Regeneration of peripheral nerves by nerve guidance conduits: Influence of design, biopolymers, cells, growth factors, and physical stimuli.

Injuries to the peripheral nervous system (PNS) cause neuropathies that lead to weakness and paralysis, poor or absent sensation, unpleasant and painful neuropathies, and impaired autonomic function. In this regard, implanted artificial nerve guidance conduits (NGCs) used to bridge an injured site may provide appropriate biochemical and biophysical guidance cues required to stimulate regeneration across a nerve gap and restore the function of PNS. Advanced conduit design and fabrication techniques have made it possible to fabricate autograft-like structures in the NGCs with incredible precision. To this end, strategies involving the use of biopolymers, cells, growth factors, and physical stimuli have been developed over the past decades and have led to the development of varying NGCs, from simple hollow tubes to complex conduits that incorporate one or more guidance cues. This paper briefly reviews the recent progress in the development of these NGCs for nerve regeneration, focusing on the design and fabrication of NGCs, as well as the influence of biopolymers, cells, growth factors, and physical stimuli. The advanced techniques used to fabricate NGCs that incorporate cells/growth factors are also discussed, along with their merits and flaws. Key issues and challenges with regard to the development of NGCs have been identified and discussed, and recommendations for future research have been included.

3D bioprinting of scaffolds with living Schwann cells for potential nerve tissue engineering applications.

Three-dimensional bioprinting of biomaterials shows great potential for producing cell-encapsulated scaffolds to repair nerves after injury or disease. For this, preparation of biomaterials and bioprinting itself are critical to create scaffolds with both biological and mechanical properties appropriate for nerve regeneration, yet remain unachievable. This paper presents our study on bioprinting Schwann cell-encapsulated scaffolds using composite hydrogels of alginate, fibrin, hyaluronic acid, and/or RGD peptide, for nerve tissue engineering applications. For the preparation of composite hydrogels, suitable hydrogel combinations were identified and prepared by adjusting the concentration of fibrin based on the morphological spreading of Schwann cells. In bioprinting, the effects of various printing process parameters (including the air pressure for dispensing, dispensing head movement speed, and crosslinking conditions) on printed structures were investigated and, by regulating these parameters, mechanically-stable scaffolds with fully interconnected pores were printed. The performance of Schwann cells within the printed scaffolds were examined in terms of viability, proliferation, orientation, and ability to produce laminin. Our results show that the printed scaffolds can promote the alignment of Schwann cells inside scaffolds and thus provide haptotactic cues to direct the extension of dorsal root ganglion neurites along the printed strands, demonstrating their great potential for applications in the field of nerve tissue engineering.

Strategic Design and Fabrication of Nerve Guidance Conduits for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) have been drawing considerable attention as an aid to promote regeneration of injured axons across damaged peripheral nerves. Ideally, NGCs should include physical and topographic axon guidance cues embedded as part of their composition. Over the past decades, much progress has been made in the development of NGCs that promote directional axonal regrowth so as to repair severed nerves. This paper briefly reviews the recent designs and fabrication techniques of NGCs for peripheral nerve regeneration. Studies associated with versatile design and preparation of NGCs fabricated with either conventional or rapid prototyping (RP) techniques have been examined and reviewed. The effect of topographic features of the filler material as well as porous structure of NGCs on axonal regeneration has also been examined from the previous studies. While such strategies as macroscale channels, lumen size, groove geometry, use of hydrogel/matrix, and unidirectional freeze-dried surface are seen to promote nerve regeneration, shortcomings such as axonal dispersion and wrong target reinnervation still remain unsolved. On this basis, future research directions are identified and discussed.

Characterization of Cell Damage and Proliferative Ability during and after Bioprinting.

When a biomaterial solution containing living cells is subject to bioprinting, the cells experience process-induced stresses, including shear and extensional stresses. These process-induced stresses breach cell membranes and can lead to cell damage, thus reducing cell viability and functioning within the printed constructs. Studies have been conducted to determine the influence of shear stress on cell damage; however, the effect of extensional stress has been typically ignored in the literature until the recently collected evidence of its importance. This paper presents a novel method to characterize and quantify the cell damage caused by both shear and extensional stresses in bioprinting. In this method, cell damage law is first established to relate cell damage to shear stress based on the experiments with a rheometer; the process-induced shear stress experienced by cells in bioprinting is represented, and the established cell damage model is applied to calculate the degree of cell damage caused by shear stress in bioprinting; then cell damage caused by extensional stress is inferred from the difference between the total cell damage and the amount of cell damage attributed to shear stress. With the obtained magnitude of extensional stress from fluidic simulation, the model that relates extensional stress to cell damage is established; the bioprinting process-induced cell damage attributed to both shear and extensional stresses is therefore presented. Schwann cells and myoblasts were used as examples to validate the models. Comparison between experimental and simulation results shows the effectiveness of the models presented in this paper. Moreover, the viability and proliferative ability of cells in the first 72 h after bioprinting is investigated, with the results illustrating that the process-induced forces affect not only cell viability but also their proliferative ability after bioprinting.

