Development of a Decellularized Hydrogel Composite and its Application in a Novel Model of Disc-associated Low Back Pain in Female Sprague Dawley Rats

Chronic low back pain is a global socioeconomic crisis compounded by an absence of reliable, curative treatments. The predominant pathology associated with chronic low back pain is degeneration of intervertebral discs in the lumbar spine. During degeneration, nerves can sprout into the intervertebral disc tissue and be chronically subjected to inflammatory and mechanical stimuli, resulting in pain. Pain arising from the intervertebral disc, or disc-associated pain, is a complex, multi-faceted disorder which necessitates valid animal models to screen therapeutics and study pathomechanisms of pain. While many research teams have created animal models of disc degeneration, the translation of these platforms to disc-associated pain models has been limited by an absence of chronic pain-like behavior. Further, the few models which measure disc-associated pain-like phenotypes have been established in mice, which are not amenable to surgical treatment procedures due to their small size. This deficiency drives the need for a new model of disc-associated pain where pain-like behavior is measurable and intervertebral discs are large enough for surgical procedures. These criteria promote rats as the optimal platform for a disc-associated model of chronic low back pain. Herein, a rat model of disc-associated pain is described that displays chronic pain-like behavior, overt disc degeneration, and nerve sprouting in the intervertebral disc. In addition to the model, a novel method for measuring disc degeneration real-time, non-invasively, is delineated which exhibits remarkable precision and accuracy. Finally, a next generation treatment, derived from decellularized, porcine nucleus pulposus tissue is described which is injectable, thermally fibrillogenic, and cytocompatible. In the rat model of disc-associated pain, this biomaterial restores degenerated disc volume and dramatically decreases pain-like behavior. In summary, this dissertation describes the development of a method for quantifying degeneration real-time, establishes a rat model of disc-associated pain, and successfully treats disc-associated pain in this model with a next-generation biomaterial. Advisor: Rebecca Wach

Equine cervical pain and dysfunction

2021 Fall.Includes bibliographical references.Cervical pain and dysfunction in horses has become more recognized in recent years. However, a horse may present with a long list of different clinical syndromes and the examination findings can be confusing, resulting in difficulty effectively treating the horse. This frequently leads to frustration by the owner, as well as the veterinarian charged with helping the horse. This body of work aims to enlighten the reader of the dearth of understanding of cervical pain and dysfunction, to highlight how dangerous behavior may be related to cervical pain, and describe the course and development of future research. There is a paucity of peer-reviewed equine literature available describing cervical pain and dysfunction in the horse. The first chapter is designed to provide a synopsis of the current state of understanding of the disease processes, diagnostic capabilities, and possible treatment strategies available to manage cervical pain and dysfunction in horses. The second chapter describes a series of horses displaying unwanted behavior that became dangerous to the rider and often times to the horse itself. The included horses all had moderate to severe ganglionitis at multiple vertebral levels. Ganglionitis has been associated with neuropathic pain in other species, and is believed to be causing a state of neuropathic pain in this series of horses. This study highlights the need for deeper understanding of pain behavior in horses. Chapter 3 describes a prospective evaluation of cervical pain and dysfunction in 12 horses. Recombinant equine interleukin-1β (reIL-1β) has been used as an acute synovitis model within the appendicular skeleton and was utilized in this study to create transient synovitis at the cervical articulation of C5-C6. This study evaluated the clinical, biomechanical and ultrasonographic features in horses with a known source of neck pain. Acute synovitis of the articular process joint (APJ) induced clinical signs of myofascial pain and neck stiffness with variable degrees of forelimb lameness. Ultrasonographic evidence of the presence and severity of APJ effusion could be readily identified and tracked over time. Utilizing this model in the future could further add to our understanding of the clinical presentations in horses experiencing cervical pain and dysfunction. Through this collection of work, we have developed collaborations to investigate many unanswered questions that have been raised. We will look to define pathways related to neuropathic pain mechanisms in order to ultimately improve the quality of life, not only for our equine patients, but potentially of other veterinary species and even the human population experiencing chronic pain

Phase II Double-blinded Randomized Controlled Clinical Trial of Chondroitinase ABC by Intraspinal Injection for Treatment of Severe Chronic Spinal Cord Injury in 60 Pet Dogs

