Browsing by Author "Reichert, William M"

Creation of novel tools for biomedical applications is critical for the improvement of patient diagnostics and therapeutics. Two particularly important needs lie in (1) improved in vitro testing and increased performance of prosthetic vascular grafts and (2) purification methods for cells that do not compromise their utility. Progress in these areas is urgently needed and would facilitate the availability of higher quality devices and treatments that raise the quality of patient care. This work focused on developing new approaches toward that goal.

A tremendous and immediate need exists for high-performance small-diameter synthetic vascular grafts, as a fifth of the 500,000 annual coronary artery bypass grafting (CABG) patients lack suitable autologous vessels for revascularization. This problem has driven intense research and development of increasingly diverse prosthetics that could be viable alternatives in the years to come. Evaluating these designs in vitro offers high-throughput, low-cost screening for promising graft technologies ahead of more stringent vetting in vivo.

Offering a fresh take on assessing vascular graft thrombogenicity in vitro, the buildup of pressure upstream to a clot was used as a metric to quantify the physical interaction between the graft lumen and a maturing thrombus. A closed tubing system was devised and continuously monitored as clotting solutions of fibrin glue, platelet-rich plasma or whole blood were cured to varying maturities and then purged from small-diameter ePTFE grafts or Tygon graft mimics. This approach provided insight into how blood flow resistance is influenced by a number of clinically relevant factors, such as the level of vessel occlusion and the physical nature of the resident coagulum.

Endothelialization of synthetic vascular grafts yields viable alternatives to native vessels and can be accomplished non-invasively using late-outgrowth endothelial progenitor cells (LO-EPCs) isolated from peripheral blood. However, the time required to amass sufficient cells to prepare a graft with current methods is risky for waiting CABG patients. An ambitious approach conceived to significantly decrease this wait period involved developing affinity ligands selective for LO-EPCs that would enable their capture directly from the circulation to facilitate rapid amassment. An in vitro directed evolution strategy to generate aptamers, the nucleic acid analogs of antibodies, that specifically bind these cells was carried out with initially promising but ultimately unsuccessful results. While the particular strategy executed here did not prevail, the high value and impact LO-EPC aptamers would deliver merit revisiting this work with a revised strategy such as the one proposed in this document.

Purification of autologous and allogenic cells is essential for their use in a variety of therapeutic and basic research applications in addition to augmenting graft performance. However, the antibody stains conventionally used to selectively purify cells are permanent and their continued presence can elicit an immune response in vivo and compromise native cell behavior. To avoid these issues, a cell purification strategy was crafted utilizing aptamers and matched oligonucleotide antidotes that enabled reversible cell staining. The reversible stains were robust enough for cell purification via fluorescence-activated cell sorting (FACS) yet subsequently able to be removed with gentle heat treatment and antidote. Importantly, cell function that was compromised without antidote was rescued to match the native behavior of non-stained cells following purification and antidote treatment.

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

Biological systems have inspired interest in developing artificial molecular self-assembly techniques that imitate nature's ability to harness chemical forces to specifically position atoms within intricate assemblies. Of the biomolecules used to mimic nature's abilities, nucleic acids have gained special attention. Specifically, deoxyribonucleic acid is a stable molecule with a readily accessible code that exhibits predictable and programmable intermolecular interactions. These properties are exploited in the revolutionary structural DNA nanotechnology method known as scaffolded DNA origami. For DNA origami to establish itself as a widely used method for creating self-assembling, complex, functional materials, current limitations need to be overcome and new methods need to be established to move forward with developing structures for diverse applications in many fields. The limitations discussed in this dissertation include 1) pushing the scale of well-formed, fully-addressable origami to two and seven times the size of conventional origami, 2) testing cost-effective staple strand synthesis methods for producing pools of oligos for a specified origami, and 3) engineering mechanical properties using non-natural nucleotides in DNA assemblies. After accomplishing the above, we're able to design complex DNA origami structures that incorporate many of the current developments in the field into a useful material with applicability in wide-ranging fields, namely cell biology and photonics.

