Two-Chambered Chitosan Nerve Guides With Increased Bendability Support Recovery of Skilled Forelimb Reaching Similar to Autologous Nerve Grafts in the Rat 10 mm Median Nerve Injury and Repair Model.

Tension-free surgical reconstruction of transected digital nerves in humans is regularly performed using autologous nerve grafts (ANGs) or bioartificial nerve grafts. Nerve grafts with increased bendability are needed to protect regenerating nerves in highly mobile extremity parts. We have recently demonstrated increased bendability and regeneration supporting properties of chitosan nerve guides with a corrugated outer wall (corrCNGs) in the common rat sciatic nerve model (model of low mobility). Here, we further modified the hollow corrCNGs into two-chambered nerve guides by inserting a perforated longitudinal chitosan-film (corrCNG[F]s) and comprehensively monitored functional recovery in the advanced rat median nerve model. In 16 adult female Lewis rats, we bilaterally reconstructed 10 mm median nerve gaps with either ANGs, standard chitosan nerve guides (CNGs), CNGs (CNG[F]s), or corrCNG[F]s (n = 8, per group). Over 16 weeks, functional recovery of each forelimb was separately surveyed using the grasping test (reflex-based motor task), the staircase test (skilled forelimb reaching task), and non-invasive electrophysiological recordings from the thenar muscles. Finally, regenerated tissue harvested from the distal part of the nerve grafts was paraffin-embedded and cross-sections were analyzed regarding the number of Neurofilament 200-immunopositive axons and the area of newly formed blood vessels. Nerve tissue harvested distal to the grafts was epon-embedded and semi-thin cross-sections underwent morphometrical analyses (e.g., number of myelinated axons, axon and fiber diameters, and myelin thicknesses). Functional recovery was fastest and most complete in the ANG group (100% recovery rate regarding all parameters), but corrCNG[F]s accelerated the recovery of all functions evaluated in comparison to the other nerve guides investigated. Furthermore, corrCNG[F]s supported recovery of reflex-based grasping (87.5%) and skilled forelimb reaching (100%) to eventually significantly higher rates than the other nerve guides (grasping test: CNGs: 75%, CNG[F]s: 62.5%; staircase test: CNGs: 66.7%, CNG[F]s: 83.3%). Histological and nerve morphometrical evaluations, in accordance to the functional results, demonstrated best outcome in the ANG group and highest myelin thicknesses in the corrCNG[F] group compared to the CNG and CNG[F] groups. We thus clearly demonstrate that corrCNG[F]s represent promising innovative nerve grafts for nerve repair in mobile body parts such as digits.

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 (ß). 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 ß 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, ß is as appropriate as commonly used EO for sterilization of CNGs; however, it may slightly increase the stiffness of the biomaterial over time.

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.

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. © 2018 Wiley Periodicals, Inc.

Investigation of cell adhesion in chitosan membranes for peripheral nerve regeneration.

Peripheral nerve injuries have produced major concerns in regenerative medicine for several years, as the recovery of normal nerve function continues to be a significant clinical challenge. Chitosan (CHT), because of its good biocompatibility, biodegradability and physicochemical properties, has been widely used as a biomaterial in tissue engineering scaffolding. In this study, CHT membranes were produced with three different Degrees of Acetylation (DA), envisioning its application in peripheral nerve regeneration. The three CHT membranes (DA I: 1%, DA II: 2%, DA III: 5%) were extensively characterized and were found to have a smooth and flat surface, with DA III membrane having slightly higher roughness and surface energy. All the membranes presented suitable mechanical properties and did not show any signs of calcification after SBF test. Biodegradability was similar for all samples, and adequate to physically support neurite outgrowth. The in vitro cell culture results indicate selective cell adhesion. The CHT membranes favoured Schwann cells invasion and proliferation, with a display of appropriate cytoskeletal morphology. At the same time they presented low fibroblast infiltration. This fact may be greatly beneficial for the prevention of fibrotic tissue formation, a common phenomenon impairing peripheral nerve regeneration. The great deal of results obtained during this work permitted to select the formulation with the greatest potential for further biological tests.

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 α5ß1 (receptor for fibronectin) and α7ß1 integrin (receptor for laminin) in spinal cord and DRG 2 days after injury. Postnatal animals showed a higher increase of α5ß1 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.

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.

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.

Poly(3-hydroxybutyrate) (PHB) patches for covering anterior skull base defects - an animal study with minipigs.

