted films, with no grooves, were prepared as a negative control. The UV-ozone treatment was performed to increase hydrophilicity. The process specifications for UV-ozone treatment were evaluated and 5 min radiation time and 6 cm distance to the UV source were suggested as the optimal practise. When cultured on grooved films, Schwann cells elongated on the V and SL shape grooves without crossing over, and grew in the direction of the grooves. However, there was less elongation with more crossing over on the SQ shape grooves. The maximum cell length (511 µm) was observed on the treated V-grooved films. The cells cultured on pitted UV-ozone treated surfaces showed random arrangements with no increase in length. We have demonstrated that the synergic effects of physical cues combined with UV-ozone treatment have the potential to enhance Schwann cell morphology and alignment.

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 Ca(2+)-imaging techniques, we have shown that stimulation of purinoceptors with adenosine 5'-triphosphate (ATP) triggers intracellular Ca(2+) 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 Ca(2+) 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.

Micro-structural geometry of thin films intended for the inner lumen of nerve conduits affects nerve repair.

Damage to peripheral nerves can cause significant motor or sensory injuries. In serious cases, a nerve is sacrificed from another part of the body to repair a damaged nerve (autograft). The development of biodegradable polymer conduits may offer an alternative to autografts. This study investigated the surface topography and mechanical properties of smooth, pitted and grooved structures of ultra-thin poly (ε-caprolactone)/poly lactic acid blended, solvent-cast films. We have investigated the effect of the groove shape on cell morphology and alignment. Photolithography and dry/wet etching was used to develop patterned silicon substrates with grooves with accurate geometries (V shaped, sloped walls and square shaped). Using a neural cell line (NG108-15), in vitro experiments confirmed good cell attachment and proliferation on all the polymer scaffolds. Imaging techniques demonstrated that there was different cellular responses and morphology according to the shape of the groove. Studies showed that the geometry, particularly the angle of the slope and the space between grooves, affected cellular responses. In addition, biomechanical studies showed that the patterned films had excellent mechanical properties and were stronger than the natural nerve. The conduit tubes were made by rolling the films around a mandrel and using a thermal welding technique to join the edges. The promising biomechanical and in vitro results demonstrate that nerve cell responses are affected by the shape of longitudinal grooves, and particularly by the angle of the slope of the groove walls.

Glial differentiation of human adipose-derived stem cells: implications for cell-based transplantation therapy.

Increasing evidence has shown that adipose-derived stem cells (ASCs) could transdifferentiate into Schwann cell (SC)-like cells to enhance nerve regeneration, suggesting potential new cell-based transplantation therapy for peripheral nerve injuries and neurodegenerative disorders. For the implementation of these results to the clinical setting, it is of great importance to establish the differentiation of human ASCs (hASCs) into a SC phenotype. In this study, we studied hASCs obtained from subcutaneous fat tissue of healthy donors. By a mixture of glial growth factors we differentiated them into Schwann cell-like cells (dhASCs). We then assessed their ability to act as Schwann cells in vitro and in vivo and also compared them with primary human Schwann cells (hSCs). Enzyme-linked immunosorbent assay showed that dhASCs secreted brain-derived neurotrophic factor (BDNF)/nerve growth factor (NGF) at a comparable level, and glial cell-derived neurotrophic factor (GDNF) at a level even higher than hSCs, whereas undifferentiated hASCs (uhASCs) secreted low levels of these neurotrophic factors. In co-culture with NG108-15 neuronal cells we found that both dhASCs and hSCs significantly increased the percentage of cells with neurites, the neurite length, and the number of neurites per neuron, whereas uhASCs increased only the percentage of cells with neurites. Finally, we transplanted green fluorescent protein (GFP)-labeled hASCs into the crushed tibial nerve of athymic nude rats. The transplanted hASCs showed a close association with PGP9.5-positive axons and myelin basic protein (MBP)-positive myelin at 8weeks after transplantation. Quantitative analysis revealed that dhASCs transplantation resulted in significantly improved survival and myelin formation rates (a 7-fold and a 10-fold increase, respectively) as compared with uhASCs transplantation. These findings suggest that hASCs took part in supporting and myelinating regenerating axons, and thus have achieved full glial differentiation in vivo. In conclusion, hASCs can differentiate into SC-like cells that possess a potent capacity to secrete neurotrophic factors as well as to form myelin in vivo. These findings make hASCs an interesting prospect for cell-based transplantation therapy for various peripheral nerve disorders.

Immobilization of cell-binding peptides on poly-ε-caprolactone film surface to biomimic the peripheral nervous system.

