Investigating the Role of Aggrecan in Intervertebral Disc Degeneration

Low back pain affects nearly 85% of the population of the United States, and is associated with a huge socioeconomic burden. One of the leading causes of low back pain is Intervertebral Disc (IVD) degeneration, which results in an ingrowth of blood vessels and neurites, as well as a sensitization of those neurites. Previous research has indicated that both Aggrecan plays a role in regulating angiogenesis in the IVD, and Aggrecan has also been shown to inhibit neurogenesis and sensitize neurites. However, it is unclear which components of Aggrecan control its inhibitory effects. The goal of this study was to determine how intact and degraded Chondroitin Sulfate groups, the dominant side-chains of Aggrecan, affect angiogenesis in vitro, as well as the sensitization of neurites. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured with either intact or degraded Chondroitin Sulfate-A (CSA), Chondroitin Sulfate-B (CSB), or Chondroitin Sulfate-C (CSC) at a concentration of either 10 µg/ml or 100µg/ml for 16 hours during a tubular formation assay. The tubular assay was quantified for total tubular length using the Angiogenesis Analyzer plugin for Image J. The assay showed that no inhibition occurred for any of the groups, and that pro-angiogenic effects were observed for the 100µg/ml concentration of degraded CSC. The protocol developed to investigate the effect of Aggrecan on neurite sensitization involved optimizing the methodology for extracting Dorsal Root Ganglia (DRGs) cells from mice for use as a primary cell model. The protocol developed is based on literature, but optimizes for simplicity, time, and reduction in the cost of surgical instruments. This protocol achieves these goals, and attains high viability for DRG cell clusters, but fails to consistently retrieve viable cells from the intact DRGs This study sought to investigate the role chondroitin sulfate side chains play in angiogenesis, and it found evidence that the degraded factors can have pro-angiogenic effects. The study also sought to develop a protocol that could be used to aggrecan in the role of sensitization, and it improved the speed and simplicity of existing procedures.No embargoAcademic Major: Biomedical Engineerin

Towards the semi-synthesis of phosphorylated mimics of glycosaminoglycans: Screening of methods for the regioselective phosphorylation of chondroitin

Glycosaminoglycan (GAG) mimics carrying phosphate rather than sulfate anionic groups have been poorly investigated, in spite of their interesting perspectives. While some GAG-mimicking phosphorylated polymers have been reported, to the best of our knowledge no phosphorylated polysaccharides having the same backbone of natural sulfated GAGs have been accessed yet. To fill this gap, in this work two standard phosphorylation protocols and two recently reported procedures have been screened on a set of polysaccharide species composed by microbial sourced chondroitin and three partially protected, semi-synthetic derivatives thereof. A detailed structural characterization by 1H, 13C and 31P NMR spectroscopy revealed the higher versatility of the innovative, biomimetic reaction employing monopotassium salt of phosphoenolpyruvate (PEP–K) with respect to standard phosphorylating agents (phosphoric acid or phosphorus oxychloride). Indeed, PEP-K and H3PO4 gave similar results in the regioselective phosphorylation of the primary hydroxyls of unprotected chondroitin, while only the former reacted on partially protected chondroitin derivatives in a controlled, regioselective fashion, affording chondroitin phosphate (CP) polysaccharides with different derivatization patterns. The reported results represent the first, key steps towards the systematic semi-synthesis of phosphorylated GAGs as a new class of GAG mimics and to the evaluation of their biological activities in comparison with native sulfated GAGs

Regulating Neuronal Growth with Structurally Defined Glycans

Chondroitin sulfate proteoglycans (CSPGs) and keratan sulfate proteoglycans (KSPGs) play an important role in neural development. Aggrecan, a CSPG, operates in the neural extracellular matrix where it negatively regulates neurite outgrowth to prevent aberrant process formation. Unfortunately, this aggrecan or CSPG-rich/KSPG-rich barrier can also prevent neuronal regeneration, which contributes to the inability to repair brain and spinal cord injuries. Removal of CSPGs and KSPGs has been shown to increase neurite outgrowth. We extend these findings by testing the ability of structurally-defined glycans to outcompete aggrecan and allow neurite outgrowth. Our overall goal is to determine if there is a particular glycan structure which can overcome inhibitory CSPGs and KSPGs without inhibiting neurite outgrowth themselves. We utilized primary cultures of rat neurons and applied polydisperse-related mixtures of CSPGs, KSPGs and newly-isolated, novel glycans in the absence and presence of aggrecan. Several of these glycans successfully removed the aggrecan-induced blockade while not inhibiting neurite outgrowth on their own. We are currently investigating the efficacy of these compounds in concentration-response curves and are testing additional glycans with different molecular weights and varying sulfation patterns to determine the structural requirements for the glycans as modulators of neurite outgrowth. By investigating the impacts of these glycans, we will increase the understanding of proteoglycan regulation of neural structure and function, and potentially identify compounds which could be used to treat spinal cord injuries

Strategies for Annulus Fibrosus Regeneration: From Biological Therapies to Tissue Engineering

Intervertebral disc (IVD) is an avascular tissue which contributes to the weight bearing, motion, and flexibility of spine. However, IVD is susceptible to damage and even failure due to injury, pathology, and aging. Annulus fibrosus (AF), the structural and functional integrity of which is critically essential to confine nucleus pulpous (NP) and maintain physiological intradiscal pressure under mechanical loading, plays a critical role in the biomechanical properties of IVD. AF degeneration commonly results in substantial deterioration of IVD. During this process, the biomechanical properties of AF and the balance between anabolism and catabolism in IVD are progressively disrupted, leading to chronic back pain, and even disability of individuals. Therefore, repairing and regenerating AF are effective treatments to degeneration-associated pains. However, they remain highly challenging due to the complexity of natural AF tissue in the aspects of cell phenotype, biochemical composition, microstructure, and mechanical properties. Tissue engineering (TE), by combining biological science and materials engineering, shed lights on AF regeneration. In this article, we review recent advances in the pro-anabolic approaches in the form of cell delivery, bioactive factors delivery, gene therapy, and TE strategies for achieving AF regeneration