Chronically implanted recording electrode arrays linked to prosthetics have the potential to make positive impacts on patients suffering from full or partial paralysis [1;2]. Such arrays are implanted into the patient's cortical tissue and record extracellular potentials from nearby neurons, allowing the information encoded by the neuronal discharges to control external devices. While such systems perform well during acute recordings, they often fail to function reliably in clinically relevant chronic settings [3]. Available evidence suggests that a major failure mode of electrode arrays is the brain tissue reaction against these implants (termed the glial scar), making the biocompatibility of implanted electrodes a primary concern in device design. Previous studies have focused on modifying the form factor of recording arrays, implanting such arrays in experimental animals, and, upon explantation, evaluating the glial scarring in response to the implant after several weeks in vivo. Because of a lack of information regarding the mechanisms involved in the tissue reaction to implanted biomaterials in the brain, it is not surprising that these in vivo studies have met with limited success. This dissertation describes the development of a simple, controlled in vitro model of glial scarring and the utilization of that model to probe the cellular and molecular mechanisms behind glial scarring.

A novel in vitro model of glial scarring was developed by adapting a primary cell-based system previously used for studying neuroinflammatory processes in neurodegenerative disease [4]. Midbrains from embryonic day 14 Fischer 344 rats were mechanically dissociated and grown on poly-D-lysine coated 24 well plates to a confluent layer of neurons, astrocytes, and microglia. The culture was injured with either a mechanical scrape or foreign-body placement (segments of 50 mm diameter stainless steel microwire), fixed at time points from 6 h to 10 days, and assessed by immunocytochemistry. Microglia invaded the scraped wound area at early time points and hypertrophied activated astrocytes repopulated the wound after 7 days. The chronic presence of microwire resulted in a glial scar forming at 10 days, with microglia forming an inner layer of cells coating the microwire, while astrocytes surrounded the microglial core with a network of cellular processes containing upregulated GFAP. Neurons within the culture did not repopulate the scrape wound and did not respond to the microwire, although they were determined to be electrically active through patch clamp recording.

This initial model recreated many of the hallmarks of glial scarring around electrodes used for recording in the brain; however, the model lacked the reproducibility necessary to establish a useful characterization tool. After the protocol was amended to resemble protocols typically used to culture neural stem/precursor cells, an intense scarring reaction was consistently seen [5]. To further optimize and characterize the reaction, six independent cell culture variables (growth media, seeding density, bFGF addition day, serum concentration in treatment media, treatment day, and duration of culture) were varied systematically and the resulting scars were quantified. The following conditions were found to give the highest level of scarring: Neurobasal medium supplemented with B27, 10% fetal bovine serum at treatment, 10 ng/ml b-FGF addition at seeding and at treatment, treatment at least 6 days after seeding and scar growth of at least 5 days. Seeding density did not affect scarring as long as at least 500,000 cells were seeded per well, but appropriate media, bFGF, and serum were essential for significant scar formation.

The optimized in vitro model was then used to help uncover the underlying molecular and cellular mechanisms behind glial scarring. A microwire coating that mimics the basal lamina present within glial scars was developed that allows cells responding to the coated microwire to be isolated and evaluated (i.e. through cell counting or cell staining). A panel of soluble factors known to be involved in glial scar formation was added to the media and the cellular response was recorded. The extent of cell accumulation on the coated microwires was significantly increased by titration of the culture with serum, the pleotropic growth factor bFGF, the inflammatory cytokines IL-1α and IL-1β, and the growth factors PDGF and BMP-2. The other fourteen soluble factors tested had little to no effect on the number of cells that attached to the coated microwires, although a specific blocker of the bFGF receptor was able to abrogate the effect of bFGF. This study proposes essential roles in glial scarring of serum, which infiltrates brain tissue upon disruption of the blood-brain barrier, and bFGF, which is a necessary growth and survival factor for the neural precursor cells that respond to injury. These insights suggest repeated rounds of implant micromotion-induced cellular damage, with the resultant neuronal death, serum release, and bFGF deposition may thicken the glial scar and lead to recording signal loss.

