Ravi V. Bellamkonda, Ph.D. - US grants

DESCRIPTION (provided by applicant): Severe traumatic injuries and invasive surgical procedures such as tumor resection often create peripheral nerve gaps, accounting for 200,000 injuries in the US annually. The clinical "gold standard" for bridging peripheral nerve gaps is autografts (typically using sensory sural nerve), where 40~50% of patients regain useful function. However, the use and effectiveness of autografts is limited by several issues, including their limited availability, collateral damage at the donor site, and the presence of inhibitory chondroitin sulfate proteoglycans within the grafts. Therefore, it is critical to develop alternative approaches that match or exceed the performance of autografts. However, the leading bioengineering strategy of using nerve guidance channels has limited efficacy and has not been successful in bridging gaps longer than 10mm in rat models. In the previous grant period, we proposed to design 3D hydrogel based scaffolds with either isotropically or anisotropically distributed ECM (laminin-1) or nerve growth factor (NGF) to bridge gaps longer than 15mm in rats. We successfully designed and fabricated isotropic and anisotropic 3D hydrogels, demonstrated that indeed growth cones extend processes significantly faster in immobilized ECM gradients in vitro and that anisotropic scaffolds with gradients of laminin-1 and NGF are able to bridge 17-20mm gaps, whereas isotropic scaffolds with uniformly distributed LN-1 and NGF do NOT. Though, unfortunately, anisotropic hydrogel scaffolds only meet success 44% of the time in bridging 17-20mm gaps in rats. Therefore, we have developed a new approach to anisotropic scaffolds - using aligned electrospun polymeric nanofibers (diameter 200- 600nm). In our preliminary data, we demonstrate that this novel oriented nanofiber-based 3D scaffold enhances peripheral nerve regeneration across long nerve gaps (17mm rat model) and matches the performance of autografts by anatomical and histological measures. A critical finding of this study was that oriented nanofibers enabled efficient Schwann cell migration into the scaffolds, which was a precursor to realizing the endogenous regenerative potential of severed peripheral nerves. The central hypothesis of this proposal is that functionalizing the polymeric nanofiber based scaffolds with factors that enhance Schwann cell migration and tropic/trophic functions will enable nanofiber based scaffolds to exceed the performance of autografts. We propose testing this hypothesis in a challenging 17mm nerve gap in rodents. In this next generation design, the oriented scaffolds will be biodegradable and `functionalized'to include biochemical cues, such as growth stimulatory extracellular matrix (laminin-1) and trophic protein (Neurotrophin 3, NT-3). We suggest that by concentrating the pro- regenerative cues (structural and biochemical) we can design engineered scaffolds that out-perform autografts in rigorous, clinically relevant animal models of peripheral nerve injury. When complete, this research will represent a significant step in the direction of providing alternatives to autografts for peripheral nerve repair. PUBLIC HEALTH RELEVANCE: 200,000 peripheral nerve injuries occur every year in the US alone. This research will advance our understanding of the mechanisms of peripheral nerve regeneration, and develops technologies that are likely to improve clinical outcomes after peripheral nerve injury.

[unreadable] DESCRIPTION (provided by applicant): Regenerative failure in the central nervous system (CNS) is a significant clinical problem. Regenerative failure is thought to occur due to the formation of astroglial scar at the site of injury , which in turn blocks regenerating or sprouting neurons. The molecular composition of glial scar, particularly the presence of chondroitin sulfate proteoglycans (CSPGs), is responsible for inhibition of growth cones and regenerative failure. However, the mechanism by which CSPGs inhibit growth cones is not known. Recent successes in various in vivo CNS injury models in eliciting regeneration by digesting the glycosaminoglycan (GAG) portion of CSPGs using chondroitinase ABC suggests that the GAGs present on CSPGs may contribute significantly to CSPG-mediated inhibition. [unreadable] [unreadable] Our central hypotheses are: i) the CS-GAG component of CSPGs contribute significantly to the inhibition of peripheral (PNS) and CNS growth cones; and that ii) this inhibition is mediated by signaling mechanisms involving an increase in intracellular calcium and a decrease in cAMP levels. The purpose of Aim 1 of this proposal is to characterize the upregulation of CS-GAGs in response to injury of the adult mammalian CNS using a very sensitive and novel technique (FACE). Additionally, Aim l introduces innovative techniques to present the upregulated CS-GAGs to growth cones in a controlled fashion to investigate the direct inhibition of growth cones by CS-GAGs. The purpose of Aim 2 of this proposal is to investigate the signaling mechanism(s) by which CS-GAGs cause growth cone inhibition, focusing specifically on growth cone concentrations of calcium and cAMP and how their levels change when growth cones contact CS-GAGs. [unreadable] [unreadable] To achieve our goals, we use