Influence of mechanical properties of alginate-based substrates on the performance of Schwann cells in culture.

In tissue engineering, artificial tissue scaffolds containing living cells have been studied for tissue repair and regeneration. Notably, the performance of these encapsulated-in-scaffolds cells in terms of cell viability, proliferation, and expression of function during and after the scaffold fabrication process, has not been well documented because of the influence of mechanical, chemical, and physical properties of the scaffold substrate materials. This paper presents our study on the influence of mechanical properties of alginate-based substrates on the performance of Schwann cells, which are the major glial cells of peripheral nervous system. Given the fact that alginate polysaccharide hydrogel has poor cell adhesion properties, in this study, we examined several types of cell-adhesion supplements and found that alginate covalently modified with RGD peptide provided improved cell proliferation and adhesion. We prepared alginate-based substrates for cell culture using varying alginate concentrations for altering their mechanical properties, which were confirmed by compression testing. Then, we examined the viability, proliferation, morphology, and expression of the extracellular matrix protein laminin of Schwann cells that were seeded on the surface of alginate-based substrates (or 2D culture) or encapsulated within alginate-based substrates (3D cultures), and correlated the examined cell performance to the alginate concentration (or mechanical properties) of hydrogel substrates. Our findings suggest that covalent attachment of RGD peptide can improve the success of Schwann cell encapsulation within alginate-based scaffolds, and provide guidance for regulating the mechanical properties of alginate-based scaffolds containing Schwann cells for applications in peripheral nervous system regeneration and repair.

An in vitro study of peptide-loaded alginate nanospheres for antagonizing the inhibitory effect of Nogo-A protein on axonal growth.

The adult mammalian central nervous system has limited ability to regenerate after injury. This is due, in part, to the presence of myelin-associated axon growth inhibitory proteins such as Nogo-A that bind and activate the Nogo receptor, leading to profound inhibition of actin-based motility within the growing axon tip. This paper presents an in vitro study of the use of a Nogo receptor-blocking peptide to antagonize the inhibitory effect of Nogo-A on axon growth. Alginate nanospheres were fabricated using an emulsion technique and loaded with Nogo receptor-blocking peptide, or with other model proteins. Protein release profiles were studied, and retention of the bioactivity of released proteins was verified. Primary dorsal root ganglion neurons were cultured and their ability to grow neurites was challenged with Nogo-A chimeric protein in the absence or presence of Nogo receptor antagonist peptide-loaded alginate nanospheres. Our results demonstrate that peptide released from alginate nanospheres could overcome the growth inhibitory effect of Nogo-A, suggesting that a similar peptide delivery strategy using alginate nanospheres might be used to improve axon regeneration within the injured central nervous system.

PLGA/alginate composite microspheres for hydrophilic protein delivery.

Poly(lactic-co-glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate.

Use of the polycation polyethyleneimine to improve the physical properties of alginate-hyaluronic acid hydrogel during fabrication of tissue repair scaffolds.

Recently alginate-based tissue repair scaffolds fabricated using 3D printing techniques have been extensively examined for use in tissue engineering applications. However, their physical and mechanical properties are unfavorable for many tissue engineering applications because these properties are poorly controlled during the fabrication process. Some improvement of alginate gel properties can be realized by addition of hyaluronic acid (HA), and this may also improve the ability of cells to interact with the gel. Here, we report improvement of the physical properties of alginate-HA gel scaffolds by the addition of the polycation polyethyleneimine (PEI) during the fabrication process in order to stabilize alginate molecular structure through the formation of a polyelectrolyte complex. We find that PEI has a significant beneficial influence on alginate-HA scaffold physical properties, including a reduction in the degree of gel swelling, a reduction in scaffold degradation rate, and an increase in the Young's modulus of the gel. Further study shows that fabrication of alginate-HA gels with PEI increases the encapsulation efficiency of bovine serum albumin, a model protein, and reduces the subsequent initial protein release rate. However, it was also found that survival of Schwann cells or ATDC-5 chondrogenic cells encapsulated during the scaffold fabrication process was modestly reduced with increasing PEI concentration. This study illustrates that the use of PEI during scaffold fabrication by plotting can provide an effective means to control alginate-based scaffold properties for tissue engineering applications, but that the many effects of PEI must be balanced for optimal outcomes in different situations.