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

Activation-induced cytidine deaminase (AID), encoded by the Aicda gene, is indispensable for secondary antibody diversification through somatic hypermutation (SHM) and class switch recombination (CSR). It is expressed predominantly in germinal center (GC) B cells, where it deaminates cytosine to uracil in the DNA of immunoglobulin (Ig) genes, triggering mutagenesis (SHM) or deletional recombination events (CSR). However, when misregulated, AID can also mutate non-Ig genes, including proto-oncogenes, thereby contributing to genomic instability and cancer. Due to its potentially deleterious effects, AID expression must be tightly controlled. At the transcriptional level, Aicda is regulated by four highly conserved cis-regulatory regions (Regions 1-4). Region 1 contains the promoter, and is responsible for basal transcription of Aicda. Region 2 contains both enhancer and silencer elements, but functions as a negative regulatory region, restricting AID expression to antigen-activated B cells. Region 3 is reported to be essential for normal AID expression, but its role is otherwise unclear. Region 4, which contains two STAT6 sites essential for its function, provides enhancer activity in response to cytokine stimulation and is responsible for the high levels of AID expression found in GC B cells. Epigenetic mechanisms could add another layer of control to the regulation of Aicda; however, little research has been done in this regard. In this study, I investigated the role of DNA methylation in the cis-regulation of Aicda transcription. Through bisulfite sequencing of splenic mouse B cell DNA, I demonstrate that Region 4 is highly methylated in naïve B cells, but becomes demethylated in activated B cells. This is in contrast to Region 2, which I found to be constitutively unmethylated, and Region 1, which only becomes partially demethylated. Using quantitative methylation-specific PCR (qMSP), I show that loss of methylation in Region 4 correlates positively with cell division number, consistent with a passive mechanism of demethylation. I also show that demethylation in Region 4 cannot be achieved by cell proliferation alone, and that it depends upon induction of AID expression (via IL-4 stimulation). However, bisulfite sequencing of CH12F3-2 cell DNA shows that demethylation by itself is insufficient to activate AID expression. Taken together, the data suggests that IL-4 induces STAT6 to bind to its cognate sites in Region 4 and inhibit DNMT1, likely through chromatin reorganization, triggering passive demethylation during the induction of Aicda transcription.

About one in four deaths in the United States are currently due to some form of heart disease [1]. Patients with cardiovascular disease usually require some form of perfusion-based surgical intervention at some point in their lives. To this end, biomaterials used in these applications elicit thrombus formation at some point after implantation. To date, accurately predicting the thrombogenecity of biomaterials remains difficult, and current testing methods are not comprehensive [2]. This is due to the fact that although blood clotting has been a topic of research for many decades, the interplay between the myriad of biochemical and physical reactions that occur in this process is not yet fully understood. One problem is the unpredictability of working with whole blood, as its precise contents vary from person to person, and its activity varies with handling methods [2]. Therefore, it would be interesting to use a bottom-up approach and build an in vitro assay with minimal and specific clotting components to determine differences in thrombogenicity between different blood-contacting surfaces that may be used on an implantable biomaterial.

By combining clotting factors Va, Xa, II, and fibrinogen and focusing on anti-thrombotic modulators antithrombin III and heparin, samples were tested to show a dose response in clotting times and fibrin clot formation. This was done by measuring turbidity in a plate reader, and by manually assessing the time at which the first fibrin strand could be pulled up with a pipette tip. R squared values of greater than 0.96 for all correlation plots demonstrated a strong correlation between manual and plate reading times in all cases. Clotting factor cocktails with thrombin and prothrombinase complex factors showed comparable clotting times at 1U, 0.5U, 0.25U, and 0.125U/mL of equivalent thrombin activity in plasma. A significant dose response to heparin sodium salt with antithrombin III and 0.1U/mL prothrombinase complex was demonstrated at 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 ug/mL of heparin sodium salt. These results show that it will be possible to construct specific microplate-based thrombogenicity assays with purified components of clotting.