novel and innovative methods to achieve high spatial control over the immobilization of CSGAGs onto glass cover slips using a micro-fluidics approach. We also use CS-GAGs coupled to Dynal beads to present CSGAGs to growth cones and study the growth cone dynamics in response to contact with inhibitory CS-GAGs. Our methods assume significance because we show that immobilized CS-GAGs are inhibitory whereas soluble GAGs are not, and it is imperative to control the manner in which CS-GAGs are presented to growth cones. This is not surprising given that in vivo, at the site of injury, growth cones encounter CS-GAGs immobilized in the scar matrix. When successfully completed, our research will clarify the extent and mechanism(s) of CS-GAG mediated inhibition of growth cones, and the potential contribution that the GAG components of CSPGs make to CNS inhibition at astroglial scars. With such elucidation, strategies will be developed to alleviate CS-GAG mediated inhibition, assisting growth cones to grow through CS-GAG/CSPG rich areas, facilitating regeneration. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Silicon microelectrode array technology holds considerable promise in advancing the goal of developing stable, electrode-brain interfaces. Chronic unit recordings from multiple neurons in the brain would significantly enhance our understanding of normal physiology and provide a valuable control signal for use in neuro-prosthetic devices. However, the current generation of silicon microelectrodes does not allow stable long-term recordings. The precise mechanisms that cause failure of silicon microelectrode mediated recordings are not known. We hypothesize that the million-fold stiffness mismatch between silicon and neural tissues generates shearing forces at the interface resulting in an astro-glial scar formation that progressively excludes neurons from the vicinity of the recording electrodes. To test our hypothesis, we propose novel and innovative methods to a) determine the strain-sensitivity of primary astrocytes in terms of their adopting a scarring phenotype; b) determine if strain-induced scar formation around Si-microelectrodes degrades their recording capabilities in organotypic hippocampal slice cultures; and c) design coatings for Si-microelectrodes that allow the sustained local release of anti-inflammatory agents to decrease scarring and increase recording stability. The above aims represent a highly inter-disciplinary investigation of an important problem in the design and development of stable neuro-prosthetic devices. Successful completion of our research goals is likely to impact the mechanical and biochemical aspects of the design of the next generation of silicon microelectrode arrays, and subsequently significantly impact the quality of life of persons with disabilities.

DESCRIPTION (provided by applicant): The ability to successfully interface the brain to external electronics would have enormous implications for our society, and facilitate a revolutionary change in the quality of life of persons with sensory and/or motor deficits. Microelectrode technology represents the initial step towards this goal and has already improved the quality of life of many patients, as is evident from the success of auditory prostheses. Much effort has been invested in the development of miniaturized silicon (Si) microelectrodes, which allows one to analyze how individual neurons function by potentially recording single-units from them. However, when these devices are implanted into brain tissue for long-term recording, they quickly (days) lose electrical connectivity. We propose two-component coatings to improve the recording stability of Si-electrode arrays in the brain-component 1 is a neuro-adhesive layer on Si; and component B is the incorporation of sustained release vehicles within component 1. This strategy enables us to promote the integration of silicon electrodes with the brain with coatings that will enhance neuronal cell attachment (therefore increasing the proximity of neurons to the recording contact) and enhance their survival and at the same time decrease any inflammatory response due to implantation procedure or Si chemistry (by sustained, local release of neurotrophic factor BDNF and anti-inflammatory anti-sense ODNs directed against NFkB mRNA). In addition, we propose to use two novel, non-invasive optical imaging techniques to assess the success of our coatings in live animals to complement conventional immuno-histochemistry and histology. The conventional techniques require one to physically remove the electrode arrays before being analyzed (because Si is a very hard substrate and difficult to section histologically), and the novel optical imaging and histology techniques we propose will be a significant advance in studying the electrode-brain interface non-invasively to enable optimization of coating composition. Studies are proposed to thoroughly characterize our coatings in vitro (physical properties and for biocompatibility); implant a number of coating compositions in vivo in a rodent model to optimize for coatings with the least inflammatory response; and finally implant the best coating into primate cortex and compare recording stability to that obtained in our earlier experiments with the similar uncoated electrode arrays.

