Trimethylene carbonate-caprolactone conduit with poly-p-dioxanone microfilaments to promote regeneration after spinal cord injury.

Spinal cord injury (SCI) is often associated with scarring and cavity formation and therefore bridging strategies are essential to provide a physical substrate for axonal regeneration. In this study we investigated the effects of a biodegradable conduit made from trimethylene carbonate and ε-caprolactone (TC) containing poly-p-dioxanone microfilaments (PDO) with longitudinal grooves on regeneration after SCI in adult rats. In vitro studies demonstrated that different cell types including astrocytes, meningeal fibroblasts, Schwann cells and adult sensory dorsal root ganglia neurons can grow on the TC and PDO material. For in vivo experiments, the TC/PDO conduit was implanted into a small 2-3 mm long cavity in the C3-C4 cervical segments immediately after injury (acute SCI) or at 2-5 months after initial surgery (chronic SCI). At 8 weeks after implantation into acute SCI, numerous 5HT-positive descending raphaespinal axons and sensory CGRP-positive axons regenerated across the conduit and were often associated with PDO microfilaments and migrated host cells. Implantation into chronically injured SCI induced regeneration mainly of the sensory CGRP-positive axons. Although the conduit had no effect on the density of OX42-positive microglial cells when compared with SCI control, the activity of GFAP-positive astrocytes was reduced. The results suggest that a TC/PDO conduit can support axonal regeneration after acute and chronic SCI even without addition of exogenous glial or stem cells. STATEMENT OF SIGNIFICANCE: Biosynthetic conduits can support regeneration after spinal cord injury but often require addition of cell therapy and neurotrophic factors. This study demonstrates that biodegradable conduits made from trimethylene carbonate and ε-caprolactone with poly-p-dioxanone microfilaments alone can promote migration of different host cells and stimulate axonal regeneration after implantation into acute and chronic spinal cord injury. These results can be used to develop biosynthetic conduits for future clinical applications.

Neuronal and glial responses to siRNA-coated nerve guide implants in vitro.

The manipulation of gene expression by RNA interference could play a key role in future neurotherapies, for example in the development of biohydrid implants to bridge nerve and spinal cord lesion gaps. Such resorbable biomaterial prostheses could serve as growth substrates together with specific siRNA to foster neuronal regeneration. To the best of our knowledge, we are the first to biofunctionalize neuronal prostheses with siRNA. We analyzed neuronal and Schwann cell responses to scrambled siRNA coated polydioxanone polymer filaments designed to imitate pro-regenerative bands of Büngner for oriented axonal regrowth. With a view to future clinical applications we were especially interested in potentially detrimental side effects. We employed a variety of in vitro methods, including a novel impedance electrode microchamber assay, fluorescence and scanning electron microscopy, metabolic labeling and RT-PCR. We found that the application of chitosan/siRNA nanoparticles (1) did not affect glial cell motility or (2) axonal growth in contrast to other formulations, (3) only slightly reduced proliferation, and (4) did not induce inflammatory responses that might hamper axonal regeneration. The data suggest that chitosan/siRNA nanoparticle-coated polymer filaments are suitable for use in biohybrid implants with no significant side effects on neuronal and glial cells.

Bioengineered glial strands for nerve regeneration.

Nerve guide implants approved for human application in the peripheral nervous system generally fail to bridge lesion gaps longer than 2 cm and cannot match the clinical performance of autologous nerve transplants. Since current synthetic implants are simply hollow tubes, we aim to recreate the native microarchitecture of nerves inside the tubular implants. Most importantly, in the regenerating nerve, dedifferentiated Schwann cells align to form thousands of long glial strands, which act as guiding structures for the regrowing axons. In order to artificially induce the formation of Schwann cell strands, 28 µm thick, endless poly-p-dioxanone filaments (PDO) were synthesized with longitudinal grooves. A polycationic coating on the PDO filaments rendered the polymer surface cell-permissive and induced the growth of highly oriented Schwann cells with polarized expression of N-cadherin at cell-cell contact sites. In vitro cell proliferation on three-dimensional PDO filaments was significantly increased in comparison to planar PDO substrates. Time lapse video recordings revealed high Schwann cell motility, which is advantageous for the repopulation of cell-free implants after implantation. In a pilot study we employed a novel microsurgical technique in vivo. All axon fascicles were selectively dissected from sciatic rat nerves, and the remaining epineural tube was filled with hundreds of PDO filaments. Histological analysis 6 weeks postoperatively showed no fibrosis or encapsulation but instead longitudinal cell alignment and axonal regrowth. The data suggest that the addition of microstructured PDO filaments to the lumen of synthetic tubular implants might significantly improve performance.