Inflammation and the formation of an avascular fibrous capsule have been identified as the key factors controlling the wound healing associated failure of implantable glucose sensors. Our aim is to guide advantageous tissue remodeling around implanted sensor leads by the temporal release of dexamethasone (Dex), a potent anti-inflammatory agent, in combination with the presentation of a stable textured surface.

First, Dex-releasing polyurethane porous coatings of controlled pore size and thickness were fabricated using salt-leaching/gas-foaming technique. Porosity, pore size, thickness, drug release kinetics, drug loading amount, and drug bioactivity were evaluated. In vitro sensor functionality test were performed to determine if Dex-releasing porous coatings interfered with sensor performance (increased signal attenuation and/or response times) compared to bare sensors. Drug release from coatings monitored over two weeks presented an initial fast release followed by a slower release. Total release from coatings was highly dependent on initial drug loading amount. Functional in vitro testing of glucose sensors deployed with porous coatings against glucose standards demonstrated that highly porous coatings minimally affected signal strength and response rate. Bioactivity of the released drug was determined by monitoring Dex-mediated, dose-dependent apoptosis of human peripheral blood derived monocytes in culture.

The tissue modifying effects of Dex-releasing porous coatings were accessed by fully implanting Tygon® tubing in the subcutaneous space of healthy and diabetic rats. Based on encouraging results from these studies, we deployed Dex-releasing porous coatings from the tips of functional sensors in both diabetic and healthy rats. We evaluated if the tissue modifying effects translated into accurate, maintainable and reliable sensor signals in the long-term. Sensor functionality was accessed by continuously monitoring glucose levels and performing acute glucose challenges at specified time points.

Sensors treated with porous Dex-releasing coatings showed diminished inflammation and enhanced vascularization of the tissue surrounding the implants in healthy rats. Functional sensors with Dex-releasing porous coatings showed enhanced sensor sensitivity over a 21-day period when compared to controls. Enhanced sensor sensitivity was accompanied with an increase in sensor signal lag and MARD score. These results indicated that Dex-loaded porous coatings were able to elicit a favorable tissue response, and that such tissue microenvironment could be conducive towards extending the performance window of glucose sensors in vivo.

The diabetic pilot animal study showed differences in wound healing patters between healthy and diabetic subjects. Diabetic rats showed lower levels of inflammation and vascularization of the tissue surrounding implants when compared to their healthy counterparts. Also, functional sensors treated with Dex-releasing porous coatings did not show enhanced sensor sensitivity over a 21-day period. Moreover, increased in sensor signal lag and MARD scores were present in porous coated sensors regardless of Dex-loading when compared to bare implants. These results suggest that the altered wound healing patterns presented in diabetic tissues may lead to premature sensor failure when compared to sensors implanted in healthy rats.

The success of implanted medical devices, such as biosensors, is dependent on the immune reaction to the surface of the implanted material. This immune reaction, termed the foreign body reaction, is potentially affected by the physical and chemical properties of the implanted material. Macrophages interact with the surface of the implanted material and secrete intercellular signals, including cytokines and growth factors, which direct the actions of immune cells in the surrounding tissue. The type and quantity of cytokines and growth factors produced by macrophages at an implant surface could be an indicator of the outcome of the foreign body reaction.

