Guidance Of Regenerating Peripheral Axon Subtypes Using Molecular Cues

Author : Alan John Man
Publisher :
ISBN 13 : 9781321019407
Total Pages : pages
Book Rating : 4.0/5 (194 download)

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Download or read book Controlled Release of Growth Factors and Axon Guidance Molecules for Peripheral Nerve Regeneration written by Alan John Man and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Therapies for peripheral nerve injury have not significantly changed in the past three decades. The implantation of autografts and coaptation of nerve stumps are still the gold standard treatments but have many disadvantages. To overcome their limitations, nerve guide conduits (NGCs) have been developed to successfully treat short nerve gaps, yet in large nerve gaps, NGCs remain ineffective. Peripheral nerve injury is characterized by the upregulation of a cascade of physical and chemical signals that mediate recovery. To provide a cell substrate for the regenerating axon, a fibrin matrix forms to bridge the nerve stumps. The upregulation of growth factors assists in the recruitment and proliferation of supporting cells. The upregulation of axon guidance molecules prevents aberrant growth into the extraperineural space and guides axons toward their target tissue. In order to improve upon existing empty NGCs, the microenvironment within the conduit lumen must contain signals that mimic the natural response to injury. The objective of this work was to investigate conduit components that can be engineered for a nerve tissue engineering application. By examining the interplay between biomaterial design and cells, new insight may be gained in understanding the factors that influence nerve regeneration. Fibrin gel stiffness was found to regulate neurite outgrowth. By modulating fibrinogen and NaCl concentrations within the hydrogels, physical properties such as porosity and the number and thickness of fiber strands were altered. Biophysical changes contributed to mechanical properties. With dorsal root ganglia (DRG) explants, neurite outgrowth correlated with substrate stiffness. As fibrinogen and NaCl concentrations were increased, making the gel stiffer, neurite outgrowth decreased. It was determined that neurite extension within fibrin gels was dependent on fibrinolysis and was mediated by serine proteases and matrix metalloproteinases, and the gene expression of these degradative enzymes was a function of gel stiffness. By altering the concentration of components of fibrin, the mechanical and physical properties can be tailored to suit different applications. To examine how the delivery of a known angiogenic factor, vascular endothelial growth factor (VEGF), affected different cell types found in the peripheral nerve, bone marrow-derived stromal cells/mesenchymal stem cells (MSCs) were engineered to overexpress VEGF and co-cultured with various cell types. Naive MSCs produce sufficient factors other than VEGF that stimulate neurite outgrowth or cell proliferation when examined in co-culture with DRG explants, Schwann cells (SCs), or endothelial cells. The primary role of VEGF-overexpressing cells was to increase the proliferation of endothelial cells, as confirmed by blocking the VEGF receptor. When embedded within fibrin gels, VEGF-overexpressing cells maintained overexpression long-term in vitro and 2 weeks post-transplantation in a murine peripheral nerve injury model. The study demonstrates the potential of VEGF secreting MSCs as a nerve therapy. Misdirectional growth is a common problem after nerve injury that results in improper reinnervation of target organs. The expression of axon guidance molecules after injury serves to reduce this aberrant axon sprouting. With the goal of directing neurite extension, poly-L-lactic acid (PLLA) membranes underwent surface modification to bind a family of axon guidance molecules, chondroitin sulfate proteoglycans (CSPGs). Surface-treated membranes directed the angular growth distribution of DRG explant neurites away from CSPG coatings. Treated-membranes did not inhibit neurite initiation or extension, while there were a smaller number of and shorter neurites when cultured on untreated membranes. Controlling neurite growth orientation with bound guidance cues has implications in preventing misdirectional growth of regenerating axons. Taken together, these data provide a novel method for improving existing conduits for treating peripheral nerve regeneration. The reductionalist approach allows for the study of the individual components within NGCs and how each affects axonal growth. By implementing the three components investigated, the natural microenvironment after injury could be mimicked. This thesis provides a basis for creating a tissue engineered NGC that contains a scaffold, supporting cells that secrete growth factors and axon guidance signals, with the goal of improving peripheral nerve regeneration.