Protein-energy malnutrition developing after global brain ischemia induces an atypical acute-phase response and hinders expression of GAP-43.

Protein-energy malnutrition (PEM) is a common post-stroke problem. PEM can independently induce a systemic acute-phase response, and pre-existing malnutrition can exacerbate neuroinflammation induced by brain ischemia. In contrast, the effects of PEM developing in the post-ischemic period have not been studied. Since excessive inflammation can impede brain remodeling, we investigated the effects of post-ischemic malnutrition on neuroinflammation, the acute-phase reaction, and neuroplasticity-related proteins. Male, Sprague-Dawley rats were exposed to global forebrain ischemia using the 2-vessel occlusion model or sham surgery. The sham rats were assigned to control diet (18% protein) on day 3 after surgery, whereas the rats exposed to global ischemia were assigned to either control diet or a low protein (PEM, 2% protein) diet. Post-ischemic PEM decreased growth associated protein-43, synaptophysin and synaptosomal-associated protein-25 immunofluorescence within the hippocampal CA3 mossy fiber terminals on day 21, whereas the glial response in the hippocampal CA1 and CA3 subregions was unaltered by PEM. No systemic acute-phase reaction attributable to global ischemia was detected in control diet-fed rats, as reflected by serum concentrations of alpha-2-macroglobulin, alpha-1-acid glycoprotein, haptoglobin, and albumin. Acute exposure to the PEM regimen after global brain ischemia caused an atypical acute-phase response. PEM decreased the serum concentrations of albumin and haptoglobin on day 5, with the decreases sustained to day 21. Serum alpha-2-macroglobulin concentrations were significantly higher in malnourished rats on day 21. This provides the first direct evidence that PEM developing after brain ischemia exerts wide-ranging effects on mechanisms important to stroke recovery.

Preparation and characterization of alginate microspheres for sustained protein delivery within tissue scaffolds.

Tissue engineering scaffolds are designed not only to provide structural support for the repair of damaged tissue, but can also serve the function of bioactive protein delivery. Here we present a study on the preparation and characterization of protein-loaded microspheres, either alone or incorporated into mock tissue scaffolds, for sustained protein delivery. Alginate microspheres were prepared by a novel, small-scale water-in-oil emulsion technique and loaded with fluorescently labeled immunoglobulin G (IgG). Microsphere size appears to be influenced by the magnitude and distribution of force generated by mechanical stirring during emulsion. Protein release studies show that sustained IgG release from microspheres could be achieved and that application of a secondary coating of chitosan could further slow the rate of protein release. Preservation of bioactivity of released IgG protein was confirmed using an immunohistochemical assay. When IgG-loaded microspheres were incorporated into mock scaffolds, initial protein release was diminished and the overall time course of release was extended. The present study demonstrates that protein-loaded microspheres can be prepared with a controlled release profile and preserved biological activity, and can be incorporated into scaffolds to achieve sustained and prolonged protein delivery in a tissue engineering application.

Strategic design and recent fabrication techniques for bioengineered tissue scaffolds to improve peripheral nerve regeneration.

Bioengineered tissue scaffolds are a potential tool for improving regenerative repair of damaged peripheral nerves. Novel modes of fabrication coupled with scaffold design strategies that are based on an understanding of the biology of nerve injury offer the prospect of intervention at a more sophisticated level. We review the etiology and incidence of peripheral nerve injury and the biological events that unfold during nerve regeneration after an injury. Newly available tissue scaffold fabrication technologies using bioplotting and laser-based techniques are described. Scaffold design strategies are also discussed, including the incorporation of living cells during scaffold fabrication, inclusion of neurotrophic gradients, use of electric stimulation, inclusion of antioxidant compounds to counteract neural apotosis, and promotion of angiogenesis. Use of these advanced fabrication techniques and incorporation of one or more of these active biological strategies may eventually lead to a greater success in peripheral nerve tissue engineering.