This study investigated the effect of the surface chemistry of an implanted device on the production of cytokines and growth factors. First, microdialysis sampling was characterized as a technique for collecting cytokines and growth factors from the tissue surrounding an implant. Based on this characterization, it was determined that a direct sampling method would be more suitable than microdialysis sampling for determining accurate tissue concentrations of cytokines and growth factors. Second, an in vitro model was developed and utilized to assess cytokine and growth factor production from monocyte/macrophage cultures seeded onto commonly implanted polymeric biomaterials with varying surface chemistries. The materials included in this study were polyethylene (PE), polyurethane (PU), polymethyl methacrylate (PMMA), expanded polytetrafluoroethylene (ePTFE), and a cytotoxic organo-tin polyvinyl chloride (ot-PVC) as a positive control. From this in vitro model, it was determined that the varying surface chemistries of these non-toxic materials, excluding ot-PVC, did not significantly affect the types and quantities of cytokines and growth factors produced. Finally, an in vivo model for evaluating the cytokine and growth factor response to an implanted biomaterial was utilized for comparison with the in vitro findings. In this model, biomaterials were implanted subcutaneously within the lumen of a stainless steel mesh cage. The mesh cage served to create a "pocket" where wound exudate fluid collected within the cage, surrounding the implanted biomaterial. The materials included in this study were PE, PU, and ot-PVC. Cytokines and growth factors produced at the material surface were sampled directly from the exudate fluid. The results from this in vivo study indicate that cytokine and growth factor production were not significantly impacted by the varying surface chemistries of the implanted biomaterials. The in vivo data support the findings from the in vitro model, suggesting that the foreign body reaction proceeds in a similar fashion for each of these non-cytotoxic, polymeric biomaterials with varying surface chemistries.

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Total joint replacement surgeries are performed on thousands of patients every year, yet these implants are subject to failure following prolonged exposure to the harsh environment of the body as well as the complex loading patterns seen in biological joints. The generation of wear debris from both the articulating surfaces and the poly(methyl methacrylate) (PMMA) bone cement used to anchor the replacements in place serves to accelerate wear and subsequent failure of the device. Self-healing approaches that employ an encapsulated healing agent embedded in a catalyst-containing matrix have been developed to restore mechanical function to materials that undergo crack damage; following capsule rupture, and healing agent release and polymerization serves to halt microcrack propagation. However, existing encapsulated systems do not adhere to biomaterials constraints. In this work, interfacial polymerization of polyurethane (PUR) in an oil-in-water emulsion was used to achieve encapsulation of octylcyanoacrylate (OCA), a medical grade adhesive used in sutureless surgeries. The optimized encapsulation procedure was determined by studying the effects of solvent, surfactant, and temperature on the final product. The average size and size distribution, capsule shell thickness, percent fill and reactivity of encapsulated agent, and shelf life of these capsules were studied and are now suitable for incorporation into PMMA and assessment as potential healing agent systems.

Over half a million coronary artery bypass operations are performed annually in the US yielding an annual health care cost of over 16 billion dollars. Only five percent of bypasses are repeat operations in spite of the procedures prevalence. Patients facing repeat coronary artery bypass operations often lack transplantable autologous arteries or veins, necessitating the use of substitutes. Unfortunately, synthetic small diameter vascular grafts have unacceptable patency rates, primarily due to lumenal thrombus formation and intimal thickening. Endothelial cells (EC) mediate the anti-thrombotic activity in healthy blood vessels, and due to the scarcity of suitable autologous vascular replacement, EC-seeded small diameter synthetic vascular grafts represent a clear, immediate, and practical solution. The fundamental goal of this project was to optimize the dual ligand (DL) system on synthetic vascular graft (SVG) surrogates to show enhanced cell adhesion, retention, and native functionality compared to fibronectin alone. Initially, two SVG surrogates were identified through characterization by x-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and 125I radiolabeling. The first modification to the DL system involved direct biotinylation of fibronectin (bFN) as a replacement for co-adsorption of FN with biotinylated bovine serum albumin (bBSA). This was analyzed with a Langmuir model using surface plasmon resonance (SPR) spectroscopy to verify the binding affinity of bFN and ELISA to detect the availability of the RGD binding motif post biotinylation. The second major change in this project examined cell binding and formation of focal adhesion after shifting from direct incubation of HUVECs with RGD-SA to sequentially adsorbing bFN(9) and RGD-SA prior to introducing unmodified HUVECs. These experiments were conducted under static seeding conditions. Next, dynamic cell seeding onto the sequentially adsorbed protein surface was examined as a function of surface immobilized protein and Trypsin/EDTA concentration. SPR results showed statistical differences in α5β1 and αvβ3 integrin binding to RGD cell binding motifs introduced by bFN(9) and RGD-SA. Increase in binding specificity through these integrins lead to rapid cell binding and retention on Teflon-AF surfaces adsorbed with this protein formulation. This system appears to be the nexus at which the DL has proven its value. These results could have broader implications in augmenting EC attachment to SVG prior to implantation.