CONCLUSION: We conclude that PHB patch material may fulfil the specific requirements that are necessary for a dural substitute, including defect closure, stability and biocompatibility. Our results support the assumed positive influence of PHB on bone regeneration. OBJECTIVES: Although many experimental and clinical studies have been performed to identify a suitable material to repair defects of the dura mater, no ideal dural substitute is currently available. PHB is a biodegradable and biocompatible polymer that might serve as dural substitute and osteosynthesis material in cranial bone defects. MATERIALS AND METHODS: Different standardized PHB patches were used in six minipigs for covering defined bone defects in the anterior skull base including a dura mater lesion as well as in the frontal sinus front wall. After a defined time of implantation of 3, 6, and 9 months PHB patches were explanted and examined for clinical findings, biodegradation, presence of microorganisms, histological findings, and electron microscopy. RESULTS: The examinations revealed an increasing closure of bone defect corresponding with time. The anterior skull base bone defect was completely closed after 9 months. The histological findings revealed a connective tissue and callus formation around the PHB patches with fibroblasts and foreign body/giant cell reaction growing through PHB membrane pores. There were no reactions or adhesions between brain and PHB or dura mater and PHB, respectively. Investigations of biodegradation and electron microscopy revealed a continuous breakdown of PHB in the course of time with variations due to different PHB structures. Microbiological investigations could not detect any florid intracranial infection.

In vitro and in vivo studies on blends of isotactic and atactic poly (3-hydroxybutyrate) for development of a dura substitute material.

Blends of semicrystalline isotactic poly(3-hydroxybutyrate) (PHB) with amorphous atactic PHB (at-PHB) were prepared by solution-casting using 30%, 50% and 70% at-PHB, and were studied for medical applications. The mechanical properties of the blends including the elastic modulus and elongation at break are strongly affected by the blend composition. The elastic modulus decreases with increasing fraction of at-PHB in the blend from 3350 MPa in the case of PHB to 170 MPa of PHB/at-PHB (30/70). In contrast, the elongation at break increases from 2% in pure PHB up to 50% in the case of the blend with 70% at-PHB. The in vitro degradation is changed as well. The molecular weight of PHB/at-PHB (30/70) is reduced to 5% after 2 years storage in phosphate buffer compared to 35% for pure PHB stored at identical conditions. The in vitro cell vitality is slightly reduced depending on the composition. PHB/at-PHB blends with 30% and 50% at-PHB were selected as dura substitute in minipigs based on the results of the in vitro investigation and the mechanical testing. Patch films with a structured surface on one side were fabricated by a dipping-leaching method. Dura defects were clinically and histologically examined 3, 6, and 9 months after implantation, confirming defect closure, prevention of adhesions to brain tissue, and no signs of inflammation or malignant degeneration. The PHB-based patch materials fulfill the requirements which are necessary for a dura substitute.

Coil-reinforced hydrogel tubes promote nerve regeneration equivalent to that of nerve autografts.

Despite spontaneous sprouting of peripheral axons after transection injury, peripheral regeneration is incomplete and limited to short gaps, even with the use of autograft tissue, which is considered to be the "gold" standard. In an attempt to obviate some of the problems associated with autografts, including limited donor tissue and donor site morbidity, we aimed to synthesize a synthetic nerve guidance channel that would perform as well as the nerve autograft. Given that the patency of the nerve guidance channel is critical for repair, we investigated a series of nerve guidance channel designs where patency and the resulting regenerative capacity were compared in a transected rat sciatic nerve injury model. Three tube designs were compared to autograft tissue: plain, corrugated and coil-reinforced tubes of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate). Of the three designs, the coil-reinforced tubes demonstrated superior performance in terms of patency. By electrophysiology and histomorphometry, the coil-reinforced tubes demonstrated outcomes that were comparable to autografts after both 8 and 16 weeks of implantation. The nerve action potential (NAP) velocity and muscle action potential (MAP) velocity for the coil-reinforced PHEMA-MMA tube was 54.6+/-10.1 and 10.9+/-1.3 m/s, respectively at 16 weeks, which was statistically equivalent to those of the autograft at 37.5+/-7.9 and 11.3+/-2.0 m/s. The axon density in the coil-reinforced tube was 2.16+/-0.61x10(4) axons/mm2, which was statistically similar to that of the autograft of 2.41+/-0.62x10(4) axons/mm2 at 16 weeks. These coil-reinforced tubes demonstrated equivalence to autografts for nerve regeneration, demonstrating the importance of channel design to regenerative capacity and more specifically the impact of patency to regeneration.

