The goal of this application is to develop an in vitro test bed capable of mimicking native and pathological extracellular matrix (ECM) to identify novel targets for treatment of traumatic neural injury. We will accomplish this goal through rational design of an ECM-based scaffold to mimic native temporal ECM damage observed after spinal cord injury (SCI). Chondroitin sulfate proteoglycans (CSPGs) are the largest component of the healthy and pathologic ECM in the central nervous system (CNS) and serve many functions. After SCI specifically, there is an increase in versican, neurocan, and brevican, and a decrease in aggrecan. There are also dynamic changes to specific proteases such at MMP-2 and -9 after SCI that will selectively cleave CSPGs. Furthermore, CSPG fragmentation has been implicated in progression of other diseases (e.g. osteoarthritis). We believe CSPG fragmentation plays a similar role in increasing unwanted glial scarring after SCI or traumatic neural injury. Currently, animal models are used to screen feasibility of biomaterials for tissue engineering. However, to examine injury and disease states an induced state must be created in the animal model that often does not represent native pathology of injury or disease. Creation of in vitro pathological ECM test beds has the potential to provide a lower cost, more relevant system for examining mechanistic responses and testing small molecule therapeutics and identifying relevant targets to treat patients with SCI. We hypothesize that neoepitopes exposed after specific proteoglycan fragmentation exacerbate progression of glial scarring after spinal cord injury, and an in vitro pathological ECM test bed is a novel platform to delineate the influence of these fragmentation profiles on glial cell phenotype.
In Aim 1 we will identify the type and degree of aggrecan fragmentation at distinct time points after spinal cord injury (SCI) using tissue from previously executed studies in a rat model.
In Aim 2 we will create 3D ECM from hyaluronan (hyaluronic acid, HA), HA-link protein 1, tenascin-R, and aggrecan (intact and fragmented) to mimic the ECM of the CNS after SCI. The final task will be to assess the temporal response of glial cells to these engineered gels by analyzing changes in their phenotype and function to assess their progression towards a reactive state. This research will enable the creation of in vitro test beds capable of isolating the role of fragmentation in the CNS after injury to help identify novel targes for clinical therapeutics. Furthermore, it will enhance our understanding of the interplay between CSPG fragmentation and pathological cell behavior after SCI. In conjunction with body-on-a-chip approaches, these biomaterials platforms hold the potential to revolutionize current screening techniques and ultimately eliminate animal screening completely. Application of these test beds could be broadened for use in diseases of the CNS and other tissues, where fragmentation of CSPGs has also been identified as a key player in disease progression (e.g., epilepsy, Parkinson's) to help isolate targets and identify novel therapies.
A quarter million people in the United States suffer from permanent deficits resulting from spinal cord injury, yet there are no treatment options to promote recovery. Current model systems used to identify new targets or evaluate new interventions have poor reproducibility and are costly to implement. Our work seeks to recreate the healthy and injured spinal cord environment in a bench-top test bed that will enhance our understanding of the interplay between the matrix and pathological cell behavior after traumatic neural injury and will enable screening of new therapeutic targets and strategies for treating patients in a cost-effective and reproducible manner.