Extending the functional lifetime of acrylic poly(methyl methacrylate) (PMMA) bone cement may reduce the number of revision total joint replacement (TJR) surgeries performed each year. We developed a system utilizing an encapsulated water-reactive, FDA-approved tissue adhesive, 2-octyl cyanoacrylate (OCA), as a healing agent to repair microcracks within a bone cement matrix. The proposed research tested the following hypotheses: (1) reactive OCA can be successfully encapsulated and the resulting capsules thoroughly characterized; (2) the static mechanical properties of the PMMA composite can be improved or maintained through inclusion of an optimal wt% of OCA-containing capsules; (3) PMMA containing encapsulated OCA has a prolonged lifetime when compared with a capsule-free PMMA control as measured by the number of cycles to failure; and (4) the addition of capsules to the PMMA does not significantly alter the biocompatibility of the material. Based on the experiments reported herein, the primary conclusions of this dissertation are as follows: (1) functional OCA can be encapsulated within polyurethane spheres and successfully incorporated into PMMA bone cement; (2) lower wt% of capsules maintained the tensile, compressive, fracture toughness, and bending properties of the PMMA; (3) inclusion of 5 wt% of OCA-containing capsules in the matrix increased the number of cycles to failure when compared to unfilled specimens and those filled with OCA-free capsules; and (4) MG63 human osteosarcoma cell proliferation and viability were unchanged following exposure to OCA-containing PMMA when compared with a capsule-free control.

Efforts to create a reliable, long–term implantable glucose sensor have been stymied by the effects of the foreign body response and wound healing that introduce delayed response times as well as unpredictable sensor performance. Loss of vascularization from fibrotic encapsulation around implanted sensors is purported as a key contributor to sensor failure, as glucose and oxygen transport to the sensor becomes impeded. Improving sensor performance by increasing angiogenesis and/or reducing capsule thickness using tissue-modifying textured coatings is attractive because texturing is not dependent upon a depletable drug reservoir. A significant range of materials and pore sizes are capable of promoting angiogenesis and reducing capsule thickness, provided pores have open-architecture with dimensions sufficiently large enough to allow inflammatory cell infiltration.

Poly–L–lactic acid was gas foamed/salt leached with ammonium bicarbonate to produce porous coatings for Medtronic MiniMed SOF–sensor glucose sensors. Coating properties included 30μm pore diameters, 90% porosity, and 50μm wall thickness. Cytotoxicity, degradation, and sensor response time studies were performed to ensure the porous coatings were non–toxic and negligibly retarded glucose diffusion prior to in vivo testing. Histology was used to evaluate angiogenesis and collagen deposition adjacent to porous coated and bare (i.e. smooth, uncoated) non–functional sensor strips after three weeks in the rat dorsal subcutis. Functional Medtronic glucose sensors, with and without porous coatings, were percutaneously implanted in the rat dorsum to assess if the angiogenic–inducing properties observed around the non–functional porous coated sensor strips translated into stable, non–attenuated sensor signals over two and three weeks. MiniLinkTM transmitters were attached to the rats, permitting continuous glucose monitoring. Vessel counts and collagen deposition adjacent to sensors were determined from histological analysis. A one–sided dorsal window model was developed to further evaluate the interplay between vascularization and sensor performance Sensors were inserted beneath the windows, allowing visualization of microvascular changes adjacent to sensor surfaces, with simultaneous evaluation of how vascular changes impacted interstitial glucose monitoring.

Porous coating did have angiogenic–inducing effects on the surrounding tissue. When fully implanted in the rat dorsum, sensor strips with porous coatings induced three–fold more vessels within 100μm2 of the sensor strip surface after three weeks and two-fold more cumulative vessel lengths within 1mm2 after two weeks, compared to bare surfaces. In contrast, when percutaneously implanted in the rat dorsum, porous coated and bare sensors were equally highly vascularized, with two–fold more vessels than fully implanted bare sensors.