Traumatic spinal cord injury is a devastating neurologic condition in both veterinary and human medicine and despite research yielding numerous potential interventions with remarkable efficacy demonstrated in rodent models, none has advanced to successful clinical translation. Pet dogs’ predilection for sustaining spinal cord injury, typically due to intervertebral disc herniation or vertebral column fracture, makes them a suitable clinical model in which putative interventions for spinal cord injury can be tested. In recent years, there has been a growing body of experimental evidence that attests to the efficacy of chondroitinase ABC in promoting axonal regeneration and functional recovery after spinal cord injury by reactivating neuroplasticity. Chondroitinase ABC is a commercialized bacterial enzyme that selectively deglycosylates chondroitin sulfate proteoglycans and thereby disrupts the perineuronal nets that limits axonal regeneration. In this 60-dog clinical trial, we examined the therapeutic effect of chondroitinase ABC on the primary outcome measure, pelvic-thoracic limb gait coordination, and several secondary parameters, including motor- and sensory-evoked potentials and urinary bladder compliance. While our study failed to detect a therapeutic effect in chronic, severe thoracolumbar spinal-injured dogs, it has established drug safety in a clinical large animal model. Thus, this study has provided a platform for future investigations in which the dose, route and timing of chondroitinase ABC administration, as well as patient selection, can be adjusted to maximize its potential therapeutic effect and benefit spinal-injured human and veterinary patients, especially if a more treatment-responsive subgroup could be identified

Intervertebral disc degeneration : symptomatic and asymptomatic distinguishers

Introduction: It is currently not possible to distinguish symptomatic (S) from asymptomatic (A) intervertebral disc degeneration (IVDD) based on patient factors or the degree of degeneration. Additionally, it is not known if age, sex, and BMI contribute to the gross/radiographic or histological progression of IVDD. These studies were performed to determine patient, gross/radiographic, histological, and protein differentiators of S-IVDD or A-IVDD. Methods: IVD tissues were recovered from 202 S clinical patients and 36 A tissue donors with IRB Approval (#2010692), informed patient consent, or a legal permit under the Uniform Anatomical Gift Act as appropriate. Age, sex, BMI, Pfirrmann grade, Thompson grade, and histological degeneration scores (HDS) were determined. Each isolated tissue was cultured for 6 days in supplemented DMEM which was collected every 3 days, and tissue culture media were assessed for their content of inflammatory cytokines/chemokines, degradative enzymes (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and growth factors. Gross/radiographic grades, histological scores, and ex vivo biomarker data were compared within and between S-IVD and A-IVDs using multivariable generalized linear models or ANOVAs while adjusting for age, sex, and BMI as covariates with significance set at p<0.05. Results: Overall, S-IVDs produced significantly increased levels of inflammatory cytokines/chemokines, specific degradative enzymes, and lower levels of TIMPs compared to A-IVDs regardless of IVDD severity. Obesity significantly affected the progression of IVDD in S and A populations. Discussion: Obesity and inflammatory cytokines may represent key factors in distinguishing S-IVDD versus A-IVDD towards earlier identification and symptom mitigation in clinical patients.Includes bibliographical references

Development of a Biomimetic, Collagen-Based Scaffold for the Repair and Regeneration of the Annulus Fibrosus

