The potential of Antheraea pernyi silk for spinal cord repair

This work was supported by the Institute of Medical Sciences of the University of Aberdeen, Scottish Rugby Union and RS McDonald Charitable Trust. We are grateful to Mr Nicholas Hawkins from Oxford University and Ms Annette Raffan from the University of Aberdeen for assistance with tensile testing. We thank Ms Michelle Gniβ for her help with the microglial response experiments. We also thank Mr Gianluca Limodio for assisting with the MATLAB script for automation of tensile testing’s data analysis.Peer reviewedPublisher PD

Machine intelligence for nerve conduit design and production

Nerve guidance conduits (NGCs) have emerged from recent advances within tissue engineering as a promising alternative to autografts for peripheral nerve repair. NGCs are tubular structures with engineered biomaterials, which guide axonal regeneration from the injured proximal nerve to the distal stump. NGC design can synergistically combine multiple properties to enhance proliferation of stem and neuronal cells, improve nerve migration, attenuate inflammation and reduce scar tissue formation. The aim of most laboratories fabricating NGCs is the development of an automated process that incorporates patient-specific features and complex tissue blueprints (e.g. neurovascular conduit) that serve as the basis for more complicated muscular and skin grafts. One of the major limitations for tissue engineering is lack of guidance for generating tissue blueprints and the absence of streamlined manufacturing processes. With the rapid expansion of machine intelligence, high dimensional image analysis, and computational scaffold design, optimized tissue templates for 3D bioprinting (3DBP) are feasible. In this review, we examine the translational challenges to peripheral nerve regeneration and where machine intelligence can innovate bottlenecks in neural tissue engineering

Understanding Molecular Pathology along Injured Spinal Cord Axis: Moving Frontiers toward Effective Neuroprotection and Regeneration

Spinal cord injury (SCI) is a severe, often life threatening, traumatic condition leading to serious neurological dysfunctions. The pathological hallmarks of SCI include inflammation, reactive gliosis, axonal demyelination, neuronal death, and cyst formation. Although much has been learned about the progression of SCI pathology affecting a large number of biochemical cascades and reactions, the roles of proteins involved in these processes are not well understood. Advances in proteomic technologies have made it possible to examine the spinal cord proteome from healthy and experimental animals and disclose a detailed overview on the spatial and temporal regionalization of these secondary processes. Data clearly demonstrated that neurotrophic molecules dominated in the segment above the central lesion, while the proteins associated with necrotic/apoptotic pathways abound the segment below the lesion. This knowledge is extremely important in finding optimal targets and pathways on which complementary neuroprotective and neuroregenerative approaches should be focused on. In terms of neuroprotection, several active substances and cell-based therapy together with biomaterials releasing bioactive substances showed partial improvement of spinal cord injury. However, one of the major challenges is to select specific therapies that can be combined safely and in the appropriate order to provide the maximum value of each individual treatment

PLG Bridge Implantation in Chronic SCI Promotes Axonal Elongation and Myelination.

Spinal cord injury (SCI) is a devastating condition that may cause permanent functional loss below the level of injury, including paralysis and loss of bladder, bowel, and sexual function. Patients are rarely treated immediately, and this delay is associated with tissue loss and scar formation that can make regeneration at chronic time points more challenging. Herein, we investigated regeneration using a poly(lactide-co-glycolide) multichannel bridge implanted into a chronic SCI following surgical resection of necrotic tissue. We characterized the dynamic injury response and noted that scar formation decreased at 4 and 8 weeks postinjury (wpi), yet macrophage infiltration increased between 4 and 8 wpi. Subsequently, the scar tissue was resected and bridges were implanted at 4 and 8 wpi. We observed robust axon growth into the bridge and remyelination at 6 months after initial injury. Axon densities were increased for 8 week bridge implantation relative to 4 week bridge implantation, whereas greater myelination, particularly by Schwann cells, was observed with 4 week bridge implantation. The process of bridge implantation did not significantly decrease the postinjury function. Collectively, this chronic model follows the pathophysiology of human SCI, and bridge implantation allows for clear demarcation of the regenerated tissue. These data demonstrate that bridge implantation into chronic SCI supports regeneration and provides a platform to investigate strategies to buttress and expand regeneration of neural tissue at chronic time points

Polycistronic Delivery of IL-10 and NT-3 Promotes Oligodendrocyte Myelination and Functional Recovery in a Mouse Spinal Cord Injury Model.

