The natural healing process for bone has multiple signaling cascades where several soluble factors are expressed at specific times to encourage regeneration. Human mesenchymal stromal cells (hMSCs) have three stages of osteogenic differentiation: an increase in cell number (day 1-4), early cell differentiation showing alkaline phosphatase (ALP) expression (day 5-14), and deposition of calcium and phosphate (day 14-28). The first two stages are of particular interest since cell adhesion peptides have been shown to have biological significance during these early stages of bone regeneration. However, far less is known about the temporal dependence of these signals. To mimic these complex systems, developing dynamic biomaterials has become a popular research area over the past decade. Advances in chemistry, materials science, and manufacturing have enabled the development of complex biomaterials that can mimic dynamic cues in the extracellular matrix. One specific area of interest is spatiotemporal control of multiple biomolecules; however, this has generally required diverse chemical approaches making the process difficult and impractical. To circumvent these issues, I developed a novel method that combines a photoresponsive hydrogel with single-stranded DNA to spatiotemporally control multiple biomolecules using a single conjugation scheme. Here, I describe a detailed protocol to manufacture a fully reversible, spatiotemporal platform using DNA handles. Norbornene-modified hyaluronic acid hydrogels were used to spatially control biomolecule presentation while single-stranded DNA was used to temporally control biomolecule presentation via toehold-mediated strand displacement. This platform was used to orthogonally control the presentation of multiple biomolecules with simple and complex spatial patterning, as well as control the cell morphology of hMSCs by tuning the presentation of the cell adhesion peptide RGDS. Then, this system was applied to study the temporal presentation of cell adhesion peptides and their effect on early osteogenic differentiation of hMSCs in vitro. The peptides used were RGDS, HAVDI, and OGP. OGP alone expressed higher ALP when presented from day 7-14 than day 0-7 or 0-14. When RGDS, HAVDI, and OGP were combined, there was an increase in ALP activity when HAVDI was presented from day 0-3 indicating that HAVDI plays an important role at earlier time points during osteogenic differentiation.

Annually, approximately 1.7 million people suffer a traumatic brain injury (TBI) in the United States. After initial insult, a TBI persists as a series of molecular and cellular events that lead to cognitive and motor deficits which have no treatment. In addition, the injured brain activates the regenerative niches of the adult brain presumably to reduce damage. The subventricular zone (SVZ) niche contains neural progenitor cells (NPCs) that generate astrocytes, oligodendrocyte, and neuroblasts. Following TBI, the injury microenvironment secretes signaling molecules like stromal cell derived factor-1a (SDF-1a). SDF-1a gradients from the injury contribute to the redirection of neuroblasts from the SVZ towards the lesion which may differentiate into neurons and integrate into existing circuitry. This repair mechanism is transient and does not lead to complete recovery of damaged tissue. Further, the mechanism by which SDF-1a gradients reach SVZ cells is not fully understood. To prolong NPC recruitment to the injured brain, exogenous SDF-1a delivery strategies have been employed. Increases in cell recruitment following stroke, spinal cord injury, and TBI have been demonstrated following SDF-1a delivery. Exogenous delivery of SDF-1a is limited by its 28-minute half-life and clearance from the injury microenvironment. Biomaterials-based delivery improves stability of molecules like SDF-1a and offer control of its release. This dissertation investigates SDF-1a delivery strategies for neural regeneration in three ways: 1) elucidating the mechanisms of spatiotemporal SDF-1a signaling across the brain, 2) developing a tunable biomaterials system for SDF-1a delivery to the brain, 3) investigating SDF-1a delivery on SVZ-derived cell migration following TBI. Using in vitro, in vivo, and in silico analyses, autocrine/paracrine signaling was necessary to produce SDF-1a gradients in the brain. Native cell types engaged in autocrine/paracrine signaling. A microfluidics device generated injectable hyaluronic-based microgels that released SDF-1a peptide via enzymatic cleavage. Microgels (±SDF-1a peptide) were injected 7 days post-TBI in a mouse model and evaluated for NPC migration 7 days later using immunohistochemistry. Initial staining suggested complex presence of astrocytes, NPCs, and neuroblasts throughout the frontoparietal cortex. Advancement of chemokine delivery was demonstrated by uncovering endogenous chemokine propagation in the brain, generating new approaches to maximize chemokine-based neural regeneration.