X-ray diffraction enhanced imaging as a novel method to visualize low-density scaffolds in soft tissue engineering.

Scaffold visualization is challenging yet essential to the success of various tissue engineering applications. The aim of this study was to explore the potential of X-ray diffraction enhanced imaging (DEI) as a novel method for the visualization of low density engineered scaffolds in soft tissue. Imaging of the scaffolds made from poly(L-lactide) (PLLA) and chitosan was conducted using synchrotron radiation-based radiography, in-line phase-contrast imaging (in-line PCI), and DEI techniques as well as laboratory-based radiography. Scaffolds were visualized in air, water, and rat muscle tissue. Compared with the images from X-ray radiography and in-line PCI techniques, DEI images more clearly show the structure of the low density scaffold in air and have enhanced image contrast. DEI was the only technique able to visualize scaffolds embedded in unstained muscle tissue; this method could also define the microstructure of muscle tissue in the boundary areas. At a photon energy of 20 KeV, DEI had the capacity to image PLLA/chitosan scaffolds in soft tissue with a sample thickness of up to 4 cm. The DEI technique can be applied at high X-ray energies, thus facilitating lower in vivo radiation doses to tissues during imaging as compared to conventional radiography.

Bioengineered scaffolds for spinal cord repair.

Spinal cord injury can lead to devastating and permanent loss of neurological function, affecting all levels below the site of trauma. Unfortunately, the injured adult mammalian spinal cord displays little regenerative capacity and little functional recovery in large part due to a tissue environment that is nonpermissive for regenerative axon growth. Artificial tissue repair scaffolds may provide a physical guide to allow regenerative axon growth that bridges the lesion cavity and restores functional neural connectivity. By integrating different strategies, including the use of various biomaterials and microstructures as well as incorporation of bioactive molecules and living cells, combined or synergistic effects for spinal cord repair through regenerative axon growth may be achieved. This article briefly reviews the development of bioengineered scaffolds for spinal cord repair, focusing on spinal cord injury and the subsequent cellular response, scaffold materials, fabrication techniques, and current therapeutic strategies. Key issues and challenges are also identified and discussed along with recommendations for future research.

Effect of needle geometry on flow rate and cell damage in the dispensing-based biofabrication process.

Biodispensing techniques have been widely applied in biofabrication processes to deliver cell suspensions and biomaterials to create cell-seeded constructs. Under identical operating conditions,two types of dispensing needles­tapered and cylindrical­can result in different flow rates of material and different cell damage percent induced by the mechanical forces. In this work, mathematical models of both flow rate and cell damage percent in biodispensing systems using tapered and cylindrical needles, respectively, were developed, and experiments were carried out to verify the effectiveness of the developed models. Both simulations and experiments show tapered needles produce much higher flow rates under the same pressure conditions than cylindrical needles. Use of a lower pressure in a tapered needle can therefore achieve the same flow rate as that in a cylindrical needle. At equivalent flow rates, cell damage in a tapered needle is lower than that in a cylindrical one. Both Schwann cells and 3T3 fibroblasts, which have been widely used in tissue engineering, were used to validate the cell damage models. Application of the developed models to specify the influence of process parameters, including needle geometry and air pressure, on the flow rate and cell damage percent represents a significant advance for biofabrication processes.The models can be used to optimize process parameters to preserve cell viability and achieve the desired cell distribution in dispensing-based biofabrication.

Modeling process-induced cell damage in the biodispensing process.

Emerging biomanufacturing processes involve incorporation of living cells into various processes and systems by employing different cell manipulation techniques. Among them, biodispensing, in which the cell suspension is extruded via a fine needle under pressurized air, is a promising technique because of its high efficiency. Cells in this process are continually subjected to mechanical forces and may be damaged if the force or manipulation time exceeds certain levels. Modeling cell injury incurred in these processes is lacking in the literature. This article presents a method to quantify the force-induced cell damage in the biodispensing process. This method consists of two steps: first is to establish cell damage laws to relate cell damage to hydrostatic pressure/shear stress; and the second is to represent the process-induced forces experienced by cells during the biodispensing process and apply the established cell damage law to represent the percentage of cell damage. Schwann cells and 3T3 fibroblasts were used to validate the model and the comparisons of experimental and simulation results show the effectiveness of the method presented in this article.

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons.

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and Talpha1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced.

Differential hypertrophy and atrophy among all types of cutaneous innervation in the glabrous skin of the monkey hand during aging and naturally occurring type 2 diabetes.