Synthesis of degradable poly(L-lactide-co-ethylene glycol) porous tubes by liquid-liquid centrifugal casting for use as nerve guidance channels.

Biodegradable nerve guidance channels are advantageous, obviating the need for their removal after regeneration; however, most channels lack the appropriate mechanical properties for soft tissue implantation and/or degrade too quickly, resulting in reduced regeneration and necessitating the need for the design of polymers with tunable degradation profiles and mechanical properties. We designed a series of biodegradable polymeric hydrogel tubes consisting of L-lactide (LLA) and polyethylene glycol (PEG) where both the ratio of LLA to PEG and PEG molar mass were varied. By adjusting the PEG:LLA ratio and the molecular weight of the PEG oligomer we were able to control degradation and mechanical properties of our polymers. By incorporating methacrylate (MA) groups on both termini of the linear oligomers, porous tubes were synthesized by a redox-initiated free radical mechanism during a liquid-liquid centrifugal casting process. The tube wall had a bead-like morphology, as determined by SEM, which was reminiscent of previous porous hydrogel tubes synthesized by the same method. Tubes swelled with degradation to 160 vol%, or 640 wt%, and an increased radius calculated at 1.26 times. Those tubes with greater PEG content and PEG molar mass degraded faster than those with greater LLA content, as was expected. Interestingly, the wall morphology changed with degradation to a fiber-like structure and the mechanical properties decreased with degradation. By correlating the accelerated degradation study to a physiologic one, these porous hydrogel tubes were stable for an equivalent of 1.5 months, after which the mechanical properties began to deteriorate. This study demonstrates how porous hydrogel tubes can be designed to meet tissue regeneration criteria by tuning the formulation chemistry and specifically how the ratio of hydrophobic/crystalline LLA and hydrophilic/amorphous PEG impact tube properties.

Controlling cell adhesion and degradation of chitosan films by N-acetylation.

As part of our ongoing effort to develop a biodegradable nerve guidance channel based on chitin/chitosan, we conducted a systematic in vitro study on the biodegradation and neural cell compatibility of chitosan and N-acetylated chitosan. The in vitro degradation (pH 7.4, 37 degrees C) in the presence of 1.5 microg/ml lysozyme showed a progressive mass loss to greater than 50% within 4 weeks for films with 30-70% acetylation. In contrast, the degradation of samples with very low or high acetylation was minimal over the 4-week period. Neural cell compatibility of chitosan and N-acetylated chitosan was tested using primary chick dorsal root ganglion (DRG) neurons. All chitosan-based films showed DRG cell adhesion after 2 days of culture. However, cell viability decreased with increasing acetylation. Chitosan that was 0.5% acetylated had the greatest cell viability, which was approximately 8 times higher than that of chitosan that was 11% acetylated. Chitosan with 0.5% and 11% acetylation showed more and longer neurites than the other samples studied. Thus chitosan amine content can be tuned for optimal biodegradation and cell compatibility, which are important for tissue engineering in the nervous system.

Chitin-based tubes for tissue engineering in the nervous system.

The purpose of this study was to investigate chitin and chitosan as potential materials for biodegradable nerve guides. Transparent chitin hydrogel tubes were synthesized, for the first time, from chitosan solutions using acylation chemistry and mold casting techniques. Alkaline hydrolysis of chitin tubes resulted in chitosan tubes, with the extent of hydrolysis controlling the resulting amine content. This, in turn, impacted compressive strength and cell adhesion. Chitosan tubes were mechanically stronger than their chitin origins, as measured by the transverse compressive test, where tubes having degrees of acetylation of 1%, 3%, 18% (i.e. chitosan) and 94% (i.e. chitin) supported loads at a 30% displacement of 40.6 +/- 4.3, 25.3 +/- 4.5, 10.6 +/- 0.8, and 8.7 +/- 0.4 g, respectively. However, the chitin processing methodology could be optimized for compressive strength, by either incorporating reinforcing coils in the tube wall, or air-drying the hydrogel tubes. Chitin and chitosan supported adhesion and differentiation of primary chick dorsal root ganglion neurons in vitro. Chitosan films showed significantly enhanced neurite outgrowth relative to chitin films, reflecting the dependence of nerve cell affinity on the amine content in the polysaccharide: neurites extended 1794.7 +/- 392.0 microm/mm(2) on chitosan films vs. 140.5 +/- 41.6 microm/mm(2) on chitin films after 2 days of culture. This implies that cell adhesion and neurite extension can be adjusted by amine content, which is important for tissue engineering in the nervous system. The methods for easy processing and modification of chitin and chitosan described herein, allow the mechanical properties and cyto-compatibility to be controlled and provide a means for a broader investigation into their use in biomedical applications.