Cell-material interactions are crucial for cell adhesion and proliferation on biomaterial surfaces. Immobilization of biomolecules leads to the formation of biomimetic substrates, improving cell response. We introduced RGD (Arg-Gly-Asp) sequences on poly-ε-caprolactone (PCL) film surfaces using thiol chemistry to enhance Schwann cell (SC) response. XPS elemental analysis indicated an estimate of 2-3% peptide functionalization on the PCL surface, comparable with carbodiimide chemistry. Contact angle was not remarkably reduced; hence, cell response was only affected by chemical cues on the film surface. Adhesion and proliferation of Schwann cells were enhanced after PCL modification. Particularly, RGD immobilization increased cell attachment up to 40% after 6 h of culture. It was demonstrated that SC morphology changed from round to very elongated shape when surface modification was carried out, with an increase in the length of cellular processes up to 50% after 5 days of culture. Finally RGD immobilization triggered the formation of focal adhesion related to higher cell spreading. In summary, this study provides a method for immobilization of biomolecules on PCL films to be used in peripheral nerve repair, as demonstrated by the enhanced response of Schwann cells.

Tacrolimus enhances the recovery of normal laryngeal muscle fibre distribution after reinnervation.

OBJECTIVES: To assess the recovery of various muscle fibre types in the posterior cricoarytenoid muscle after laryngeal reinnervation in the rat, and to determine the influence of tacrolimus on this process. METHODS: Four groups of rats underwent resection and anastomosis of the left vagus nerve, and were administered either tacrolimus at a low dose or an immunosuppressive dose, or cyclosporin A at a low dose or an immunosuppressive dose. A fifth group received surgery alone, and a sixth group received neither surgery nor drug treatment (healthy group). Muscles were removed for immunohistochemical analysis 45 days after surgery. RESULTS: There was no difference in the proportion of types 1, 2a and 2b muscle fibres, comparing the immunosuppressive tacrolimus group and the healthy group, whereas there were fewer type 1 fibres in the group receiving surgery alone, compared with the healthy group (7 vs 12.1 per cent, respectively; p = 0.0303). CONCLUSION: Tacrolimus enhanced the recovery of normal laryngeal muscle fibres after reinnervation in the rat, indicating a possible role in laryngeal transplantation.

Subluxation-related ulnar neuropathy (SUN) syndrome related to distal radioulnar joint instability.

Ulnar neuropathy coexistent with distal radioulnar joint (DRUJ) instability has previously been observed in our practice. The aim of this study was to define this phenomenon and investigate the hypothesis that the cause of this intermittent, positional ulnar neuropathy is related to kinking of the ulnar nerve about the DRUJ. Ulna neuropathy was present in 10/51 (19.6%) of a historical cohort of patients who presented with DRUJ instability. Nine subsequent patients with DRUJ instability and coexistent ulnar neuropathy underwent 3-T magnetic resonance imaging to better understand the mechanism of the observed syndrome. Both 3D qualitative and quantitative analyses were used to assess the presence of nerve 'kinking', displacing the nerve from its normal course and causing nerve compression/distraction in the distal forearm and Guyon's canal. Results of the quantitative analysis were statistically significant (p < 0.05). The clinical features of the condition have been delineated and termed subluxation-related ulnar neuropathy or SUN syndrome. The imaging study was a level II diagnostic study.

Nerve repair with adipose-derived stem cells protects dorsal root ganglia neurons from apoptosis.

Novel approaches are required in the clinical management of peripheral nerve injuries because current surgical techniques result in deficient sensory recovery. Microsurgery alone fails to address extensive cell death in the dorsal root ganglia (DRG), in addition to poor axonal regeneration. Incorporation of cultured cells into nerve conduits may offer a novel approach in which to combine nerve repair and enhance axonal regeneration with neuroprotective therapies. We examined apoptotic mediator expression in rat DRG neurons following repair of a 10-mm sciatic nerve gap using a novel synthetic conduit made of poly ε-caprolactone (PCL) and primed with adipose-derived stem cells (ADSC) differentiated towards a Schwann cell phenotype or with primary adult Schwann cells. Differentiated ADSC expressed a range of neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and neurotrophin-4 (NT4). Incorporation of either differentiated ADSC or Schwann cells significantly increased anti-apoptotic Bcl-2 mRNA expression (P<0.001) in the DRG, while significantly decreasing pro-apoptotic Bax (P<0.001) and caspase-3 mRNA (P<0.01) expression. Cleaved caspase-3 protein was observed in the DRG following nerve injury which was attenuated when nerve repair was performed using conduits seeded with cells. Cell incorporation into conduit repair of peripheral nerves demonstrates experimental promise as a novel intervention to prevent DRG neuronal loss.

Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts.

Although autologous nerve graft is still the first choice strategy in nerve reconstruction, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to improve nerve regeneration. Nerve fibrin conduits were seeded with various cell types: primary Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC). Two further control groups were fibrin conduits without cells and autografts. Conduits were used to bridge a 1 cm rat sciatic nerve gap in a long term experiment (16 weeks). Functional and morphological properties of regenerated nerves were investigated. A reduction in muscle atrophy was observed in the autograft and in all cell-seeded groups, when compared with the empty fibrin conduits. SC showed significant improvement in axon myelination and average fiber diameter of the regenerated nerves. dASC were the most effective cell population in terms of improvement of axonal and fiber diameter, evoked potentials at the level of the gastrocnemius muscle and regeneration of motoneurons, similar to the autografts. Given these results and other advantages of adipose derived stem cells such as ease of harvest and relative abundance, dASC could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Laryngeal transplantation in minipigs: vascular, myologic and functional outcomes.

There is no effective way of replacing all the functions of the larynx in those requiring laryngectomy. Regenerative medicine offers promise, but cannot presently deliver implants with functioning neuromuscular units. A single well-documented laryngeal transplant in man was a qualified success, but more information is required before clinical trials may be proposed. We studied the early response of the larynx to laryngeal transplantation between 17 pairs of NIH minipigs full matched at the MHC2 locus. Following iterative technical improvements, pigs had good swallowing and a patent airway at 1 week. No significant changes in mucosal blood flux were observed compared with pre-operative measurements. Changes in muscle morphology and fibre phenotype were observed in transplant muscles retrieved after 7 days: the levels of fast and slow myosin heavy chain (MyHC) protein were reduced and embryonic MyHC was up regulated consistent with denervation induced atrophy. At 1 week laryngeal transplantation can result in good swallowing, and is not associated with clinical evidence of ischemia-reperfusion injury in MHC-matched pigs.

Regeneration potential and survival of transplanted undifferentiated adipose tissue-derived stem cells in peripheral nerve conduits.

Adipose tissue-derived stem cells (ADSCs) have shown potential for the treatment of nerve injuries. Most previous efforts have aimed at stimulating regeneration by using neural-differentiation protocols, but the potential of undifferentiated ADSCs to enhance axonal growth as well as their ability to transdifferentiate in situ have been poorly investigated. In this study, using a rat sciatic nerve model we show that ADSCs, transplanted in an artificial nerve conduit, stimulate axonal outgrowth from the proximal nerve stump and evoke greater Schwann cell (SC) proliferation/intrusion in the distal stump. To track the fate of the transplanted cells, we used green fluorescent protein (GFP)-labelling and polymerase chain reaction (PCR) for the detection of the sex determining region Y (SRY) gene in the donor male cells. Both methods indicated a lack of significant quantities of viable cells 14 days after transplantation. These results suggest that any regenerative effect of transplanted ADSCs is more likely to be mediated by an initial boost of released growth factors and/or by an indirect effect on endogenous SCs activity. Future studies need to address long-term cell survival in tissue-engineered nerve conduits to improve the neuroregenerative potential of ADSCs.

In vitro and in vivo testing of novel ultrathin PCL and PCL/PLA blend films as peripheral nerve conduit.

In an attempt to obviate the drawbacks of nerve autograft, ultrathin microporous biodegradable PCL and PCL/PLA films were tested for their compatibility with motor neuron-like NG108-15 cells and primary Schwann cells. Data obtained from MTS colorimetric and DNA fluorimetric assays showed that both cell lines readily attached and proliferated on these materials. Images taken using scanning electron microscope and fluorescence microscope confirmed these observations. Enhanced cell-surface interaction was achieved by pretreating the films in NaOH solution. Importantly, NG108-15 cells could be induced into differentiated phenotype with long, un-branched neurites growing across the surface of the materials. The bipolar spindle-shaped phenotype of Schwann cells was also retained on these scaffolds. Positive immunochemical staining using antibodies against neurofilament for NG108-15 cells and S100 for Schwann cells indicated the expression of these marker proteins. In a small-scaled pilot testing, the performance of PCL conduits in bridging up a 10 mm gap in rat sciatic nerve model was assessed. Immunohistochemical staining showed that regenerated nerve tissue and penetrated Schwann cells have the potential to span the whole length of the conduit in 2 weeks.