Intraoperative diagnosis in neurosurgery has traditionally relied on frozen and formalin-fixed, paraffin-embedded section analysis of biopsied tissue samples. Although this technique is considered to be the “gold standard” for establishing a histopathologic diagnosis, it entails a number of significant limitations such as invasiveness and the time required for processing and interpreting the tissue. Rapid intraoperative diagnosis has become possible with a handheld confocal laser endomicroscopy (CLE) system. Combined with appropriate fluorescent stains or labels, CLE provides an imaging technique for real-time intraoperative visualization of histopathologic features of the suspected tumor and healthy tissues.

This thesis scrutinizes CLE technology for its ability to provide real-time intraoperative in vivo and ex vivo visualization of histopathological features of the normal and tumor brain tissues. First, the optimal settings for CLE imaging are studied in an animal model along with a generational comparison of CLE performance. Second, the ability of CLE to discriminate uninjured normal brain, injured normal brain and tumor tissues is demonstrated. Third, CLE was used to investigate cerebral microvasculature and blood flow in normal and pathological conditions. Fourth, the feasibility of CLE for providing optical biopsies of brain tumors was established during the fluorescence-guided neurosurgical procedures. This study established the optimal workflow and confirmed the high specificity of the CLE optical biopsies. Fifth, the feasibility of CLE was established for endoscopic endonasal approaches and interrogation of pituitary tumor tissue. Finally, improved and prolonged near wide-field fluorescent visualization of brain tumor margins was demonstrated with a scanning fiber endoscopy and 5-aminolevulinic acid.

These studies suggested a novel paradigm for neurosurgery-pathology workflow when the noninvasive intraoperative optical biopsies are used to interrogate the tissue and augment intraoperative decision making. Such optical biopsies could shorten the time for obtaining preliminary information on the histological composition of the tissue of interest and may lead to improved diagnostics and tumor resection. This work establishes a basis for future in vivo optical biopsy use in neurosurgery and planning of patient-related outcome studies. Future studies would lead to refinement and development of new confocal scanning technologies making noninvasive optical biopsy faster, convenient and more accurate.

Traumatic brain injury (TBI) affects an estimated 1.7 million people in the United States each year and is a leading cause of death and disability for children and young adults in industrialized countries. Unfortunately, the molecular and cellular mechanisms of injury progression have yet to be fully elucidated. Consequently, this complexity impacts the development of accurate diagnosis and treatment options. Biomarkers, objective signatures of injury, can inform and facilitate development of sensitive and specific theranostic devices. Discovery techniques that take advantage of mining the temporal complexity of TBI are critical for the identification of high specificity biomarkers.

Domain antibody fragment (dAb) phage display, a powerful screening technique to uncover protein-protein interactions, has been applied to biomarker discovery in various cancers and more recently, neurological conditions such as Alzheimer’s Disease and stroke. The small size of dAbs (12-15 kDa) and ability to screen against brain vasculature make them ideal for interacting with the neural milieu in vivo. Despite these characteristics, implementation of dAb phage display to elucidate temporal mechanisms of TBI has yet to reach its full potential.

My dissertation employs a unique target identification pipeline that entails in vivo dAb phage display and next generation sequencing (NGS) analysis to screen for temporal biomarkers of TBI. Using a mouse model of controlled cortical impact (CCI) injury, targeting motifs were designed based on the heavy complementarity determining region (HCDR3) structure of dAbs with preferential binding to acute (1 day) and subacute (7 days) post-injury timepoints. Bioreactivity for these two constructs was validated via immunohistochemistry. Further, immunoprecipitation-mass spectrometry analysis identified temporally distinct candidate biological targets in brain tissue lysate.

The pipeline of phage display followed by NGS analysis demonstrated a unique approach to discover motifs that are sensitive to the heterogeneous and diverse pathology caused by neural injury. This strategy successfully achieves 1) target motif identification for TBI at distinct timepoints and 2) characterization of their spatiotemporal specificity.

