Native Nerve Research Articles

Development of a magnetically aligned regenerative tissue-engineered electronic nerve interface for peripheral nerve applications

Peripheral nerve injuries can be debilitating to motor and sensory function, with severe cases often resulting in complete limb amputation. Over the past two decades, prosthetic limb technology has rapidly advanced to provide users with crude motor control of up to 20° of freedom; however, the nerve-interfacing technology required to provide high movement selectivity has not progressed at the same rate. The work presented here focuses on the development of a magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI) that combines polyimide “threads” encapsulated within a magnetically aligned hydrogel scaffold. The technology exploits tissue-engineered strategies to address concerns over traditional peripheral nerve interfaces including poor axonal sampling through the nerve and rigid substrates. A magnetically templated hydrogel is used to physically support the polyimide threads while also promoting regeneration in close proximity to the electrode sites on the polyimide. This work demonstrates the utility of magnetic templating for use in tuning the mechanical properties of hydrogel scaffolds to match the stiffness of native nerve tissue while providing an aligned substrate for Schwann cell migration in vitro. MARTEENI devices were fabricated and implanted within a 5-mm-long rat sciatic-nerve transection model to assess regeneration at 6 and 12 weeks. MARTEENI devices do not disrupt tissue remodeling and show axon densities equivalent to fresh tissue controls around the polyimide substrates. Devices are observed to have attenuated foreign-body responses around the polyimide threads. It is expected that future studies with functional MARTEENI devices will be able to record and stimulate single axons with high selectivity and low stimulation regimes.

Decellularized peripheral nerve as an injectable delivery vehicle for neural applications.

Damage to the nervous system can result in loss of sensory and motor function, paralysis, or even death. To facilitate neural regeneration and functional recovery, researchers have employed biomaterials strategies to address both peripheral and central nervous system injuries. Injectable hydrogels that recapitulate native nerve extracellular matrix are especially promising for neural tissue engineering because they offer more flexibility for minimally invasive applications and provide a growth-permissive substrate for neural cell types. Here, we explore the development of injectable hydrogels derived from decellularized rat peripheral nerves (referred to as "injectable peripheral nerve [iPN] hydrogels"), which are processed using a newly developed sodium deoxycholate and DNase (SDD) decellularization method. We assess the gelation kinetics, mechanical properties, cell bioactivity, and drug release kinetics of the iPN hydrogels. The iPN hydrogels thermally gel when exposed to 37°C in under 20 min and have mechanical properties similar to neural tissue. The hydrogels demonstrate in vitro biocompatibility through support of Schwann cell viability and metabolic activity. Additionally, iPN hydrogels promote greater astrocyte spreading compared to collagen I hydrogels. Finally, the iPN is a promising delivery vehicle of drug-loaded microparticles for a combinatorial approach to neural injury therapies.

"Off-the-Shelf" Nerve Matrix Preservation.

Background: Decellularized human nerves overcome the limitations of the current treatments for large peripheral nerve injuries. However, the use of decellularized nerves requires an "off-the-shelf" availability for useful and actual clinical application. In this study, we addressed the preservation of the native and decellularized human nerve matrix in an integrative approach for tissue scaffold production. Materials and Methods: For native nerve matrix preservation analysis, we used histological examination and immunofluorescence to examine the structure, biomechanical assays to evaluate the tensile strength and Young's modulus, and analyzed the extracellular matrix (ECM) composition using enzyme-linked immunosorbent assay (ELISA) and biochemical assays for laminin, collagen and sulfated glycosaminoglycans (sGAG). After decellularization, nuclear remnants and DNA content were evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining and the picogreen quantification assay, as well as immunofluorescence or ELISA for cell rests (S100 protein and myelin staining) evaluation. Decellularized cryopreserved scaffolds were assayed for biomechanics, ECM composition, and structural maintenance. Cytotoxicity assays were performed to evaluate the biocompatibility of the nerve matrix extracts after cryopreservation. Results: We compared different strategies for native nerve storage and found that preservation up to 7 days at 4°C in Roswell Park Memorial Institute medium maintained biomechanical properties, such as Young's modulus and tensile strength, along with the structure and ECM composition, regarding laminin, collagen, and sGAG. After a successful decellularization process, that eliminated cell remnants, nerve scaffolds were frozen in an "in house" formulated cryoprotectant, using an automatic controlled rate freezer. Nerve structure, ECM composition, and biomechanical properties were maintained before and after the freezing process in comparison with native nerves. The extracts of the nerve scaffolds after thawing were not cytotoxic and the freezing process sustained good viability in 3T3 cells (graphical abstract). Conclusion: Since our approach facilitates transport, storage, and provide a ready-to-use alternative, it could be used in a clinical application for the treatment of long-gap peripheral nerve injuries in regenerative medicine.