One million estimated cases of spinal cord injury (SCI) have been reported in the United States and repairing an injury has constituted a difficult clinical challenge. The complex, dynamic, inhibitory microenvironment postinjury, which is characterized by proinflammatory signaling from invading leukocytes and lack of sufficient factors that promote axonal survival and elongation, limits regeneration. Herein, we investigated the delivery of polycistronic vectors, which have the potential to coexpress factors that target distinct barriers to regeneration, from a multiple channel poly(lactide-co-glycolide) (PLG) bridge to enhance spinal cord regeneration. In this study, we investigated polycistronic delivery of IL-10 that targets proinflammatory signaling, and NT-3 that targets axonal survival and elongation. A significant increase was observed in the density of regenerative macrophages for IL-10+NT-3 condition relative to conditions without IL-10. Furthermore, combined delivery of IL-10+NT-3 produced a significant increase of axonal density and notably myelinated axons compared with all other conditions. A significant increase in functional recovery was observed for IL-10+NT-3 delivery at 12 weeks postinjury that was positively correlated to oligodendrocyte myelinated axon density, suggesting oligodendrocyte-mediated myelination as an important target to improve functional recovery. These results further support the use of multiple channel PLG bridges as a growth supportive substrate and platform to deliver bioactive agents to modulate the SCI microenvironment and promote regeneration and functional recovery. Impact statement Spinal cord injury (SCI) results in a complex microenvironment that contains multiple barriers to regeneration and functional recovery. Multiple factors are necessary to address these barriers to regeneration, and polycistronic lentiviral gene therapy represents a strategy to locally express multiple factors simultaneously. A bicistronic vector encoding IL-10 and NT-3 was delivered from a poly(lactide-co-glycolide) bridge, which provides structural support that guides regeneration, resulting in increased axonal growth, myelination, and subsequent functional recovery. These results demonstrate the opportunity of targeting multiple barriers to SCI regeneration for additive effects

Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration

Neural tissue repair and regeneration strategies have received a great deal of attention because it directly affects the quality of the patient's life. There are many scientific challenges to regenerate nerve while using conventional autologous nerve grafts and from the newly developed therapeutic strategies for the reconstruction of damaged nerves. Recent advancements in nerve regeneration have involved the application of tissue engineering principles and this has evolved a new perspective to neural therapy. The success of neural tissue engineering is mainly based on the regulation of cell behavior and tissue progression through the development of a synthetic scaffold that is analogous to the natural extracellular matrix and can support three-dimensional cell cultures. As the natural extracellular matrix provides an ideal environment for topographical, electrical and chemical cues to the adhesion and proliferation of neural cells, there exists a need to develop a synthetic scaffold that would be biocompatible, immunologically inert, conducting, biodegradable, and infection-resistant biomaterial to support neurite outgrowth. This review outlines the rationale for effective neural tissue engineering through the use of suitable biomaterials and scaffolding techniques for fabrication of a construct that would allow the neurons to adhere, proliferate and eventually form nerves