[unreadable] DESCRIPTION (provided by applicant): The field of Biomaterials significantly impacts the quality of life of thousands of people. Biomaterials have traditionally been used in medical devices such as hip implants, vascular grafts, and stents with new evolving applications in scaffolds/drug delivery devices for tissue engineering and regenerative medicine. However, significant challenges remain with the ability of Biomaterials to integrate seamlessly with the body, to respond and remodel with time in vivo. The full potential of Biomaterials has not yet been realized, in part, because most materials that are used today are by-products of other processes, not rationally designed with the specific medical requirements in mind. Biomedical engineering as a discipline offers a unique opportunity to address this shortcoming. Georgia Institute of Technology has had an excellent, interdisciplinary graduate program in Bioengineering for 15 years with a high quality of pre-doctoral students. The goal of GTBioMAT (Graduate Training for Rationally Designed, Integrative Biomaterials) is to leverage the existing inter-departmental and inter-university ties and provide a structured, focused, integrative training for the rational design and application of the next generation of biomimetic, integrative materials. The GTBioMAT training program will focus on integrating four important skill sets that are critical to training the future leaders in Biomaterials science and engineering: 1) the ability to elucidate Biomaterials design criteria through strong clinical interactions; 2) the ability to synthesize and characterize new materials whose design is driven by an understanding of the underlying clinical and basic science issues; 3) the ability to functionalize and apply these materials such that they integrate appropriately into living systems; and 4) develop leadership skills in the trainees such that they lead the next generation of Biomaterials Science and Engineering research through their innovation and research. The GTBioMAT training program proposes to introduce novel problem based learning approaches that promote self-directed inquiry and collaborative problem solving of complex but authentic biomaterials related problems. It leverages the strong research strength of the faculty at Georgia Institute of Technology and Emory University and will significantly impact the training of the future leaders in Biomaterials. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Severe traumatic injuries and surgical procedures like tumor resection often create peripheral nerve gaps, accounting for over 250,000 injuries in the US annually. The clinical "gold standard" for repair is autografts, with which 40~50% of patients regain useful function. The effectiveness and use of autografts is limited by issues including limited availability and collateral damage at the donor site. So, it is critical to develop alternative bioengineered approaches that match or exceed autograft performance. We recently reported a breakthrough in bridging critically sized nerve gas (=15mm) in rats using a thin film-based intra-luminal scaffold carried in a standard nerve guidance channel. This finding gives rise to two important questions. (1) How does intra-luminal presentation of minimal thin film-based cues (occupying just 0.3% of intra-luminal volume) have a dramatic effect on regeneration? (2) If we understood the mechanism underlying this effect, could we influence the process, and further enhance regeneration to match or exceed autograft performance? An understanding of the mechanistic interplay between polymer fiber-based topography (what may be termed the endogenous 'regenerative processes/sequence') is necessary for the rational design of intra-luminal scaffolds. This process spontaneously occurs during the successful bridging of short gaps (<10mm in rats), but fails to occur in the bridging of longer gaps (=15mm in rats). It involves a fibrin cable formation, extracellular matrix deposition/remodeling (e.g., fibronectin), glial/support cell (fibroblasts and Schwann cells) and axonal infiltration into the gap. Our central hypothesis is that the mechanism by which thin films with topographical cues enhance regeneration is by serving as physical 'organizing templates'for Schwann cell infiltration, Schwann cell orientation, extra-cellular matrix deposition/organization, and axon infiltration, which in turn leads to successful regeneration. Our specific aims are as follows: Aim 1: Investigate the interplay between polymer fiber-based thin film topographyand fibrin cable/ECM organization and glial cell migration during repair of critically sized nerve gaps in vivo. Aim 2: Determine the effect of local delivery of diffusible biochemical factors that influence the regenerative sequence to synergistically enhance the regeneration when combined with topographical cues. The innovation here is that we will investigate the previously under-explored interplay between early events of the regenerative/wound healing sequence and intra-luminal thin-film scaffolds that present topographical cues. In addition to this physical template that modulates the regenerative sequence, we further propose to give it a 'biochemical boost'with the sustained local delivery of neurotrophin-3 [Aim 2]. We therefore address a significant clinical problem through the rational design of minimalist, intra-luminal film-based scaffolds that should a) enhance our understanding of intra-luminal scaffold design and b) result in significantly better performance than previously attainable from nerve guidance channels in bridging critically sized nerve gaps. PUBLIC HEALTH RELEVANCE: Over 250,000-300,000 peripheral nerve injuries occur every year in the US alone. This research will advance our understanding of the mechanisms of peripheral nerve regeneration that is promoted by intra-luminal scaffolds, and will develop technologies that are likely to improve clinical outcomes after peripheral nerve injury.

