Mingzhu Sun

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.

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.

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.

In vitro evaluation of polyester-based scaffolds seeded with adipose derived stem cells for peripheral nerve regeneration

To overcome the disadvantages of autografts for peripheral nerve repair, different methods such as artificial nerve conduits have been investigated for an alternative approach. This study demonstrated that solvent casting is a simple but efficient method to create thin polyester-based scaffolds for stem cell delivery. Using poly (ε-caprolactone) and poly (D,L-lactic acid), we produced scaffold films containing heterogenous depressions (pits) on the air surface with a size ranging from 0.5 to 30 μm(2). These scaffolds were moderately hydrophobic; however, they supported the differentiation of adipose derived stem cells (ADSC) into a Schwann cell-like phenotype. The differentiated ADSC (dADSC) expressed S100 protein and glial fibrillary acidic protein and readily adhered to the films and proliferated at a similar rate to those cultured on tissue culture polystyrene. Cells were also positive for proliferating cell nuclear antigen. Furthermore, dADSC retained functional activity and significantly enhanced neurite outgrowth from dorsal root ganglia neurons. This study suggests polymer scaffolds combined with dADSCs could be a promising therapy for peripheral nerve injuries.

Solvent-cast PCL films support the regeneration of NG108-15 nerve cells

The reconstruction of peripheral nerve defects over a short distance (10-20mm) could benefit from the development of novel biomaterials. Bio-degradable and bio-compatible materials are being pursued to replace the currently used nerve autografts. We hypothesize that physical properties, particularly surface texture, could have substantial effects on the hydrophilicity of some synthetic polymers and subsequently the compatibility of them with cells. In this study, poly (ε-caprolactone) (PCL) films have been cast using four solvents from different chemical families and evaluated for their suitability and potential use as a nerve conduit substratum. The following solvents: dichloromethane (DCM, Halocarbon), methyl acetate (MA, Ester), tetrahydrofuran (THF, Ether) and acetic acid (AA, organic acid) were used to produce PCL films. The physical properties of these PCL films, average surface roughness (Ra) and wettability were measured. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) techniques were applied to obtain information on the surface texture. Motor neuron-like NG108-15 cells were used as a model to evaluate the biocompatibility of the films. It was found that same polymer when processed using different solvents could produce materials with markedly different physical and biochemical properties. Importantly, all PCL films were supportive for the growth and differentiation of NG108-15 cells.