Peripheral Nerve Matrix Hydrogel Promotes Recovery after Nerve Transection and Repair.

BACKGROUND: Nerve transection is the most common form of peripheral nerve injury. Treatment of peripheral nerve injury has primarily focused on stabilization and mechanical cues to guide extension of the regenerating growth cone across the site of transection. The authors investigated the effects of a peripheral nerve matrix (PNM) hydrogel on recovery after nerve transection. METHODS: The authors used rodent models to determine the effect of PNM on axon extension, electrophysiologic nerve conduction, force generation, and neuromuscular junction formation after nerve transection and repair. The authors complemented this work with in vivo and in vitro fluorescence-activated cell sorting and immunohistochemistry approaches to determine the effects of PNM on critical cell populations early after repair. RESULTS: Extension of axons from the proximal stump and overall green fluorescent protein-positive axon volume within the regenerative bridge were increased in the presence of PNM compared with an empty conduit ( P < 0.005) 21 days after repair. PNM increased electrophysiologic conduction (compound muscle action potential amplitude) across the repair site ( P < 0.05) and neuromuscular junction formation ( P = 0.04) 56 days after repair. PNM produced a shift in macrophage phenotype in vitro and in vivo ( P < 0.05) and promoted regeneration in a murine model used to characterize the early immune response to PNM ( P < 0.05). CONCLUSION: PNM, delivered by subepineural injection, promoted recovery after nerve transection with immediate repair, supporting a beneficial macrophage response, axon extension, and downstream remodeling using a range of clinically relevant outcome measures. CLINICAL RELEVANCE STATEMENT: This article describes an approach for subepineural injection at the site of nerve coaptation to modulate the response to injury and improve outcomes.

Safety and efficacy of an injectable nerve-specific hydrogel in a rodent crush injury model.

INTRODUCTION/AIMS: While the peripheral nervous system has the inherent ability to recover following injury, results are often unsatisfactory, resulting in permanent functional deficits and disability. Therefore, methods that enhance regeneration are of significant interest. The present study investigates an injectable nerve-tissue-specific hydrogel as a biomaterial for nerve regeneration in a rat nerve crush model. METHODS: Nerve-specific hydrogels were injected into the subepineurial space in both uninjured and crushed sciatic nerves of rats to assess safety and efficacy, respectively. The animals were followed longitudinally for 12 wk using sciatic functional index and kinematic measures. At 12 wk, electrophysiologic examination was also performed, followed by nerve and muscle histologic assessment. RESULTS: When the hydrogel was injected into an uninjured nerve, no differences in sciatic functional index, kinematic function, or axon counts were observed. A slight reduction in muscle fiber diameter was observed in the hydrogel-injected animals, but overall muscle area and kinematic function were not affected. Hydrogel injection following nerve crush injury resulted in multiple modest improvements in sciatic functional index and kinematic function with an earlier return to normal function observed in the hydrogel treated animals as compared to untreated controls. While no improvements in supramaximal compound motor action potential were observed in hydrogel treated animals, increased axon counts were observed on histologic assessment. DISCUSSION: These improvements in functional and histologic outcomes in a rapidly and fully recovering model suggest that injection of a nerve-specific hydrogel is safe and has the potential to improve outcomes following nerve injury.

Decellularization techniques and their applications for the repair and regeneration of the nervous system.

A variety of surgical and non-surgical approaches have been used to address the impacts of nervous system injuries, which can lead to either impairment or a complete loss of function for affected patients. The inherent ability of nervous tissues to repair and/or regenerate is dampened due to irreversible changes that occur within the tissue remodeling microenvironment following injury. Specifically, dysregulation of the extracellular matrix (i.e., scarring) has been suggested as one of the major factors that can directly impair normal cell function and could significantly alter the regenerative potential of these tissues. A number of tissue engineering and regenerative medicine-based approaches have been suggested to intervene in the process of remodeling which occurs following injury. Decellularization has become an increasingly popular technique used to obtain acellular scaffolds, and their derivatives (hydrogels, etc.), which retain tissue-specific components, including critical structural and functional proteins. These advantageous characteristics make this approach an intriguing option for creating materials capable of stimulating the sensitive repair mechanisms associated with nervous system injuries. Over the past decade, several diverse decellularization methods have been implemented specifically for nervous system applications in an attempt to carefully remove cellular content while preserving tissue morphology and composition. Each application-based decellularized ECM product requires carefully designed treatments that preserve the unique biochemical signatures associated within each tissue type to stimulate the repair of brain, spinal cord, and peripheral nerve tissues. Herein, we review the decellularization techniques that have been applied to create biomaterials with the potential to promote the repair and regeneration of tissues within the central and peripheral nervous system.

Conduits harnessing spatially controlled cell-secreted neurotrophic factors improve peripheral nerve regeneration.

An essential structure in nerve regeneration within engineered conduits is the "nerve bridge" initiated by centrally migrating Schwann cells in response to chemokine gradients. Introducing exogenous cells secreting neurotrophic factors aims to augment this repair process, but conventional cell-seeding methods fail to produce a directional chemokine gradient. We report a versatile method to encapsulate cells within conduit walls, allowing for reproducible control of spatial distribution along the conduit. Conduits with stem cells encapsulated within the central third possessed markedly different cell distribution and retention over 6 weeks in vivo, compared to standard cell lumen injection. Such a construct promoted Schwann cell migration centrally, and at 16 weeks rats presented with significantly enhanced function and axonal myelination. The method of utilizing a spatially restricted cell secretome departs from traditional homogeneous cell loading, and presents new approaches for studying and maximizing the potential of cell application in peripheral nerve repair.

Nerve-specific, xenogeneic extracellular matrix hydrogel promotes recovery following peripheral nerve injury.

Peripheral nerve possesses the inherent ability to regrow and recover following injury. However, nerve regeneration is often slow and incomplete due to limitations associated with the local microenvironment during the repair process. Manipulation of the local microenvironment at the site of nerve repair, therefore, represents a significant opportunity for improvement in downstream outcomes. Macrophages and Schwann cells play a key role in the orchestration of early events after peripheral nerve injury. We describe the production, characterization, and use of an injectable, peripheral nerve-specific extracellular matrix-based hydrogel (PNSECM) for promoting modulation of the local macrophage and Schwann cell responses at the site of nerve repair in a rodent model of sciatic nerve injury. We show that PNSECM hydrogels largely maintain the matrix structure associated with normal native peripheral nerve tissue. PNSECM hydrogels were also found to promote increased macrophage invasion, higher percentages of M2 macrophages and enhanced Schwann cell migration when used as a lumen filler in a rodent model of nerve gap repair using an inert nerve guidance conduit. These results suggest that an injectable PNSECM hydrogel can provide a supportive, bioactive scaffold which promotes repair of peripheral nerve in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 450-459, 2018.

Preparation of Decellularized Biological Scaffolds for 3D Cell Culture.

The biggest challenge of designing and implementing an in vitro study is developing a microenvironment that most closely represents the interactions observed in vivo. Decellularization of tissues and organs has been shown to be an effective method for the removal of potentially immunogenic constituents while preserving essential growth factors and extracellular matrix (ECM ) proteins necessary for proper cell function. Enzymatic digestion of decellularized tissues allows these tissue-specific components to be reconstituted into bioactive hydrogels through a physical crosslinking of collagen. In the following protocol, we describe unique decellularization methods for both dermis and urinary bladder matrix (UBM) derived from porcine tissues. We then provide details for hydrogel formation and subsequent three-dimensional (3D) culture of two cell types: NIH 3T3 fibroblasts and C2C12 myoblasts .