Morphine-sensitive synaptic transmission emerges in embryonic rat microphysiological model of lower afferent nociceptive signaling.

Debilitating chronic pain resulting from genetic predisposition, injury, or acquired neuropathy is becoming increasingly pervasive. Opioid analgesics remain the gold standard for intractable pain, but overprescription of increasingly powerful and addictive opioids has contributed to the current prescription drug abuse epidemic. There is a pressing need to screen experimental compounds more efficiently for analgesic potential that remains unmet by conventional research models. The spinal cord dorsal horn is a common target for analgesic intervention, where peripheral nociceptive signals are relayed to the central nervous system through synaptic transmission. Here, we demonstrate that coculturing peripheral and dorsal spinal cord nerve cells in a novel bioengineered microphysiological system facilitates self-directed emergence of native nerve tissue macrostructure and concerted synaptic function. The mechanistically distinct analgesics-morphine, lidocaine, and clonidine-differentially and predictably modulate this microphysiological synaptic transmission. Screening drug candidates for similar microphysiological profiles will efficiently identify therapeutics with analgesic potential.

Microfluidic hollow fiber with improved stiffness repairs peripheral nerve injury through non-invasive electromagnetic induction and controlled release of NGF

Peripheral nerve injury can lead to paralysis, chronic pain, muscle atrophy and disability due to interrupted bioelectric signal communications between spinal cord and the innervated bodies. Bioelectric materials, particularly conductive hydrogels, have shown the increasing potentials in neural engineering because they serve as tissue scaffolds that can support cell growth and differentiation, and restore functional electric conduction. In this work, we fabricated size-tunable microfluidic hollow fibers (HF) with improved stiffness and elasticity based on a triple network called SCPAPPy, which was composed of sodium alginate/Ca2+ (SC), polyacrylamide (PA), and polypyrrole (PPy). This HF showed a higher conductivity of 0.32 S/m than that of native sciatic nerve. Based on finite element analysis and digital oscilloscope measurement, the conductive HF was demonstrated to generate electromotive forces through electromagnetic induction by using a figure-eight coil. This creative approach could non-invasively facilitate myelination by improving Schwann cell’s growth and migration. Meanwhile, the electromotive forces drove the secretion of endogenous nerve growth factor (NGF) from Schwann cells. Moreover, exogenous nerve growth factor-7S (NGF-7S) was loaded in the HF, which was released gradually and promoted the differentiation of neurons in a controlled manner. By applying the pulsed magnetic field (MF) and NGF-7S together, it was found that the rats with 5-mm sciatic nerve defects achieved a more rapid nerve regeneration and functional recovery. In a word, our study demonstrated the practical role of electromagnetic induction on nerve repair, and opened the window of synergistic therapy combining the bioelectricity and neurotrophic factors towards peripheral nerve injury.

Acellular Nerve Allografts in Major Peripheral Nerve Repairs: An Analysis of Cases Presenting With Limited Recovery.