Diabetic neuropathy (DN) is a common severe complication of type 2 diabetes. The symptoms of chronic pain, tingling, and numbness are generally attributed to small fiber dysfunction. However, little is known about the pathology among innervation to distal extremities, where symptoms start earliest and are most severe, and where the innervation density is the highest and includes a wide variety of large fiber sensory endings. Our study assessed the immunochemistry, morphology, and density of the nonvascular innervation in glabrous skin from the hands of aged nondiabetic rhesus monkeys and from age-matched monkeys that had different durations of spontaneously occurring type 2 diabetes. Age-related reductions occurred among all types of innervation, with epidermal C-fiber endings preferentially diminishing earlier than presumptive Adelta-fiber endings. In diabetic monkeys epidermal innervation density diminished faster, became more unevenly distributed, and lost immunodetectable expression of calcitonin gene-related peptide and capsaicin receptors, TrpV1. Pacinian corpuscles also deteriorated. However, during the first few years of hyperglycemia, a surprising hypertrophy occurred among terminal arbors of remaining epidermal endings. Hypertrophy also occurred among Meissner corpuscles and Merkel endings supplied by Abeta fibers. After longer-term hyperglycemia, Meissner corpuscle hypertrophy declined but the number of corpuscles remained higher than in age-matched nondiabetics. However, the diabetic Meissner corpuscles had an abnormal structure and immunochemistry. In contrast, the expanded Merkel innervation was reduced to age-matched nondiabetic levels. These results indicate that transient phases of substantial innervation remodeling occur during the progression of diabetes, with differential increases and decreases occurring among the varieties of innervation.

Developmental down-regulation of GAP-43 expression and timing of target contact in rat corticospinal neurons.

During early nervous system development axons grow toward the target tissue that they will innervate. As axons invade target tissue, growth slows and ceases. Neurons express high levels of the growth-associated protein GAP-43 during developmental axon growth, declining with maturation. It has been suggested that target contact provides a signal which down-regulates GAP-43 expression. To study this issue in more detail, we used in situ hybridization to quantify relative changes in GAP-43 mRNA in corticospinal tract neurons identified by Fast Blue retrograde labeling. We also used anterograde transport of biotinylated dextran amine to study the invasion of target by corticospinal axons. We find that GAP-43 mRNA is high during the first postnatal week and then declines in two phases. Approximately half of the initial level of GAP-43 expression in corticospinal neurons is lost by P12; then expression remains at a plateau until P21. Between P21 and P28, GAP-43 expression again declines by half and then remains steady at the adult level (one fourth of initial level). Corticospinal axons initially invade spinal gray matter during the first 2 postnatal weeks, in a rostrocaudal gradient. Varicosities suggestive of terminal boutons become numerous during the third and fourth week, and the morphology of corticospinal axon terminals achieves the mature form at the end of the fourth week. These data suggest that the first phase of down-regulation of GAP-43 in corticospinal neurons is coincident with initial target contact and that the second phase is coincident with final maturation of terminal arborization.

Retrograde repression of growth-associated protein-43 mRNA expression in rat cortical neurons.

Corticospinal neurons support rapid growth of axons toward spinal cord targets in the perinatal period. Initial axon growth is accompanied by elevated expression of growth-associated protein-43 (GAP-43), which then declines in postnatal development. To investigate whether expression of GAP-43 mRNA is regulated by retrograde signals, we injected colchicine into the corticospinal tract to block retrograde axonal transport during a time when GAP-43 is normally declining in corticospinal neurons. Colchicine caused a prolongation of high GAP-43 mRNA expression in neurons located in layer V (but not other layers) of sensorimotor cortex. We next used osmotic minipumps to infuse soluble adult spinal cord extract into the sensorimotor cortex. This resulted in a premature downregulation of GAP-43 mRNA in identified corticospinal neurons. GAP-43 repressive activity was found in extracts of the spinal cord tissue as young as postnatal day 8. The effect of spinal cord extract in vivo was not mimicked by adult cerebellar or muscle extracts. Cultures of postnatal cortical neurons also underwent downregulation of GAP-43 mRNA when treated with spinal cord extract. Activation of cAMP signaling also repressed GAP-43 mRNA in cortical cultures, and the repressive effect of spinal cord extract was diminished by an adenyl cyclase inhibitor. Thus, GAP-43 mRNA may be downregulated late in development by a target-derived retrograde repressive factor, and this effect may be mediated by cAMP second messenger signaling.