Thomas Freier

Chitosan tubes of varying degrees of acetylation for bridging peripheral nerve defects

Biosynthetic nerve grafts are desired as alternative to autologous nerve grafts in peripheral nerve reconstruction. Artificial nerve conduits still have their limitations and are not widely accepted in the clinical setting. Here we report an analysis of fine-tuned chitosan tubes used to reconstruct 10 mm nerve defects in the adult rat. The chitosan tubes displayed low, medium and high degrees of acetylation (DAI: ≈ 2%, DA: ≈ 5%, DAIII: ≈ 20%) and therefore different degradability and microenvironments for the regenerating nerve tissue. Short and long term investigations were performed demonstrating that the chitosan tubes allowed functional and morphological nerve regeneration similar to autologous nerve grafts. Irrespective of the DA growth factor regulation demonstrated to be the same as in controls. Analyses of stereological parameters as well as the immunological tissue response at the implantation site and in the regenerated nerves, revealed that DAI and DAIII chitosan tubes displayed some limitations in the support of axonal regeneration and a high speed of degradation accompanied with low mechanical stability, respectively. The chitosan tubes combine several pre-requisites for a clinical acceptance and DAII chitosan tubes have to be judged as the most supportive for peripheral nerve regeneration.

The use of chitosan-based scaffolds to enhance regeneration in the nervous system.

Various biomaterials have been proposed to build up scaffolds for promoting neural repair. Among them, chitosan, a derivative of chitin, has been raising more and more interest among basic and clinical scientists. A number of studies with neuronal and glial cell cultures have shown that this biomaterial has biomimetic properties, which make it a good candidate for developing innovative devices for neural repair. Yet, in vivo experimental studies have shown that chitosan can be successfully used to create scaffolds that promote regeneration both in the central and in the peripheral nervous system. In this review, the relevant literature on the use of chitosan in the nervous tissue, either alone or in combination with other components, is overviewed. Altogether, the promising in vitro and in vivo experimental results make it possible to foresee that time for clinical trials with chitosan-based nerve regeneration-promoting devices is approaching quickly.

Tubulization with chitosan guides for the repair of long gap peripheral nerve injury in the rat.

Biosynthetic guides can be an alternative to nerve grafts for reconstructing severely injured peripheral nerves. The aim of this study was to evaluate the regenerative capability of chitosan tubes to bridge critical nerve gaps (15 mm long) in the rat sciatic nerve compared with silicone (SIL) tubes and nerve autografts (AGs). A total of 28 Wistar Hannover rats were randomly distributed into four groups (n = 7 each), in which the nerve was repaired by SIL tube, chitosan guides of low (∼2%, DAI) and medium (∼5%, DAII) degree of acetylation, and AG. Electrophysiological and algesimetry tests were performed serially along 4 months follow‐up, and histomorphometric analysis was performed at the end of the study. Both groups with chitosan tubes showed similar degree of functional recovery, and similar number of myelinated nerve fibers at mid tube after 4 months of implantation. The results with chitosan tubes were significantly better compared to SIL tubes (P < 0.01), but lower than with AG (P < 0.01). In contrast to AG, in which all the rats had effective regeneration and target reinnervation, chitosan tubes from DAI and DAII achieved 43 and 57% success, respectively, whereas regeneration failed in all the animals repaired with SIL tubes. This study suggests that chitosan guides are promising conduits to construct artificial nerve grafts.

Substratum preferences of motor and sensory neurons in postnatal and adult rats.

After peripheral nerve injuries, damaged axons can regenerate but functional recovery is limited by the specific reinnervation of targets. In this study we evaluated if motor and sensory neurites have a substrate preference for laminin and fibronectin in postnatal and adult stages. In postnatal dorsal root ganglia (DRG) explants, sensory neurons extended longer neurites on collagen matrices enriched with laminin (~50%) or fibronectin (~35%), whereas motoneurons extended longer neurites (~100%) in organotypic spinal cord slices embedded in fibronectin‐enriched matrix. An increased percentage of parvalbumin‐positive neurites (presumptive proprioceptive) vs. neurofilament‐positive neurites was also found in DRG in fibronectin‐enriched matrix. To test if the different preference of neurons for extracellular matrix components was maintained in vivo, these matrices were used to fill a chitosan guide to repair a 6‐mm gap in the sciatic nerve of adult rats. However, the number of regenerating motor and sensory neurons after 1 month was similar between groups. Moreover, none of the retrotraced sensory neurons in DRG was positive for parvalbumin, suggesting that presumptive proprioceptive neurons had poor regenerative capabilities compared with other peripheral neurons. Using real‐time PCR we evaluated the expression of α5β1 (receptor for fibronectin) and α7β1 integrin (receptor for laminin) in spinal cord and DRG 2 days after injury. Postnatal animals showed a higher increase of α5β1 integrin, whereas both integrins were similarly expressed in adult neurons. Therefore, we conclude that motor and sensory axons have a different substrate preference at early postnatal stages but this difference is lost in the adult.