Strategies for inducing the formation of bands of Büngner in peripheral nerve regeneration.

Peripheral human nerves fail to regenerate across longer tube implants (>2 cm), most likely because implants lack the microarchitecture of native nerves, including bands of Büngner. Bands of Büngner comprise longitudinally aligned Schwann cell strands that guide selectively regrowing axons. We aim to optimize tubular implants by integrating artificial bands of Büngner. Three principle strategies for inducing the formation of bands of Büngner were investigated: (a) an aligned extracellular matrix, (b) polarizing differentiation factors, and (c) microstructured biomaterial filaments. In vitro oriented collagen and a combination of differentiation factors (NGF, neuregulin-1, TGF-beta) induced Schwann cell alignment to some extent. The most pronounced Schwann cell alignment was evident on ultrathin, endless poly-epsilon-caprolactone (PCL) filaments with longitudinal microgrooves. Precoated PCL filaments proved to be non-cytotoxic, displayed good cell attachment, and supported Schwann cell proliferation as well as guided axonal outgrowth. In vitro on PCL filaments Schwann cells displayed a polarized expression of the cell adhesion molecule L1 similar to that seen in vivo in bands of Büngner after sciatic nerve crush in adult rats. In summary, the integration of bioengineered bands of Büngner based on microstructured polymer filaments in nerve conduits promises to be the most valuable approach to initiating a more efficient regeneration across longer nerve lesions.

Long nerve gaps limit the regenerative potential of bioartificial nerve conduits filled with Schwann cells.

PURPOSE: Recently we successfully used a conduit of epsilon-caprolactone-co-trimethylene carbonate filled with Schwann cells (SC) across a 20 mm gap in a rat median nerve. In this study we applied the tubes with SC across a 40 mm gap in order to analyse the regenerative potential of the tubes in long nerve defects. METHODS: To augment the nerve defect a cross-chest procedure was used and the tubes were implanted with injected isogeneic SCs inside (group 3). Both ulnar nerves were used for a 40 mm autograft (group 2). For control group non-operated animals were used (group 1). The grasping test, histology (S-100, PAM), electrophysiology, and the muscle weight were used to assess regeneration. RESULTS: After 12 months, grasping was seen only in three animals of group 3 (3.6 g [95% CI: 0 to 7.6 g]). However, in group 2 all rats had a partial functional regeneration (42.8 g [95% CI: 39.1 to 46.6 g]). The grasping force of the non-operated animals (group 1) was 240.9 g [95% CI: 237.2 to 244.7 g] at the time. Histology from group 3 confirmed an irregular arrangement of fibres in contrast to more organized structures in group 2. Electrophysiology in group 3 displayed potentials only in the three animals with functional regeneration. In group 2 all animals exhibited potentials. A significant decrease of muscle weight was observed in groups 2 and 3, most prominent in the latter. CONCLUSION: Regeneration was not successful across the 40 mm gap using the applied tube in combination with SC. For future experiments further consideration should be taken in optimizing the cellular and material components that are critical for a successful application to overcome very large nerve gaps.

Neuro tissue engineering of glial nerve guides and the impact of different cell types.

The aim of neuro tissue engineering is to imitate biological features in order to enhance regeneration. Following lesions of peripheral nerves, Schwann cells (SCs) reorganize to form longitudinal bands of Büngner (boB) which function as guides for regrowing axons. In order to imitate boB in synthetic implants designed to bridge nerve lesions, we developed resorbable, semipermeable nerve guide conduits with microstructured internal polymer filaments. We utilized a novel microcell chip and identified three extracellular matrix components conducive for coating non-permissive polymer surfaces. In order to maximize SC alignment, seven different microtopographies were investigated via the silicon chip technology. Special longitudinal microgrooves directed SC orientation and growing axons of dorsal root ganglia (DRG), thus achieving stereotropism. When these results were applied to microgrooved polymer filaments inside nerve guide conduits, we observed highly oriented axon growth without meandering. Since scar-forming fibroblasts could potentially interfere with axonal regrowth, triple cultures with fibroblasts, SC and DRG were conducted. Fibroblasts positioned on the outer nanopore containing conduit wall, did not hamper neuronal and glial differentiation inside the tube. In summary, for more rapid regrowth, functional boB can be induced by guided microtissue engineering. By considering both the negative and positive effects of cell interactions, a more rational design of nerve implants becomes feasible.