Acellular nerve allografts have been used successfully and with increasing frequency to reconstruct nerve injuries. As their use has been expanded to treat longer gap, larger diameter nerve injuries, some failed cases have been reported. We present the histomorphometry of 5 such cases illustrating these limitations and review the current literature of acellular nerve allografts. Between 2014 and 2019, 5 patients with iatrogenic nerve injuries to the median or ulnar nerve reconstructed with an AxoGen AVANCE nerve allograft at an outside hospital were treated in our center with allograft excision and alternative reconstruction. These patients had no clinical or electrophysiological evidence of recovery, and allograft specimens at the time of surgery were sent for histomorphological examination. Three patients with a median and 2 with ulnar nerve injury were included. Histology demonstrated myelinated axons present in all proximal native nerve specimens. In 2 cases, axons failed to regenerate into the allograft and in 3 cases, axonal regeneration diminished or terminated within the allograft. The reported cases demonstrate the importance of evaluating the length and the function of nerves undergoing acellular nerve allograft repair. In long length, large-diameter nerves, the use of acellular nerve allografts should be carefully considered.

Three-Dimensional Engineered Peripheral Nerve: Toward a New Era of Patient-Specific Nerve Repair Solutions.

Reconstruction of peripheral nerve injuries (PNIs) with substance loss remains challenging because of limited treatment solutions and unsatisfactory patient outcomes. Currently, nerve autografting is the first-line management choice for bridging critical-sized nerve defects. The procedure, however, is often complicated by donor site morbidity and paucity of nerve tissue, raising a quest for better alternatives. The application of other treatment surrogates, such as nerve guides, remains questionable, and it is inefficient in irreducible nerve gaps. More importantly, these strategies lack customization for personalized patient therapy, which is a significant drawback of these nerve repair options. This negatively impacts the fascicle-to-fascicle regeneration process, critical to restoring the physiological axonal pathway of the disrupted nerve. Recently, the use of additive manufacturing (AM) technologies has offered major advancements to the bioengineering solutions for PNI therapy. These techniques aim at reinstating the native nerve fascicle pathway using biomimetic approaches, thereby augmenting end-organ innervation. AM-based approaches, such as three-dimensional (3D) bioprinting, are capable of biofabricating 3D-engineered nerve graft scaffolds in a patient-specific manner with high precision. Moreover, realistic in vitro models of peripheral nerve tissues that represent the physiologically and functionally relevant environment of human organs could also be developed. However, the technology is still nascent and faces major translational hurdles. In this review, we spotlighted the clinical burden of PNIs and most up-to-date treatment to address nerve gaps. Next, a summarized illustration of the nerve ultrastructure that guides research solutions is discussed. This is followed by a contrast of the existing bioengineering strategies used to repair peripheral nerve discontinuities. In addition, we elaborated on the most recent advances in 3D printing and biofabrication applications in peripheral nerve modeling and engineering. Finally, the major challenges that limit the evolution of the field along with their possible solutions are also critically analyzed. Impact statement Complex nerve injuries, including critical-sized gaps (>3 cm loss of substance), gaps involving nerve bifurcations, and those associated with ischemic environments, are difficult to manage. A biomimetic, personalized peripheral nerve tissue surrogate to address these injuries is lacking. The peripheral nerve repair market currently represents a multi-billion-dollar industry that is projected to expand. Given the clinical and economical dilemmas posed by this medical condition, it is crucial to devise novel and effective nerve substitutes. In this review article, we discuss progress in three-dimensional printing technologies, including biofabrication and nerve computer-aided design modeling, toward achieving a patient-specific and biomimetic nerve repair solution.