Despite increased angiogenesis adjacent to percutaneous sensors, sensor signal attenuation occurred over 14 days, suggesting that angiogenesis plays a secondary role in maintaining sensor function. Percutaneously implanted porous coated sensors had greater reductions in baseline current (20 to 50+%) over two weeks than bare sensors (10 to 30%). Mechanical stresses imposed by percutaneous tethering may override the beneficial effects of porous coatings. Furthermore, integration of the porous coating with the surrounding tissue may have increased tissue tearing at the porous coating–tissue, increasing inflammation and collagen deposition resulting in greater signal attenuation compared with bare sensors. Future investigations of the role mechanical irritation has on wound healing around percutaneous glucose sensors are warranted.

Road accidents are the leading cause of death within the 15-29 years age range worldwide and the risk of death for motorcyclists is 20 times that of car occupants. As such, 31% of over 10,250 annual road traffic deaths in Uganda are due to either 2 or 3-wheeler motorists’ accidents. Another study in Uganda revealed that 71% of its motorcycle crash victims sustained a head injury while more research shows that helmets can reduce risk of death by 37% and risk of head injury by 69% in the event of a crash. Unfortunately, helmet-use compliance is 30.8% and 1% compliance for riders and passengers respectively in Uganda. Market research by Design without Borders and the Uganda Helmet Vaccine Initiative, attributes this low helmet-use to discomfort, poor helmet ventilation and the prohibitive price of the helmets. A large part of the prohibitive helmet price is due to onerous performance requirements which drive up the development and manufacturing costs. One such requirement is ensuring the helmet's optimal performance in temperatures as low as -20o Celsius which are atypical in tropical climate regions. Another is that the helmet withstands multiple identical impacts at exactly the same location which is extremely rare in a crash. This Masters research is concerned with investigating the effect of continuity and thickness of motorcycle helmet shells on performance. Helmeted head impacts were simulated at three impact points using two different impact surfaces while varying shell thicknesses and continuity using LS DYNA, a Finite Element Analysis software. Increase in shell thickness reduced motorcycle helmet performance while splitting the shell in halves did not significantly affect motorcycle helmet performance. Insights from this research will inform and guide the engineering design of affordable market approved, better ventilated motorcycle helmets under 10 USD that will be suited for the Tropics.

As an increasingly prevalent chronic disease, diabetes represents one of the fastest growing health burdens to both the developed and developing world. In an effort to improve the management and treatment of diabetes, implantable sensors that continuously monitor glucose levels have become popular alternatives to patient-administered finger prick measurements of blood glucose. However, following implantation, the performance of these implants suffers from inaccurate and erratic readings that compromise their useful lives. As a result, implantable glucose sensors remain limited as a platform for the reliable management of diabetes. While the interaction between the sensor and its surrounding tissue has been posited as a culprit for erroneous in vivo sensor performance, there remains little evidence to support that theory.

This dissertation describes the effects that implant-associated tissue reactions have on implantable sensor function. Since tissue response to an implant changes over time, the overall effect of these tissue reactions is broken into two temporal phases: (1) the phase of weeks to months following implantation when a mature foreign body capsule is present around the sensor and (2) the phase of days to weeks immediately following sensor implantation when a provisional matrix of proteins and inflammatory cells envelops the sensor.

Late stage sensor responses to implantation are marked by both an attenuated sensor signal and a significant time lag relative to blood glucose readings. For this later stage of sensor response, a computational model of glucose transport through the interstitial space and foreign body capsule was derived and implemented. Utilizing physiologically relevant parameters, the model was used to mechanistically study how each constituent part of the capsular tissue could affect sensor response with respect to signal attenuation and lag. Each parameter was then analyzed using logarithmic sensitivity analysis to study the effects of different transport variables on both lag and attenuation. Results identified capsule thickness as the strongest determinant of sensor time lag, while subcutaneous vessel density and capsule porosity had the largest effects on attenuation of the sensor signal.