Fundamentals and current strategies for Peripheral Nerve Repair and Regeneration

A body of evidence indicates that peripheral nerves have an extraordinary yet limited capacity to regenerate after an injury. Peripheral nerve injuries have confounded professionals in this field, from neuroscientists to neurologists, plastic surgeons, and the scientific community. Despite all the efforts, full functional recovery is still seldom. The inadequate results attained with the â gold standardâ autograft procedure still encourage a dynamic and energetic research around the world for establishing good performing tissue engineered alternative grafts. Resourcing to nerve guidance conduits, a variety of methods have been experimentally used to bridge peripheral nerve gaps of limited size, up to 30-40 mm in length, in humans. Herein, we aim to summarize the fundamentals related to peripheral nerve anatomy and overview the challenges and scientific evidences related to peripheral nerve injury and repair mechanisms. The most relevant reports dealing with the use of both synthetic and natural-based biomaterials used in tissue engineering strategies when treatment of nerve injuries is envisioned are also discussed in depth, along with the state-of-the-art approaches in this field.This work was supported by Cristiana Carvalho PhD scholarship (Norte-08-5369-FSE-000037). J. M. Oliveira also thanks the FCT for the funds provided under the program Investigador FCT 2015 (IF/01285/2015).The authors are also thankful to the FCT funded project NanoOptoNerv(ref. PTDC/NAN-MAT/29936/2017).The authors would also like to acknowledge the project: “Nano-accelerated nerve regeneration and optogenetic empowering of neuromuscular functionality” (ref.PTDC/NAN-MAT/29936/2017)

Scaffolds for Peripheral Nerve Regeneration, the Importance of In Vitro and In Vivo Studies for the Development of Cell-Based Therapies and Biomaterials: State of the Art

Human adult peripheral nerve injuries are a high incidence clinical problem that greatly affects patients’ quality of life. Although peripheral nervous system has intrinsic regenerative capacity, this occurs in an incomplete or poorly functional manner. When a nerve fiber loses its continuity with consequent damage of the basal lamina tubes, axon spontaneous regeneration is disorganized and mismatched. These phenomena translate in an inadequate nerve functional recovery and consequent musculoskeletal incapacity. Nerve grafts still remain the gold standard in peripheral injuries treatment. However, this approach contains its disadvantages such as the necessity of primary surgery to harvest the autografts, loss of a functional nerve, donor site morbidity and longer surgery procedures. Therefore, biomaterials and tissue engineering can provide efficient resources and alternatives to nerve injury repair not only by the development of biocompatible structures but also, introducing neurotrophic factors and cellular systems to stimulate optimum clinical outcome. In this chapter, a comprehensive state-of-the art picture of tissue-engineered nerve grafts scaffolds, their application in nerve regeneration along with latest advances in peripheral nerve repair and future perspectives will be discussed, including our own large experience in this field of knowledge

Doctor of Philosophy

dissertationSpinal cord injury (SCI) is extremely debilitating to patients and costly to our healthcare system. Since it is an important contributor to mortality and morbidity, various therapeutic strategies have been investigated, either experimentally or clinically, to improve patients' quality of life. Studies utilizing pharmacological methods to mitigate the inhibitory components of the glial scar and facilitate axonal regeneration have been the primary experimental approaches in the field. However, the results are still not satisfactory. In this research, we aimed to tackle the issue from a novel perspective by developing cell derived, tissue engineered biomaterials that can be used in combination with other therapeutic approaches to improve the efficacy of current treatments. In this dissertation, a simple method to create either cellularized or acellular ECM biomaterial constructs is described. In particular, by utilizing patterned surface ligands, organized orientation can be introduced to the entire astrocyte derived construct morphologically and with regard to its associated matrix proteins, which mimics the native astrocyte framework within the spinal cord fiber tracts and provides these constructs the ability to guide axonal regeneration in vitro. In addition, meningeal fibroblast based biomaterial constructs are also developed taking advantage of the same engineering approach. It has been demonstrated that repairing damaged dura mater with allografts also benefits the regeneration process of the damaged spinal cord. In particular, acellular meningeal ECM constructs preserve a similar matrix protein profile as the native rat dura mater and support allogeneic meningeal cell adhesion and promote proliferation. The results suggest these engineered biomaterial constructs derived particularly from cells residing within tissue targeted for repair may carry appropriate tissue specific biological cues and hold therapeutic potentials for spinal cord injury repair as well as dual defect reconstruction