ANATOMY OF RENAL DENERVATION DURABILITY

Objective: Safety and efficacy of catheter-based renal denervation (RDN) have been demonstrated in previous randomized, sham-controlled trials. Ongoing registries also suggest long-term durability of the RDN procedure. However, the exact mechanisms for RDN durability have not been previously elucidated. This report aimed to characterize the pathophysiological processes post radiofrequency (RF) RDN that translate into procedural durability utilizing animal models. Design and method: Animal studies were conducted in accordance with published guidelines. RDN was performed using the Symplicity RDN system (Medtronic, Santa Rosa, CA, USA) and the multielectrode RF Symplicity Spyral catheter in 164 healthy swine. Serial histological tissue samples were obtained 7, 28, 60, and 180 days post-procedure and followed by immunohistochemical and histopathologic analysis of RF-treated regions, sympathetic nerves and renal cortical axons. Results: At 7 days post-RDN, inflammation and swelling predominated the nerve lesions at the treatment site. A progressive nerve deterioration accompanied by Schwann cell reaction occurred over time leading to terminal atrophy and fibrosis at 180 days post-RDN. Nerve atrophy also became present distal to the treatment site. Although new Schwann cells appeared post-RDN, they failed to form the characteristic nerve fascicles and bundles which would reconstitute normal nerve morphology and support functional axons. Quantitative comparison of nerve necrosis and nerve atrophy indicated that proximal nerve necrosis peaks in the treated segments of the vessel, whereas nerve atrophy and low-grade nerve inflammation originates at the region of peak necrosis and extends distally beyond the RDN lesion. Thus, despite anatomic Schwann cell regeneration, the functional axonal nerve component did not recover. Conclusions: Histological analyses of porcine tissues demonstrated that terminal atrophy and fibrosis of the renal nerves developed within 180 days post-RF RDN procedure. Although new Schwan cell formation occurred, it did not reconstitute native nerve morphology suggesting functional nerve recovery is unlikely.

Bioresorbable and Mechanically Optimized Nerve Guidance Conduit Based on a Naturally Derived Medium Chain Length Polyhydroxyalkanoate and Poly(ε-Caprolactone) Blend.

Severe peripheral nerve injuries represent a large clinical problem with relevant challenges such as the development of successful synthetic scaffolds as substitutes to autologous nerve grafting. Numerous studies have reported the use of polyesters and type I collagen-based nerve guidance conduits (NGCs) to promote nerve regeneration through critical nerve defects while providing protection from external factors. However, none of the commercially available hollow bioresorbable NGCs have demonstrated superior clinical outcomes to an autologous nerve graft. Hence, new materials and NGC geometries have been explored in the literature to mimic the native nerve properties and architecture. Here, we report a novel blend of a natural medium chain length polyhydroxyalkanoate (MCL-PHA) with a synthetic aliphatic polyester, poly(ε-caprolactone) (PCL), suitable for extrusion-based high-throughput manufacturing. The blend was designed to combine the excellent ability of PHAs to support the growth and proliferation of mammalian cells with the good processability of PCL. The material exhibited excellent neuroregenerative properties and a good bioresorption rate, while the extruded porous tubes exhibited similar mechanical properties to the rat sciatic nerve. The NGCs were implanted to treat a 10 mm long sciatic nerve defect in rats, where significant differences were found between thin and thick wall thickness implants, and both electrophysiological and histological data, as well as the number of recovered animals, provided superior outcomes than the well-referenced synthetic Neurolac NGC.

Histological, Biomechanical, and Biological Properties of Genipin-Crosslinked Decellularized Peripheral Nerves.

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

Biomimetic electrospun tubular PLLA/gelatin nanofiber scaffold promoting regeneration of sciatic nerve transection in SD rat

The micro- or nanoscale surface morphology of the tissue engineering nerve guidance scaffold (NGS) will affect different cell behaviors, such as their growth rate, migration, and matrix secretion. Although different technologies for manufacturing scaffolds with biomimetic topography have been established, most of them tend to be high cost and long preparation time. Here we have prepared a biomimetic NGS with physical properties to simulate native nerve tissue more accurately. We used poly(l-lactic acid) (PLLA) nanofibers doped with gelatin to prepare a biomimetic NGS whose structure mimics the native epineurium layer. By adjusting the doping ratio of gelatin and PLLA in the tubular scaffold, the bionic scaffold's surface morphology and mechanical properties are closer to native tissues. In vitro cell scaffold interaction experiments demonstrated that the PLLA/gelatin nanofibers could significantly promote the elongation, proliferation, and the secretion of glial cell-derived neurotrophic factor (GDNF) of RSC96 Schwann cells (SCs), as well as the diffusion of GDNF. In vivo scaffold replacement of SD rat, sciatic nerves showed that the nerve guide scaffold composed of PLLA/gelatin nanofibers was helpful to the myelination of SCs and the remolding of epineurium in the injured area, which could effectively rehabilitate the motor and sensory functions of the injured nerve and prevent the atrophy of the target muscle tissue. This study showed that the synergistic impact of nano topographical and biochemical clues on designing biomimetic scaffolds could efficiently promote regenerating nerve tissue.