Medulloblastoma is the most common malignant pediatric brain cancer and is classified into four different subgroups based on genetic profiling: sonic hedgehog (SHH), WNT, Group 3 and 4. Changes in gene expression often alter the progression and development of cancers. One way to control gene expression is through the acetylation and deacetylation of histones. More specifically in medulloblastoma SHH and Group 3, there is an increased deacetylation, and histone deacetylase inhibitors (HDACi) can be used to target this change. Not only can HDACi target increases in deacetylation, they are also known to induce cell cycle arrest and apoptosis. The combination of these factors has made HDACi a promising cancer therapeutic. Panobinostat, a hydrophobic, small molecule HDACi was recently identified as a potent molecule of interest for the treatment of medulloblastoma. Furthermore, panobinostat has already been FDA approved for treatment in multiple myeloma and is being explored in clinical trials against various solid tumors. The laboratory is interested in developing strategies to encapsulate panobinostat within nanoparticles composed of the biodegradable and biocompatible polymer poly(lactic acid)-poly(ethylene glycol) (PLA-PEG). Nanoparticles are formed by single emulsion, a process in which hydrophobic drugs can be trapped within the hydrophobic nanoparticle core. The goal was to determine if the molecular weight of the hydrophobic portion of the polymer, PLA, has an impact on loading of panobinostat in PLA-PEG nanoparticles. Nanoparticles formulated with PLA of varying molecular weight were characterized for loading, size, zeta potential, controlled release, and in vivo tolerability. The results of this work demonstrate that panobinostat loaded nanoparticles are optimally formulated with a 20:5kDa PLA-PEG, enabling loading of ~3.2 % w/w panobinostat within nanoparticles possessing an average diameter of 102 nm and surface charge of -8.04 mV. Panobinostat was released from nanoparticles in a potentially biphasic fashion over 72 hours. Nanoparticles were well tolerated by intrathecal injection, although a cell culture assay suggesting reduced bioactivity of encapsulated drug warrants further study. These experiments demonstrate that the molecular weight of PLA influences loading of panobinostat into PLA-PEG nanoparticles and provide basic characterization of nanoparticle properties to enable future in vivo evaluation.

Traumatic brain injury (TBI) is a leading cause of disability worldwide with 1.7 million TBIs reported annually in the United States. Broadly, TBI can be classified into focal injury, associated with cerebral contusion, and diffuse injury, a widespread injury pathology. TBI results in a host of pathological alterations and may lead to a transient blood-brain-barrier (BBB) breakdown. Although the BBB dysfunction after TBI may provide a window for therapeutic delivery, the current drug delivery approaches remains largely inefficient due to rapid clearance, inactivation and degradation. One potential strategy to address the current therapeutic limitations is to employ nanoparticle (NP)-based technology to archive greater efficacy and reduced clearance compared to standard drug administration. However, NP application for TBI is challenging not only due to the transient temporal resolution of the BBB breakdown, but also due to the heterogeneous (focal/diffuse) aspect of the disease itself. Furthermore, recent literature suggests sex of the animal influences neuroinflammation/outcome after TBI; yet, the influence of sex on BBB integrity following TBI and subsequent NP delivery has not been previously investigated. The overarching hypothesis for this thesis is that TBI-induced compromised BBB and leaky vasculature will enable delivery of systemically injected NPs to the injury penumbra. This study specifically explored the feasibility and the temporal accumulation of NPs in preclinical mouse models of focal and diffuse TBI. Key findings from these studies include the following. (1) After focal TBI, NPs ranging from 20-500nm exhibited peak accumulation within the injury penumbra acutely (1h) post-injury. (2) A smaller delayed peak of NP accumulation (40nm) was observed sub-acutely (3d) after focal brain injury. (3) Mild diffuse TBI simulated with a mild closed head injury model did not display any measurable NP accumulation after 1h post-injury. (4) In contrast, a moderate diffuse model (fluid percussion injury) demonstrated peak accumulation at 3h post-injury with up to 500 nm size NPs accumulating in cortical tissue. (5) Robust NP accumulation (40nm) was found in female mice compared to the males at 24h and 3d following focal brain injury. Taken together, these results demonstrate the potential for NP delivery at acute and sub-acute time points after TBI by exploiting the compromised BBB. Results also reveal a potential sex dependent component of BBB disruption leading to altered NP accumulation. The applications of this research are far-reaching ranging from theranostic delivery to personalized NP delivery for effective therapeutic outcome.

