Dataset Information

A New Preparation Method for Anisotropic Silk Fibroin Nerve Guidance Conduits and Its Evaluation In Vitro and in a Rat Sciatic Nerve Defect Model.

ABSTRACT: Over the past decade, silk fibroin (SF) has been emergently used in peripheral nerve tissue engineering. Current approaches aiming at producing SF-based nerve guidance conduits (SF-NGCs) used dissolved silk based on either aqueous solutions or organic solvents. In this study, we describe a novel procedure to produce SF-NGCs: A braided tubular structure of raw Bombyx mori silk is subsequently processed with the ternary solvent CaCl2/H2O/ethanol, formic acid, and methanol to improve its mechanical and topographical characteristics. Topographically, the combination of the treatments results in a fusion of the outer single silk fibers to a closed layer with a thickness ranging from about 40 to 75??m. In contrast to the outer wall, the inner lumen (not treated with processing solvents) still represents the braided structure of single fibers. Mechanical stability, elasticity, and kink characteristics were evaluated with a custom-made test system. The modification procedure described here drastically improved the elastic properties of our tubular raw scaffold, favoring its use as a NGC. A cell migration assay with NIH/3T3-fibroblasts revealed the impermeability of the SF-NGC wall for possible invading and scar-forming cells. Moreover, the potential of the SF-NGC to serve as a substratum for Schwann cells has been demonstrated by cytotoxicity tests and live-dead stainings of Schwann cells grown on the inner surface of the SF-NGC. In vivo, the SF-NGC was tested in a rat sciatic nerve injury model. In short-term in vivo studies, it was proved that SF-NGCs are not triggering host inflammatory reactions. After 12 weeks, we could demonstrate morphological and functional reinnervation of the distal targets. Filled with collagen, a higher number of axons could be found in the distal to the graft (1678±303), compared with the empty SF-NGC (1274±146). The novel SF-NGC presented here shows promising results for the treatment of peripheral nerve injuries. The modification of braided structures to adapt their mechanical and topographical characteristics may support the translation of SF-based scaffolds into the clinical setting. However, further improvements and the use of extracellular matrix molecules and Schwann cells are suggested to enable silk tube based conduits to bridge long-distance nerve gaps.

SUBMITTER: Teuschl AH

PROVIDER: S-EPMC4553371 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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A New Preparation Method for Anisotropic Silk Fibroin Nerve Guidance Conduits and Its Evaluation In Vitro and in a Rat Sciatic Nerve Defect Model.

Teuschl Andreas Herbert AH Schuh Christina C Halbweis Robert R Pajer Krisztián K Márton Gábor G Hopf Rudolf R Mosia Shorena S Rünzler Dominik D Redl Heinz H Nógrádi Antal A Hausner Thomas T

Tissue engineering. Part C, Methods 20150518 9

Over the past decade, silk fibroin (SF) has been emergently used in peripheral nerve tissue engineering. Current approaches aiming at producing SF-based nerve guidance conduits (SF-NGCs) used dissolved silk based on either aqueous solutions or organic solvents. In this study, we describe a novel procedure to produce SF-NGCs: A braided tubular structure of raw Bombyx mori silk is subsequently processed with the ternary solvent CaCl2/H2O/ethanol, formic acid, and methanol to improve its mechanical ...[more]

PMID: 25819471

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Project description:BackgroundNerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration.Methods3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 106 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve.ResultsThe inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair.Conclusions3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats.

Project description:Rationale: Peripheral nerve injury (PNI) is a great challenge for regenerative medicine. Nerve autograft is the gold standard for clinical PNI repair. Due to its significant drawbacks, artificial nerve guidance conduits (NGCs) have drawn much attention as replacement therapies. We developed a combinatorial NGC consisting of longitudinally aligned electrospun nanofibers and porcine decellularized nerve matrix hydrogel (pDNM gel). The in vivo capacity for facilitating nerve tissue regeneration and functional recovery was evaluated in a rat sciatic nerve defect model. Methods: Poly (L-lactic acid) (PLLA) was electrospun into randomly oriented (PLLA-random) and longitudinally aligned (PLLA-aligned) nanofibers. PLLA-aligned were further coated with pDNM gel at concentrations of 0.25% (PLLA-aligned/0.25% pDNM gel) and 1% (PLLA-aligned/1% pDNM gel). Axonal extension and Schwann cells migration were evaluated by immunofluorescence staining of dorsal root ganglia cultured on the scaffolds. To fabricate implantable NGCs, the nanofibrous scaffolds were rolled and covered with an electrospun protection tube. The fabricated NGCs were then implanted into a 5 mm sciatic nerve defect model in adult male Sprague-Dawley rats. Nerves treated with NGCs were compared to contralateral uninjured nerves (control group), injured but untreated nerves (unstitched group), and autografted nerves. Nerve regeneration was monitored by an established set of assays, including T2 values and diffusion tensor imaging (DTI) derived from multiparametric magnetic resonance imaging (MRI), histological assessments, and immunostaining. Nerve functional recovery was evaluated by walking track analysis. Results: PLLA-aligned/0.25% pDNM gel scaffold exhibited the best performance in facilitating directed axonal extension and Schwann cells migration in vitro due to the combined effects of the topological cues provided by the aligned nanofibers and the biochemical cues retained in the pDNM gel. Consistent results were obtained in animal experiments with the fabricated NGCs. Both the T2 and fractional anisotropy values of the PLLA-aligned/0.25% pDNM gel group were the closest to those of the autografted group, and returned to normal much faster than those of the other NGCs groups. Histological assessment indicated that the implanted PLLA-aligned/0.25% pDNM gel NGC resulted in the largest number of axons and the most extensive myelination among all fabricated NGCs. Further, the PLLA-aligned/0.25% pDNM gel group exhibited the highest sciatic nerve function index, which was comparable to that of the autografted group, at 8 weeks post-surgery. Conclusions: NGCs composed of aligned PLLA nanofibers decorated with 0.25% pDNM gel provided both topological and biochemical guidance for directing and promoting axonal extension, nerve fiber myelination, and functional recovery. Moreover, T2-mapping and DTI metrics were found to be useful non-invasive monitoring techniques for PNI treatment.

Dataset Information

A New Preparation Method for Anisotropic Silk Fibroin Nerve Guidance Conduits and Its Evaluation In Vitro and in a Rat Sciatic Nerve Defect Model.

Publications

A New Preparation Method for Anisotropic Silk Fibroin Nerve Guidance Conduits and Its Evaluation In Vitro and in a Rat Sciatic Nerve Defect Model.

Similar Datasets

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