Histomorphometry in Peripheral Nerve Regeneration: Experimental Comparison of Different Axon Counting Methods

PURPOSE: Histomorphometry is a common tool to quantitatively evaluate outcomes of peripheral nerve regeneration by measuring axonal parameters such as count, diameter, and myelination. Manual measurement across the entire nerve cross-section is the most accurate technique of histomorphometric analysis but is extremely labor intensive. Thus, most researchers have opted for analysis of just a sample of the cross-section. Currently, automated sampled analysis is the most common method. However, no study has been performed to compare the accuracy of these techniques, making it difficult to compare results across the literature. METHODS: Rat sciatic nerves were transected and repaired with a 20 mm nerve isograft. A total of 34 sections were stained with toluidine blue and digitized: 24 from native nerve distal to the graft, 5 from nerve proximal to the graft, and 5 from the graft itself. Three blinded researchers manually counted total myelinated fibers in each full cross-section. Total myelinated fiber counts were also extrapolated from sampled fields representing 20% of the cross-sectional area. Sampled counts were performed both manually and automatically with the software ImageJ (National Institutes of Health). Myelinated fiber diameter (FD), axon diameter (AD), and myelin sheath thickness (MTh) were measured manually in the full cross-sections and the sampled fields. Repeated measures MANOVA, Spearman’s correlation, and Wilcoxon signed-rank tests were performed. RESULTS: Results are expressed in mean ± standard deviation. Full manual axon count was 12,504 ± 4,195 overall and 13,506 ± 4,217 distally. Sampled axon counts were significantly higher than full manual, especially distally (sampled manual: 15,316 ± 4,613, P < 0.001; sampled automated: 16,297 ± 7,733, P = 0.037). All three methods showed strong, significant correlation with each other, especially for distal sections (full manual and sampled manual: rs = 0.912, P < 0.001; full manual and sampled automated: rs = 0.599, P = 0.002; sampled manual and sampled automated: rs = 0.708, P < 0.001). Overall full manual fiber diameter, axon diameter, and myelin sheath thickness were 5.42 ± 1.18 µm, 3.61 ± 0.78 µm, and 0.93 ± 0.28 µm, respectively. They did not differ from sampled measurements (P = 0.144, P = 0.059, and P = 0.817, respectively). CONCLUSIONS: Manual count of sampled nerve sections produces highly correlated, reliable results when using standardized and systematic sampling methods. Automated data should be regarded with more caution as correlation was not as high. However, the significant correlation indicates it is an acceptable technique if manual sampled analysis is not possible. Although the methods explored in this study correlated highly with one another, the accuracy of both sampled techniques differed significantly from full manual analysis. Therefore, comparison of numerical results between papers reporting these parameters (ie, for a metanalysis) may not be possible. Researchers must be cognizant of the wide variety of techniques reported in the literature and exercise caution when comparing data between studies.