According to sources of the Centers for Disease Control and Prevention, approximately 1.7 million traumatic brain injury (TBI) cases occur annually in the United States. TBI results in 50 thousand deaths, nearly 300 thousand hospitalizations and 2.2 million emergency room visits causing a $76 billion economic burden in direct and indirect costs. Furthermore, it is estimated that over 5 million TBI survivors in the US are struggling with long-term disabilities. And yet, a point-of-care TBI diagnostic has not replaced the non-quantitative cognitive and physiological methods used today. Presently, pupil dilation and the Glasgow Coma Scale (GCS) are clinically used to diagnose TBI. However, GSC presents difficulties in detecting subtle patient changes, oftentimes leaving mild TBI undiagnosed. Given the long-term deficits associated with TBIs, a quantitative method that enables capturing of subtle and changing TBI pathologies is of great interest to the field.

The goal of this research is to work towards a test strip and meter point-of-care technology (similar to the glucose meter) that will quantify several TBI biomarkers in a drop of whole blood simultaneously. It is generally understood that measuring only one blood biomarker may not accurately diagnose TBI, thus this work lays the foundation to develop a multi-analyte approach to detect four promising TBI biomarkers: glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), S-100β protein, and tumor necrosis factor-α (TNF-α). To achieve this, each biomarker was individually assessed and modeled using sensitive and label-free electrochemical impedance techniques first in purified, then in blood solutions using standard electrochemical electrodes. Next, the biomarkers were individually characterized using novel mesoporous carbon electrode materials to facilitate detection in blood solutions and compared to the commercial standard Nafion coating. Finally, the feasibility of measuring these biomarkers in the same sample simultaneously was explored in purified and blood solutions. This work shows that a handheld TBI blood diagnostic is feasible if the electronics can be miniaturized and large quantity production of these sensors can be achieved.

Prosthetic joint infection (PJI) is a devastating complication associated with total joint arthroplasty that results in high cost and patient morbidity. There are approximately 50,000 PJIs per year in the US, imposing a burden of about $5 billion on the healthcare system. PJI is especially difficult to treat because of the presence of bacteria in biofilm, often highly tolerant to antimicrobials. Treatment of PJI requires surgical debridement of infected tissues, and local, sustained delivery of antimicrobials at high concentrations to eradicate residual biofilm bacteria. However, the antimicrobial concentrations required to eradicate biofilm bacteria grown in vivo or on tissue surfaces have not been measured. In this study, an experimental rabbit femur infection model was established by introducing a variety of pathogens representative of those found in PJIs [Staphylococcus Aureus (ATCC 49230, ATCC BAA-1556, ATCC BAA-1680), Staphylococcus Epidermidis (ATCC 35984, ATCC 12228), Enterococcus Faecalis (ATCC 29212), Pseudomonas Aeruginosa (ATCC 27853), Escherichia Coli (ATCC 25922)]. Biofilms of the same pathogens were grown in vitro on biologic surfaces (bone and muscle). The ex vivo and in vitro tissue minimum biofilm eradication concentration (MBEC; the level required to eradicate biofilm bacteria) and minimum inhibitory concentration (MIC; the level required to inhibit planktonic, non-biofilm bacteria) were measured using microbiological susceptibility assays against tobramycin (TOB) and vancomycin (VANC) alone or in 1:1 weight combination of both (TOB+VANC) over three exposure durations (6 hour, 24 hour, 72 hour). MBECs for all treatment combinations (pathogen, antimicrobial used, exposure time, and tissue) were compared against the corresponding MIC values to compare the relative susceptibility increase due to biofilm formation. Our data showed median in vitro MBEC to be 100-1000 times greater than the median MIC demonstrating the administration of local antimicrobial doses at MIC level would not kill the persisting bacteria in biofilm. Also, administering dual agent (TOB+VANC) showed median MBEC values to be comparable or lower than the single agents (TOB or VANC)