Surface modification of tricalcium phosphate for improvement of the interfacial compatibility with biodegradable polymers.

The surface of tricalcium phosphate (TCP) filler particles was activated by treatment with dilute aqueous phosphoric acid. ATR-IR spectra indicated the formation of calcium hydrogen phosphate dihydrate at the surface. Oligo(lactone)s were formed by the subsequent reaction of the activated TCP with L-lactide and epsilon -caprolactone, respectively, at 150 degrees C without any additional catalysts. After extraction of the oligo(lactide), the residue of modified TCP-included calcium lactate whereas the water of crystallization of the dihydrate disappeared as shown by ATR-IR spectroscopy. Owing to the insolubility of TCP in common solvents, the analogous reaction between water-soluble disodium hydrogen phosphate dihydrate and L-lactide was used to study the kind of chemical bonds by high-resolution NMR spectroscopy. The 1H and 13C NMR spectra of the reaction product also pointed out the presence of calcium lactate. Additionally, signals were found indicating a covalent attachment of lactic acid units onto the phosphorus. For the preparation of composites, poly(L,DL-lactide) was mixed with TCP and modified TCP, respectively, in a ratio of 75/25 (w/w) and directly injection moulded into tensile test specimens at a barrel temperature of 180 degrees C. Although mechanical properties were not improved, scanning electron microscopy (SEM) indicated a better interfacial phase interaction in the composite with the modified TCP. Chemical bonds between filler and polymer matrix are assumed to be formed by transesterification reactions.

In vitro and in vivo degradation studies for development of a biodegradable patch based on poly(3-hydroxybutyrate).

For the development of a resorbable gastrointestinal patch, the in vitro degradation of solution-cast films of poly(3-hydroxybutyrate) (PHB), modifications of PHB expected to influence its degradation time, as well a poly(L-lactide) (PLLA) was examined. The molecular weight of pure PHB decreased by one-half after 1 year in buffer solution (pH 7.4, 37 degrees C). Acceleration in molecular weight decrease was observed by blending with atactic PHB, whereas no influence was found with low-molecular weight PHB. Leaching of a water-soluble additive led to a slight acceleration of PHB degradability. In contrast, a deceleration in degradation rate was observed with the addition of a hydrophobic plasticizer. In vitro tests indicated an accelerating effect of pancreatin on PHB degradation, whereas PLLA degradation remained essentially uninfluenced. In comparison to simple hydrolysis, the degradation rate of PHB was accelerated about threefold. From the in vitro results, a PHB/atactic PHB blend was selected for repair of a bowel defect in Wistar rats. A patch film was fabricated by a dipping/leaching method. Twenty-six weeks post-implantation, material remnants were found in only one of four animals. The bowel defects were closed in all cases. It could be assessed that the patch material resists the intestinal secretions for a sufficiently long time but that it finally degrades completely.

Anti-inflammatory prodrugs as plasticizers for biodegradable implant materials based on poly(3-hydroxybutyrate).

Salicylic and acetylsalicylic acid esters were tested as plasticizers for biodegradable poly(3-hydroxybutyrate) (PHB). The aim is the combination of plasticizing and anti-inflammatory properties in the fabrication of implant materials. Solution-cast films made of mixtures of PHB and 30% ester showed plasticization accompanied by a decrease of elastic modulus and an increase in elongation at break in comparison with pure PHB films. However, the number of usable plasticizers from the group of the salicylic acid and acetylsalicylic acid esters is limited. Short-chain derivatives are volatile while long-chain compounds tend to crystallize. In both cases PHB films embrittle within short time. Moreover, some derivatives show a fast release in an aqueous environment. As alternative nonsteroidal anti-inflammatory prodrugs arylpropionic acid esters were tested as plasticizers. The addition of ketoprofen ethyl ester led to PHB films with decreased brittleness. In summary, various esters of anti-inflammatory drugs show plasticizing effects on solution-cast PHB films comparable with those of commonly used citric acid esters.