Biomimetic and hierarchical nerve conduits from multifunctional nanofibers for guided peripheral nerve regeneration

Development of a functional nerve conduit to replace autografts remains a significant challenge particularly considering the compositional complexity and structural hierarchy of native peripheral nerves. In the present study, a multiscale strategy was adopted to fabricate 3D biomimetic nerve conduit from Antheraea pernyi silk fibroin (ApF)/(Poly(L-lactic acid-co-caprolactone)) (PLCL)/graphene oxide (GO) (ApF/PLCL/GO) nanofibers via nanofiber dispersion, template-molding, freeze-drying and crosslinking. The resultant conduits exhibit parallel multichannels (ϕ = 125 µm) surrounded by biomimetic fibrous fragments with tailored degradation rate and improved mechanical properties in comparison with the scaffold without GO. In vitro studies showed that such 3D biomimetic nerve scaffolds had the ability to offer an effective guiding interface for neuronal cell growth. Furthermore, these conduits showed a similarity to autografts in vivo repairing sciatic nerve defects based on a series of analysis (walking track, triceps weight, morphogenesis, vascularization, axonal regrowth and myelination). The conduits almost completely degraded within 12 weeks. These findings demonstrate that the 3D hierarchical nerve guidance conduit (NGC) with fascicle-like structure have great potential for peripheral nerve repair.

Micro-grooved nerve guidance conduits combined with microfiber for rat sciatic nerve regeneration

In order to accelerate the regeneration of injured nerves, highly aligned structures that mimic native tissues are essential for nerve guidance conduits (NGC). In this study, a biodegradable porous micro-patterned NGCs were manufactured by use of capillary force lithography and salt leaching method, which filled as wrapped with aligned electrospun microfiber (PA-NGC) using. These combined NGC showed 5μm aligned groove pattern on the interior wall and were filled with 4–5μm align fibers for a fascicular structure to mimic the native nerve. The highly aligned structure has increased PC12 migration and morphological elongation compared with random microfiber. For the in vivo study, we bridged in the 10mm sciatic nerve defect margins with NGC. The PA-NGC were compared to flat NGCs filled with random (FR-NGC) or aligned fiber (FA-NGC) and patterned NGCs filled with random fiber (PR-NGC). The PA-NGC showed as dramatically re-bridged neurofilament and higher sciatic function index, onset-to-peak of amplitude, muscle weight and fiber diameter compared to other groups after 8 weeks of implantation. In our results, the PA-NGC not only can serve as a design for functional NGC without growth factor but also can be used in clinical application for peripheral nerve regeneration.

Effects of nitrous oxide on glycinergic transmission in rat spinal neurons

We investigated the effects of nitrous oxide (N2O) on glycinergic inhibitory whole-cell and synaptic responses using a “synapse bouton preparation,” dissociated mechanically from rat spinal sacral dorsal commissural nucleus (SDCN) neurons. This technique can evaluate pure single- or multi-synaptic responses from native functional nerve endings and enable us to accurately quantify how N2O influences pre- and postsynaptic transmission. We found that 70 % N2O enhanced exogenous glycine-induced whole-cell currents (IGly) at glycine concentrations lower than 3 × 10–5 M, but did not affect IGly at glycine concentrations higher than 10–4 M. N2O did not affect the amplitude and 1/e decay-time of both spontaneous and miniature glycinergic inhibitory postsynaptic currents recorded in the absence and presence of tetrodotoxin (sIPSCs and mIPSCs, respectively). The decrease in frequency induced by N2O was observed in sIPSCs but not in mIPSCs, which was recorded in the presence of both tetrodotoxin and Cd2+, which block voltage-gated Na+ and Ca2+ channels, respectively. N2O also decreased the amplitude and increased the failure rate and paired-pulse ratio of action potential-evoked glycinergic inhibitory postsynaptic currents. N2O slightly decreased the Ba2+ currents mediated by voltage-gated Ca2+ channels in SDCN neurons. We found that N2O suppresses glycinergic responses at synaptic levels with presynaptic effect having much more predominant role. The difference between glycinergic whole-cell and synaptic responses suggests that extrasynaptic responses seriously modulate whole-cell currents. Our results strongly suggest that these responses may thus in part explain analgesic effects of N2O via marked glutamatergic inhibition by glycinergic responses in the spinal cord.