Stromal cell-derived factor-1α (SDF-1α) and its key receptor, CXCR4 are ubiquitously expressed in systems across the body (e.g. liver, skin, lung, etc.). This signaling axis regulates a myriad of physiological processes that range from maintaining of organ homeostasis in adults to, chemotaxis of stem/progenitor and immune cell types after injury. Given its potential role as a therapeutic target for diverse applications, surprisingly little is known about how SDF-1α mediated signaling propagates through native tissues. This limitation ultimately constrains rational design of interventional biomaterials that aim to target the SDF-1α/CXCR4 signaling axis. One application of particular interest is traumatic brain injury (TBI) for which, there are currently no means of targeting the underlying biochemical pathology to improve prognosis.

Growing evidence suggests a relationship between SDF-1α/CXCR4 signaling and endogenous neural progenitor/stem cells (NPSC)-mediated regeneration after neural injury. Long-term modulation of the SDF-1α/CXCR4 signaling axis is thus hypothesized as a possible avenue for harnessing and amplifying endogenous regenerative mechanisms after TBI. In order to understand how the SDF-1α/CXCR4 signaling can be modulated in vivo, we first developed and characterized a sustained protein delivery platform in vitro. We were the first, to our knowledge, to demonstrate that protein release profiles from poly(D,L,-lactic-co-glycolic) acid (PLGA) particles can be tuned independent of particle fabrication parameters via centrifugal fractioning. This process of physically separating the particles altered the average diameter of a particle population, which is in turn was correlated to critical release characteristics. Secondly, we demonstrated sustained release of SDF-1α from PLGA/fibrin composites (particles embedded in fibrin) with tunable burst release as a function of fibrin concentration. Finally, we contrasted the spatiotemporal localization of endogenous SDF-1α and CXCR4 expression in response to either bolus or sustained release of exogenous SDF-1α. Sustained release of exogenous SDF-1α induced spatially diffuse endogenous SDF-1/CXCR4 expression relative to bolus SDF-1 administration; however, the observed effects were transient in both cases, persisting only to a maximum of 3 days post injection. These studies will inform future systematic evaluations of strategies that exploit SDF-1α/CXCR4 signaling for diverse applications.

Traumatic brain injury (TBI) is a significant public health concern in the U.S., where approximately 1.7 million Americans sustain a TBI annually, an estimated 52,000 of which lead to death. Almost half (43%) of all TBI patients report experiencing long-term cognitive and/or motor dysfunction. These long-term deficits are largely due to the expansive biochemical injury that underlies the mechanical injury traditionally associated with TBI. Despite this, there are currently no clinically available therapies that directly address these underlying pathologies. Preclinical studies have looked at stem cell transplantation as a means to mitigate the effects of the biochemical injury with moderate success; however, transplants suffer very low retention and engraftment rates (2-4%). Therefore, transplants need better tools to dynamically respond to the injury microenvironment.

One approach to develop new tools for stem cell transplants may be to look towards the endogenous repair response for inspiration. Specifically, activated cell types surrounding the injury secrete the chemokine stromal cell-derived factor-1α (SDF-1α), which has been shown to play a critical role in recruiting endogenous neural progenitor/stem cells (NPSCs) to the site of injury. Therefore, it was hypothesized that improving NPSC response to SDF-1α may be a viable mechanism for improving NPSC transplant retention and migration into the surrounding host tissue. To this end, work presented here has 1. identified critical extracellular signals that mediate the NPSC response to SDF-1α, 2. incorporated these findings into the development of a transplantation platform that increases NPSC responsiveness to SDF-1α and 3. observed increased NPSC responsiveness to local exogenous SDF-1α signaling following transplantation within our novel system. Future work will include studies investigating NSPC response to endogenous, injury-induced SDF-1α and the application of this work to understanding differences between stem cell sources and their implications in cell therapies.