Adipose-derived stem cells enhance peripheral nerve regeneration.

Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like phenotype) for repair of sciatic nerve injury. Two weeks after implantation, the conduits were removed and examined by immunohistochemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell presence (detected by S100 expression). The results show a significant increase in axonal regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in a similar manner to differentiated bone marrow mesenchymal stem cells. These observations suggest that adipose-derived stem cells may provide an effective cell population, without the limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Schwann cell strip for peripheral nerve repair.

Many strategies have been investigated to provide an ideal substitute to treat a nerve gap injury. Initially, silicone conduits were used and more recently conduits fabricated from natural materials such as poly-3-hydroxybutyrate (PHB) showed good results but still have their limitations. Surgically, a new concept optimising harvested autologous nerve graft has been introduced as the single fascicle method. It has been shown that a single fascicle repair of nerve grafting is successful. We investigated a new approach using a PHB strip seeded with Schwann cells to mimic a small nerve fascicle. Schwann cells were attached to the PHB strip using diluted fibrin glue and used to bridge a 10-mm sciatic nerve gap in rats. Comparison was made with a group using conventional PHB conduit tubes filled with Schwann cells and fibrin glue. After 2 weeks, the nerve samples were harvested and investigated for axonal and Schwann cell markers. PGP9.5 immunohistochemistry showed a superior nerve regeneration distance in the PHB strip group versus the PHB tube group (> 10 mm, crossed versus 3.17+/- 0.32 mm respectively, P<0.05) as well as superior Schwann cell intrusion (S100 staining) from proximal (> 10 mm, crossed versus 3.40+/- 0.36 mm, P<0.01) and distal (> 10 mm, crossed versus 2.91+/- 0.31 mm, P<0.001) ends. These findings suggest a significant advantage of a strip in rapidly connecting a nerve gap lesion and imply that single fascicle nerve grafting is advantageous for nerve repair in rats.

[Fibrin matrix enhances adherence of peripheral nerve regenerative cells]. / Fibrinmatrix erhöht adhärenz von regenerativen peripheren nervenzellen.

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.

Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit.

Peripheral nerve injury presents with specific problems of neuronal reconstructions, and from a clinical viewpoint a tissue engineering approach would facilitate the process of repair and regeneration. We have previously used artificial nerve conduits made from bioresorbable poly-3-hydroxybutyrate (PHB) in order to refine the ways in which peripheral nerves are repaired and reconnected to the target muscles and skin. The addition of Schwann cells (SC) or differentiated mesenchymal stem cells (dMSC) to the conduits enhances regeneration. In this study, we have used a matrix based on fibrin (Tisseel) to fill optimally the nerve-conduits with cells. In vitro analysis showed that both SC and MSC adhered significantly better to PHB in the presence of fibrin and cells continued to maintain their differentiated state. Cells were more optimally distributed throughout the conduit when seeded in fibrin than by delivery in growth medium alone. Transplantation of the nerve conduits in vivo showed that cells in combination with fibrin matrix significantly increased nerve regeneration distance (using PGP9.5 and S100 distal and proximal immunohistochemistry) when compared with empty PHB conduits. This study shows the beneficial combinatory effect of an optimised matrix, cells and conduit material as a step towards bridging nerve gaps which should ultimately lead to improved functional recovery following nerve injury.

Analysis of the dose-response of N-acetylcysteine in the prevention of sensory neuronal loss after peripheral nerve injury.

BACKGROUND: N-Acetylcysteine (NAC) is a safe pharmaceutical agent known to protect cells from oxidative damage. Following peripheral nerve transection, NAC has been found to eliminate sensory neuronal loss. This study examines the dose-response relationship of NAC in preventing neuronal death. METHODS AND FINDINGS: The rat sciatic nerve transection model was used, and stereological quantification of sensory neuron survival carried out at two weeks post-axotomy. NAC was administered systemically as an intraperitoneal injection to five groups of rats at a range of doses (1-300 mg/kg/day). Significant neuronal loss was observed in the 1 mg/kg/day dosage group (18.5% loss, p = 0.067 vs. sham treatment). A degree of neuroprotection occurred with 10 mg/kg/day (9.1% loss, p < 0.005 vs. control), whilst there was no significant loss with either 150 or 300 mg/kg/day. CONCLUSIONS: The prevention of sensory neuronal loss with NAC is dose dependent and effective over a wide therapeutic range. This analysis confirms the efficacy of systemic administration and provides a dose framework with which NAC has clinical potential to improve outcome after peripheral nerve trauma.