Nanocomposite-Coated Silk-Based Artificial Conduits: The Influence of Structures on Regeneration of the Peripheral Nerve.

In this study, silk filaments are coated with different concentrations (5, 7.5, and 10% w/w) of carbon nanofibers (CNFs) dispersed in poly-ε-caprolactone. The nanocomposite-coated silk filaments are subjected to knitting, braiding, and twisting. The tubular structures are covered with a silk fibroin/polyvinyl film for the nerve conduit application. Physical characterization of the developed nerve conduits demonstrates suitable mechanical properties comparable to native nerve tissue. Cell proliferation is confirmed through in vitro cell culture studies using Neuro 2a and rat primary cortical neural progenitor cells, which show that the proliferation happens along the interconnected macrochannels of the internal structure of the nerve conduit. The knitted structure presents better biological properties than the nerve conduits with other internal structures. The in vivo sciatic nerve implantation is performed in a rabbit model using the best conduit, i.e., 10% CNF-based nanocomposite-coated silk with a knitted inner structure without any biomolecules or tube filling gels. Regeneration of a 2 cm gap excised sciatic nerve is investigated by immunohistochemistry and histology of implanted nerve conduits removed after 30 days. Results suggest that the CNF-based conducting nanocomposite coating in this well-defined architecture of the conduit helps in signal transmission and neural growth during the regeneration of the transected nerve.

UV-Casting on Methacrylated PCL for the Production of a Peripheral Nerve Implant Containing an Array of Porous Aligned Microchannels.

Peripheral nerves are basic communication structures guiding motor and sensory information from the central nervous system to receptor units. Severed peripheral nerve injuries represent a large clinical problem with relevant challenges to successful synthetic nerve repair scaffolds as substitutes to autologous nerve grafting. Numerous studies reported the use of hollow tubes made of synthetic polymers sutured between severed nerve stumps to promote nerve regeneration while providing protection for external factors, such as scar tissue formation and inflammation. Few approaches have described the potential use of a lumen structure comprised of microchannels or microfibers to provide axon growth avoiding misdirection and fostering proper healing. Here, we report the use of a 3D porous microchannel-based structure made of a photocurable methacrylated polycaprolactone, whose mechanical properties are comparable to native nerves. The neuro-regenerative properties of the polymer were assessed in vitro, prior to the implantation of the 3D porous structure, in a 6-mm rat sciatic nerve gap injury. The manufactured implants were biocompatible and able to be resorbed by the host’s body at a suitable rate, allowing the complete healing of the nerve. The innovative design of the highly porous structure with the axon guiding microchannels, along with the observation of myelinated axons and Schwann cells in the in vivo tests, led to a significant progress towards the standardized use of synthetic 3D multichannel-based structures in peripheral nerve surgery.

3D bioprinted multiscale composite scaffolds based on gelatin methacryloyl (GelMA)/chitosan microspheres as a modular bioink for enhancing 3D neurite outgrowth and elongation

The integration of multiscale micro- and macroenvironment has been demonstrated as a critical role in designing biomimetic scaffolds for peripheral nerve tissue regeneration. While it remains a remarkable challenge for developing a biomimetic multiscale scaffold for enhancing 3D neuronal maturation and outgrowth. Herein, we present a 3D bioprinted multiscale scaffold based on a modular bioink for integrating the 3D micro- and macroenvironment of native nerve tissue. Gelatin methacryloyl (GelMA)/Chitosan Microspheres (GC-MSs) were prepared by a microfluidic approach, and the effect of these microspheres on enhancing neurite outgrowth and elongation of PC12 cells was demonstrated. The 3D multiscale composite scaffolds were bioprinted based on microspheres and hydrogel as the modular bioink. The co-culture of PC12 cells and RSC96 Schwann cells within these 3D biomimetic scaffolds were investigated to evaluate such a 3D multiscale environment for neurite outgrowth and Schwann cell proliferation. These results indicate that such multiscale composite scaffold with hydrogel microspheres provided a suitable 3D microenvironment for enhancing neurite growth, and the 3D printed hydrogel network provided a 3D macroenvironment mimicking the epineurium layer for Schwann cells proliferation and nerve cell organization, which is promising for the great potential applications in nerve tissue engineering.