For the phase of early stage tissue response, human whole blood was used as a simple ex vivo experimental system. The impacts of protein accumulation at the sensor surface (biofouling effects) and cellular consumption of glucose in both the biofouling layer and in the bulk (metabolic effects) on sensor response were assessed. Medtronic Minimed SofSensor glucose sensors were incubated in whole blood, plasma diluted whole blood, and cell-free platelet poor plasma (PPP) to analyze the effects of different blood constituents on sensor function. Experimental conditions were then simulated using MATLAB to predict the relative impacts of biofouling and metabolic effects on the observed sensor responses. It was found that the physical barrier to glucose transport presented by protein biofouling did not hinder glucose movement to the sensor surface. Instead, glucose consumption by inflammatory cells was identified as the major culprit for generating poor sensor performance immediately following implantation.

Lastly, a novel, biomimetic construct was designed to mimic the in vivo 3D cellular setting around the sensor for the focused in vitro investigation of early stage effects of implantation on glucose sensor performance. Results with this construct demonstrate similar trends in sensor signal decline to the ex vivo cases described above, suggesting this construct could be used as an in vitro platform for assessing implantable glucose sensor performance.

In total, it may be concluded from this dissertation that instead of sensors "failing" in vivo, as is often reported, that different physiological factors mediate long term sensor function by altering the environment around the implant. For times immediately following implantation, sensor signals are mediated by the presence of inflammatory macrophages adhered on the surface. However, at longer times post-implantation, sensor signals are mediated not by the consumptive capacity of macrophages, but instead by the subcutaneous vessel density surrounding the sensor as well as the porosity and thickness of the foreign body capsule itself. Taken in concert, the results of this dissertation provide a temporal framework for outlining the effects of tissue response on sensor performance, hopefully informing more biocompatible glucose sensor designs in the future.

Synthetic small diameter vascular grafts fail clinically due to thrombosis and intimal hyperplasia. The attachment of endothelial cells (ECs) onto the inner lumen of synthetic small diameter vascular grafts can improve graft patency; however, significant challenges remain that prevent wide clinical adoption. These issues include difficulties in the autologous sourcing of ECs, the lack of attachment, growth and retention of the layer of ECs to the graft lumen, and the maintenance of an anti-thrombotic and anti-inflammatory profile by the layer of ECs.

This dissertation describes the isolation, characterization, and use of endothelial progenitor cells (EPCs) to improve the performance of small diameter vascular grafts. First, EPC isolation efficiency and expression of critical EC markers was compared between young healthy volunteers and patients with documented coronary artery disease (CAD). EPCs were isolated and expanded from patients with CAD and had a similar phenotype to EPCs isolated from healthy donors, and a control population of human aortic ECs. Second, we assessed the ability to enhance the anti-thrombotic activity of patient derived EPCs through the over expression of thrombomodulin (TM). In vitro testing showed TM-transfected EPCs had significantly increased production of key anti-thrombotic molecules, reduced platelet adhesion, and extended clotting times over untransfected EPCs. Finally, native and TM-transfected EPCs were seeded onto small diameter vascular grafts and tested for their ability to improve graft performance. EPCs sodded onto the lumen of small diameter ePTFE vascular grafts had strong adhesion and remained adherent during graft clamping and exposure to flow. TM-transfected EPCs improved graft anti-thrombotic performance significantly over bare grafts and grafts seeded with native EPCs. Based on these promising in vitro results, grafts were implanted bilaterally into the femoral arteries of athymic rats. Bare grafts and grafts with air removed clotted and had only 25% patency at 7 days. In contrast, graft sodded with native EPCs or TM-transfected EPCs had 87% and 89% respective patency rates. High patency rates continued with 28 day implant testing with EPC sodded grafts (88% Native; 75% TM). There were no significant differences in patency rates at 7 or 28 days between native and TM-transfected grafts. These in vivo data suggest patient blood-derived EPCs can be used to improve the performance of small diameter vascular grafts.