DESCRIPTION (provided by applicant): Medulloblastomas are highly invasive primitive neuroectodermal tumors of the cerebellum and the most common childhood malignant brain tumor, constituting 20-40% of pediatric brain tumors. Treating invasive intracranial brain tumors in children represents a significant challenge that is complicated further due to confined space and the need to preserve as much non-tumor, normal tissue as possible to avoid long-term cognitive dysfunction. In such cases, surgery is complicated and chemotherapy is prone to major side effects because cytotoxic drugs cannot differentially kill invading tumor cells surrounded by normal cells. In this EUREKA application, we present a highly innovative and unorthodox solution to this problem. We exploit the invasive nature of pediatric medulloblastomas by engineering a path of least resistance that moves tumors from the cerebellum to a pre-determined sub-dural location where they are killed. In this context, we introduce the term exvasion to mean the opposite of invasion - the tumor cells migrate and proliferate in a direction away from the primary tumor site, instead of invading deeper into the brain, and are thus directed to migrate to a safer , pre-determined sub-dural region to be killed. Our approach exploits the tumor s invasive character to move it away from the primary site by offering a path of least resistance specifically engineered to compete with its natural migratory pathway. Medulloblastoma migration and invasion along the leptomeningial pathway is facilitated by two elements: a) topographical cues presented by leptomeningial white matter tracts, and b) collagen rich extracellular matrix expressed along the leptomeningeal tract. Our design criteria to engineer a system to excavate tumors incorporates both of these elements;we propose to use aligned nanofiber-based polymeric thin films to mimic the topographical cues, and we coat these 10 micron-thin films with collagen I to mimic the ECM cues of the leptomeningial pathway. In addition to moving tumors out we propose to direct them to an engineered apoptosis-inducing hydrogel that will be implanted in a relatively safe sub-dural location. By directing tumor cell migration and invasion to an external sink, we will deliver tumor cells to the drug, rather than the current strategy of delivering the drug to the tumor, which is problematic due to the irregular vasculature and poor diffusivity of the tumor tissue. We have assembled a highly qualified, inter-disciplinary team consisting of a bioengineer/tumor drug delivery expert, Prof. Bellamkonda (PI), the Director of the Pediatric Oncology program at Emory School of Medicine and Children s Healthcare of Atlanta (CHOA), Prof. Macdonald, and a practicing pediatric neurosurgeon at Emory/CHOA who treats children with Medulloblastoma in his clinical practice, Prof. Brahma. We suggest that the proposed research is highly innovative, has the potential to open a new avenue for the treatment of solid tumors located intracranially, and represents significantly unorthodox research with a reasonably high chance of success from a highly qualified team worthy of EUREKA support. PUBLIC HEALTH RELEVANCE: Medulloblastomas are highly invasive primitive neuroectodermal tumors (PNETs) of the cerebellum and the most common malignant brain tumor of childhood, constituting 20-40% of all pediatric brain tumors. Currently, there exist no effective therapies to safely manage or treat invasive medulloblastomas in children. This application aims to exploit the invasive nature of tumors to exvade tumors out of the brain;and, if successful, it will dramatically enhance therapeutic options for patients diagnosed with these aggressive tumors.

DESCRIPTION (provided by applicant): Approximately 250,000 Americans currently suffer from spinal cord injury or disease, which can cause paralysis and other deteriorations in quality of life. Our project's objective is to build a prosthetic device that has potential to help Spinal Cord Injury patients regain their ability to control their body's movements and, in particular, their ability to walk. Multiple labs have shown that it is possible to evoke walking patterns in the spinal cord directly, via electrodes that stimulate the surface of the spinal cord. Additionally, stimulating the cord electrically at certain cites have been shown to increase or decrease the speed of walking patterns. These studies have potential to be incorporated into a multiple-electrode prosthetic that can evoke and control walking output directly from the spinal cord, bypassing injury sites that block commands from the brain. Our proposed work uses a biocompatible, conformable array of electrodes that can activate walking output when stimulating electrically the surface of the mammalian spinal cord (we use the neonatal rat spinal cord as our model). Our proposed experiments are designed to identify optimal multi-site locations on the spinal cord surface that, when electrically stimulated at low amplitudes and frequencies, initiate and control spinal cord locomotor (i.e. walking) output. Identification of such sites and stimulation patterns is necessary to our overall objective of creating a multi-electrode, implantable prosthetic for restoration of walking capability in Spinal Cord Injury (SCI) patients.