BIOHYBRID – Biohybrid templates for peripheral nerve regeneration

Dear Editor, Peripheral nerve injuries represent a major cause for morbidity and disability in affected patients and cause substantial costs for society in a global perspective. It has been estimated that peripheral nerve injuries affect 2.8% of trauma patients, many of whom acquire life-long disability (Noble et al., 1998). With respect to an incidence of nerve injuries of 13.9/100,000 inhabitants per year (Asplund et al., 2009) and the number of inhabitants in the EU (495,000,000 inhabitants in 2007), the number of peripheral nerve injuries requiring repair and reconstruction, excluding nerve injuries by amputations, may be 70,000 annually only in EU countries. Related to peripheral nerve injuries, the costs for society are substantial and consist of direct (costs for surgery, outpatient visits and rehabilitation) and indirect (lost production) costs. Individual median and ulnar nerve injuries in the forearm have total costs of EUR 51,000 and 31,000, respectively, where around 85% of the costs consist of loss of production (Rosberg et al., 2005), still excluding costs for adjusted quality of life (Eriksson et al., 2011). Thus, one may estimate that the annual costs only in the EU may be as high as EUR 2.2 billion, indicating that improved treatment strategies for peripheral nerve injuries may not only improve the situation for patients, but may also significantly reduce costs for society. Based on these premises, the EU granted EUR 6,000,000 fund as part of the seventh Framework Program to the BIOHYBRID consortium that was built with the overall aim to develop, in a pre-clinical perspective, an innovative biohybrid artificial nerve device for improving the regenerative treatment of severe traumatic injuries of peripheral nerves. BIOHYBRID is a 4-year collaborative project that started on October 1, 2011, and involves 10 partners from 5 European Countries (Germany, Italy, Portugal,

Comparative Evaluation of Chitosan Nerve Guides with Regular or Increased Bendability for Acute and Delayed Peripheral Nerve Repair: A Comprehensive Comparison with Autologous Nerve Grafts and Muscle-in-Vein Grafts: BENDABLE NERVE GUIDANCE CONDUITS

Reconstruction of joint‐crossing digital nerves requires the application of nerve guides with a much higher flexibility than used for peripheral nerve repair along larger bones. Nevertheless, collapse‐resistance should be preserved to avoid secondary damage to the regrowing nerve tissue. In recent years, we presented chitosan nerve guides (CNGs) to be highly supportive for the regeneration of critical gap length peripheral nerve defects in the rat. Now, we evidently increased the bendability of regular CNGs (regCNGs) by developing a wavy wall structure, that is, corrugated CNGs (corrCNGs). In a comprehensive in vivo study, we compared both types of CNGs with clinical gold standard autologous nerve grafts (ANGs) and muscle‐in‐vein grafts (MVGs) that have recently been highlighted in the literature as a suitable alternative to ANGs. We reconstructed rat sciatic nerves over a critical gap length of 15 mm either immediately upon transection or after a delay period of 45 days. Electrodiagnostic measurements were applied to monitor functional motor recovery at 60, 90, 120, and 150 (only delayed repair) days postreconstruction. Upon explanation, tube properties were analyzed. Furthermore, distal nerve ends were evaluated using histomorphometry, while connective tissue specimens were subjected to immunohistological stainings. After 120 days (acute repair) or 150 days (delayed repair), respectively, compression–stability of regCNGs was slightly increased while it remained stable in corrCNGs. In both substudies, regCNGs and corrCNGs supported functional recovery of distal plantar muscles in a similar way and to a greater extent when compared with MVGs, while ANGs demonstrated the best support of regeneration. Anat Rec, 301:1697–1713, 2018.

Long-Term In Vivo Evaluation of Chitosan Nerve Guide Properties with respect to Two Different Sterilization Methods

Severe peripheral nerve injuries are reconstructed either with autologous nerve grafts (gold standard) or alternatively with clinically approved artificial nerve guides. The most common method used to sterilize these medical products is ethylene oxide gassing (EO). However, this method has several disadvantages. An alternative, which has been barely studied so far, represents beta irradiation (β). In previous studies, we developed an artificial nerve guide made of chitosan (chitosan nerve guide, CNG), a biomaterial that is known to potentially retain toxic residues upon EO sterilization. Therefore, we analyzed the long-term regeneration-supporting and mechanical properties of CNGs upon their sterilization with EO or β and their following application in unilateral repair of 12 mm gaps of the rat sciatic nerve. Over a period of 76 weeks, we serially evaluated the recovery of motor functions, the possible emergence of an inflammation in the surrounding connective tissue, the regrowth of axons into the distal nerve, and possible changes in the material properties. Our first long-term evaluation did not reveal significant differences between both sterilization methods. Thus, β is as appropriate as commonly used EO for sterilization of CNGs; however, it may slightly increase the stiffness of the biomaterial over time.