Annually, over 5.7 million Americans are diagnosed with two IVD-associated pathologies: IVD herniation (IVDH- a mechanical disruption of the concentric fibrous layers of the annulus fibrosus (AF)) and/or degeneration (IVDD- a multifactorial process which initiates within the inner gelatinous core (NP), and results in a biochemical degradation of NP tissue), with over 2.7 million requiring surgical interventions. Although both underlying pathologies are different, quite often they both lead to a decrease in IVD height, impaired mechanical function, and increased pain and disability. These pain symptoms affect approximately 80% of the adult population during their lifetime with estimated expenditures exceeding $85.9 billion. Current surgical procedures for IVDH and IVDD are palliative and suffer from drawbacks. While they are performed to address patient symptoms, they fail to address the underlying pathology of a focal defect remaining within the subsequent outer layers of the AF. It is hypothesized that an effective AF closure/repair device in conjunction with a less aggressive discectomy for IVDH and/or NP arthroplasty for IVDD may result in improved patient outcomes, decreased pain, and provide fewer revision surgeries via lower re-herniation and expulsion rates. Therefore, an intact AF must be re-established to prevent implant expulsion or re-herniation, thus addressing the two major spinal pathologies directly associated with an IVD. Currently, within the medical device market, no tissue engineering biomaterials are available for AF closure/repair. Current market AF closure devices (Intrinsic Barricaid®, Anulex X-Close® Tissue Repair System, and Anulex Inclose® Surgical Mesh System) are synthetic materials focused solely on preserving and reinforcing the native tissue and lack effective strategies for implantation, fixation, and regeneration. Therefore, there has been an increase in tissue engineering and regenerative therapeutic approaches aiming for structural and biological AF repair investigated over the last decade using in vitro and in vivo experimentation. It is proposed that the optimum AF tissue engineering scaffold should reproduce the native AF microarchitecture and native mechanical properties. Recent articles illustrate several novel sutures, sealants, and barrier techniques currently under development, resulting in an increasing attention at scientific workshops and conferences. To develop a tissue engineering biomaterial that is suitable for AF closure we propose it must first meet the following criteria: (1) mimic the structural angle-ply architecture of the native AF, (2) fundamentally demonstrate mechanical properties mimicking the native functional characteristics, and (3) demonstrate cytocompatibility while promoting tissue regeneration. Current biomaterials gaining attention in the tissue engineering academic field, electrospinning, polymers, glue, silk scaffolds, and honeycomb-scaffolds, require complex manufacturing procedures and typically work to address two of the three criteria (mimicking the biological or structural characteristics). Therefore, the use of a decellularized tissue from a xenogeneic source may be ideal due to its advantage of maintaining native extracellular matrix (ECM) while also removing all potential harmful xenogeneic factors. Although, the mechanical advantage of closing annular focal defects to retain NP material seems intuitive, only recently have AF closure devices begun to examined in human cadaveric or animal tissues for their ability to withstand in situ IDP or flexibility testing. We propose to address all three criteria with the development of a biomimetic, collagen-based angle-ply annulus fibrosus repair patch (AFRP) comprised of the decellularized porcine pericardium. The porcine pericardium was chosen due to its innate type I collagen content, mechanical strength, and cytocompatibility. The objectives of this research were to investigate the development of this biomimetic AFRP to biologically augment AF repair by (1) mimicking and characterizing the micro-architecture of the multi-laminate angle-ply AFRP, (2) mechanically evaluating the AFRP’s mechanical properties and attachment strength in situ, (3) evaluating the ability of the AFRPs to support AF tissue regeneration in the context of a healthy and inflammatory environment, and (4) evaluating the in vivo mechanical strength, biocompatibility, and tissue regeneration capacity of the AFRP in a large animal model for intervertebral disc degeneration/herniation

Identification of compounds that cause axonal dieback without cytotoxicity in dorsal root ganglia explants and intervertebral disc cells with potential to treat pain via denervation

Low back pain, knee osteoarthritis, and cancer patients suffer from chronic pain. Aberrant nerve growth into intervertebral disc, knee, and tumors, are common pathologies that lead to these chronic pain conditions. Axonal dieback induced by capsaicin (Caps) denervation has been FDA-approved to treat painful neuropathies and knee osteoarthritis but with shortterm efficacy and discomfort. Herein, we propose to evaluate pyridoxine (Pyr), vincristine sulfate (Vcr) and ionomycin (Imy) as axonal dieback compounds for denervation with potential to alleviate pain. Previous literature suggests Pyr, Vcr, and Imy can cause undesired axonal degeneration, but no previous work has evaluated axonal dieback and cytotoxicity on adult rat dorsal root ganglia (DRG) explants. Thus, we performed axonal dieback screening using adult rat DRG explants in vitro with Caps as a positive control and assessed cytotoxicity. Imy inhibited axonal outgrowth and slowed axonal dieback, while Pyr and Vcr at high concentrations produced significant reduction in axon length and robust axonal dieback within three days. DRGs treated with Caps, Vcr, or Imy had increased DRG cytotoxicity compared to matched controls, but overall cytotoxicity was minimal and at least 88% lower compared to lysed DRGs. Pyr did not lead to any DRG cytotoxicity. Further, neither Pyr nor Vcr triggered intervertebral disc cell death or affected cellular metabolic activity after three days of incubation in vitro. Overall, our findings suggest Pyr and Vcr are not toxic to DRGs and intervertebral disc cells, and there is potential for repurposing these compounds for axonal dieback compounds to cause local denervation and alleviate pain