Repairing Volumetric Muscle Loss in the Ovine Peroneus Tertius Following a 3-Month Recovery.

Much effort has been made to fabricate engineered tissues on a scale that is clinically relevant to humans; however, scale-up remains one of the most significant technological challenges of tissue engineering to date. To address this limitation, our laboratory has developed tissue-engineered skeletal muscle units (SMUs) and engineered neural conduits (ENCs), and modularly scaled them to clinically relevant sizes for the treatment of volumetric muscle loss (VML). The goal of this study was to evaluate the SMUs and ENCs in vitro, and to test the efficacy of our SMUs and ENCs in restoring muscle function in a clinically relevant large animal (sheep) model. The animals received a 30% VML injury to the peroneus tertius muscle and were allowed to recover for 3 months. The animals were divided into three experimental groups: VML injury without a repair (VML only), repair with an SMU (VML+SMU), or repair with an SMU and ENC (VML+SMU+ENC). We evaluated the SMUs before implantation and found that our single scaled-up SMUs were characterized by the presence of contracting myotubes, linearly aligned extracellular matrix proteins, and Pax7+ satellite cells. Three months after implantation, we found that the repair groups (VML+SMU and VML+SMU+ENC) had restored muscle mass and tetanic force production to a level that was statistically indistinguishable from the uninjured contralateral muscle after 3 months in vivo. Furthermore, we demonstrated the ability of our ENCs to effectively bridge the gap between native nerve and the repair site by eliciting a muscle contraction through direct electrical stimulation of the re-routed nerve. Impact statement The fabrication of tissues of clinically relevant sizes is one of the largest obstacles preventing engineered tissues from achieving widespread use in the clinic. This study aimed to combat this limitation by developing a fabrication method to scale-up tissue-engineered skeletal muscle for the treatment of volumetric muscle loss in a large animal (sheep) model and evaluating the efficacy of the tissue-engineered constructs after a 3-month recovery.

Magnetic particle templating of hydrogels: engineering naturally derived hydrogel scaffolds with 3D aligned microarchitecture for nerve repair

Objective. Hydrogel scaffolds hold promise for a myriad of tissue engineering applications, but often lack tissue-mimetic architecture. Therefore, in this work, we sought to develop a new technology for the incorporation of aligned tubular architecture within hydrogel scaffolds engineered from the bottom-up. Approach. We report a platform fabrication technology—magnetic templating—distinct from other approaches in that it uses dissolvable magnetic alginate microparticles (MAMs) to form aligned columnar structures under an applied magnetic field. Removal of the MAMs yields scaffolds with aligned tubular microarchitecture that can promote cell remodeling for a variety of applications. This approach affords control of microstructure diameter and biological modification for advanced applications. Here, we sought to replicate the microarchitecture of the native nerve basal lamina using magnetic templating of hydrogels composed of glycidyl methacrylate hyaluronic acid and collagen I. Main results. Magnetically templated hydrogels were characterized for particle alignment and micro-porosity. Overall MAM removal efficacy was verified by 96.8% removal of iron oxide nanoparticles. Compressive mechanical properties were well-matched to peripheral nerve tissue at 0.93 kPa and 1.29 kPa, respectively. In vitro, templated hydrogels exhibited approximately 36% faster degradation over 12 h, and were found to guide axon extension from dorsal root ganglia. Finally, in a pilot in vivo study utilizing a 10 mm rat sciatic nerve defect model, magnetically templated hydrogels demonstrated promising results with qualitatively increased remodeling and axon regeneration compared to non-templated controls. Significance. This simple and scalable technology has the flexibility to control tubular microstructure over long length scales, and thus the potential to meet the need for engineered scaffolds for tissue regeneration, including nerve guidance scaffolds.