DESCRIPTION (provided by applicant): Medulloblastomas are highly invasive primitive neuroectodermal tumors of the cerebellum and the most common childhood malignant brain tumor, constituting 20-40% of pediatric brain tumors. Treating invasive intracranial brain tumors in children represents a significant challenge that is complicated further due to confined space and the need to preserve as much non-tumor, normal tissue as possible to avoid long-term cognitive dysfunction. In such cases, surgery is complicated and chemotherapy is prone to major side effects because cytotoxic drugs cannot differentially kill invading tumor cells surrounded by normal cells. In this EUREKA application, we present a highly innovative and unorthodox solution to this problem. We exploit the invasive nature of pediatric medulloblastomas by engineering a path of least resistance that moves tumors from the cerebellum to a pre-determined sub-dural location where they are killed. In this context, we introduce the term exvasion to mean the opposite of invasion - the tumor cells migrate and proliferate in a direction away from the primary tumor site, instead of invading deeper into the brain, and are thus directed to migrate to a safer , pre-determined sub-dural region to be killed. Our approach exploits the tumor s invasive character to move it away from the primary site by offering a path of least resistance specifically engineered to compete with its natural migratory pathway. Medulloblastoma migration and invasion along the leptomeningial pathway is facilitated by two elements: a) topographical cues presented by leptomeningial white matter tracts, and b) collagen rich extracellular matrix expressed along the leptomeningeal tract. Our design criteria to engineer a system to excavate tumors incorporates both of these elements; we propose to use aligned nanofiber-based polymeric thin films to mimic the topographical cues, and we coat these 10 micron-thin films with collagen I to mimic the ECM cues of the leptomeningial pathway. In addition to moving tumors out we propose to direct them to an engineered apoptosis-inducing hydrogel that will be implanted in a relatively safe sub-dural location. By directing tumor cell migration and invasion to an external sink, we will deliver tumor cells to the drug, rather than the current strategy of delivering the drug to the tumor, which is problematic due to the irregular vasculature and poor diffusivity of the tumor tissue. We have assembled a highly qualified, inter-disciplinary team consisting of a bioengineer/tumor drug delivery expert, Prof. Bellamkonda (PI), the Director of the Pediatric Oncology program at Emory School of Medicine and Children s Healthcare of Atlanta (CHOA), Prof. Macdonald, and a practicing pediatric neurosurgeon at Emory/CHOA who treats children with Medulloblastoma in his clinical practice, Prof. Brahma. We suggest that the proposed research is highly innovative, has the potential to open a new avenue for the treatment of solid tumors located intracranially, and represents significantly unorthodox research with a reasonably high chance of success from a highly qualified team worthy of EUREKA support.

DESCRIPTION (provided by applicant): The center for disease control reports that 1.7 million Americans suffer traumatic brain injuries, half a million of whom are children. Clinical management of traumatic brain injury (TBI) is challenging due to the complexity of the injury as well as due to the paucity of effective treatment options. Due to the complex nature of the injury involving inflammation, cell loss and edema, cell therapies may be necessary as small molecular anti-inflammatory or neuroprotective strategies have not been very successful. Specifically stem cell therapy has the potential to significantly improve outcomes after TBI. However, one major impediment to successful neural stem cell (NSC) therapy is their poor survival after transplantation due to host T cells and Natural Killer (NK) cell mediated apoptosis of transplanted NSCs. Here, we propose to design and engineer in situ gelling hydrogel carriers for NSCs such that they confer immune-privilege for a period of days to weeks in vivo. We propose to exploit the Fas-ligand mediated cell death of T cells and NK cells to generate an immune privilege zone for NSCs. We posit that enhancing NSC survival is a critical and necessary condition to evaluating NSC therapy's potential for treating TBI. Our aims are designed to design the appropriate hydrogel carriers for NSC, test their ability to promote NSC survival in vivo in an experimental model of TBI, and finally investigate whether indeed enhanced NSC survival results in motor and cognitive improvement in a rodent model of TBI relative to untreated cohorts. Successful completion of the proposed studies would have significant impact on improvement of the quality of life of individuals with TBI, and have implications for stem cell therapies in other organ systems.