Cancer metastasis directly accounts for an estimated 90% of all cancer related deaths and is correlated with the presence of malignant cells in systemic circulation. This observed relationship has prompted efforts to develop a fluid biopsy, with the goal of detecting these rare cells in patient peripheral blood as surrogate markers for metastatic disease as a partial replacement or supplement to tissue biopsies. Numerous platforms have been designed, yet these have generally failed to support a reliable fluid biopsy due to poor performance parameters such as low throughput, low purity of enriched antigen positive cells, and insufficiently low detection thresholds to detect poor expressed surface markers of target cell populations. This work describes the development of a rapid cell sorting technology called Antigen Specific Lysis (ASL) based on photo-crosslinked polymer encapsulation to isolate tumor cells in suspension. In the first study, we characterize the chemical and structural properties of the surface-initiated polymer films formed directly on mammalian cell surfaces. Coated populations are shown to remain highly viable after coating formation. Biomolecular transport is examined though film coatings on cellular substrates using fluorescent, time-resolved confocal microscopy and diffusivity estimates are generated for these materials. In the next study, a lysis-based cell isolation platform is described in which marker positive cells can be specifically coated in a heterogeneous cell suspension. Anionic surfactants lyse virtually 100% of uncoated cells while fully encapsulated cells remain protected, and are then easily collected by centrifugation. We report that purified cells are released from polymeric coatings to yield viable and functional populations. We monitor cell response throughout the isolation process by multiple techniques, and report viability >80% after the sorting process. Lastly, we examine the response of process yield on the level of photoinitiator loading on target populations. Streptavidin-fluorochrome loading was quantitatively assessed on a panel of markers, both epithelial and mesenchymal, on representative model breast and lung cancer cells. We report that ASL is fundamentally capable of achieving 50-60% yield which is promising for fluid biopsy applications. Finally, both EpCAM and metastatic targeting strategies are then compared to covalently biotinylated samples to inform future robust targeting strategies.

Bone and cartilage constructs are often plagued with mechanical failure, poor nutrient transport, poor tissue ingrowth, and necrosis of embedded cells. However, advances in computer aided design (CAD) and computational modeling enable the design of scaffolds with complex internal michroarchitectures and the a priori prediction of their transport and mechanical properties, such that the design of constructs satisfying the needs of the tissue environment can be optimized. The goal of this research is to investigate the capability of additive manufacturing technologies to create designed microarchitectured tissue engineering scaffolds for bone and cartilage regeneration. This goal will be achieved by pursuing the following two objectives: (1) the manufacture of bioresorbable thermoplastic scaffolds by selective laser sintering (SLS) (2) and the manufacture of hydrogel scaffolds by large area maskless photopolymerization (LAMP). SLS is a laser based additive manufacturing method in which an object is built layer-by-layer by fusing powdered material using a computer-controlled scanning laser. LAMP is a massively parallel ultraviolet curing-based process that can be used to create hydrogels from a photomonomer on a large-scale (558x558mm) while maintaining extremely high feature resolution (20µm). In this research, SLS is used to process polycaprolactone (PCL) and composites of PCL with hydroxyapatite (HA) for bone tissue engineering applications while LAMP is used to process polyethylene glycol diacrylate (PEGDA) which can be used for hard and soft tissue applications.

<p>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. </p><p>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. </p><p>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. </p><p>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.</p><p>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.</p> / Dissertation

Cancerous solid tumors can migrate and lead to metastatic bone disease. Drugs prescribed to reduce bone resorption from metastasis, such as zoledronic acid and the RANKL-inhibitory antibody Denosumab, cause side effects such as osteonecrosis of the jaw when delivered systemically. This project used two biocompatible materials, acrylic bone cement (PMMA) and poly(lactic-co-glycolic acid) (PLGA), to incorporate and sustain release of anti-resorptive agents. Results showed similar mechanical properties for acrylic bone cements loaded up to 6.6% drug by weight. Results showed sustained zoledronic acid release for 8 weeks from both systems, with PMMA releasing up to 22% of loaded drug and PLGA films releasing over 95%. The antibody release rate was lower, with the majority of antibody still inside the PLGA films after 8 weeks. In vitro bioactivity remained above 50% for zoledronic acid eluted from both materials at early, middle, and late time points. This study sheds light on the behavior of these biocompatible polymers at high drug weight percent loadings compared to previous studies. PLGA demonstrated superior release kinetics but inferior bioactivity of eluted drug. By incorporating anti-resorptive drugs into locally implantable materials, this work could lead to a treatment offering improved quality of life for cancer patients.

Creating effective cellular interfaces that can provide specific cellular signals is important for a number of fields ranging from tissue engineering to biosensors. Electroconducting polymers, especially polypyrrole (PPy), have attracted much attention for use in numerous biomedical applications since they provide a potential platform for local delivery of electrical stimuli to target tissues. To effectively modulate cellular functions at neural interfaces, it is essential to incorporate a range of extracellular cues into conducting polymers according to specific applications, such as nerve guidance conduits and implantable neural probes. For nerve regeneration scaffolds, three dimensional forms are desired for control of critical properties, such as porosity, mechanical strength, and topography. However, most researchers have worked on conventional two-dimensional PPy films, which cannot mimic a native three-dimensional architecture. Thus, a portion of my work has focused on introducing three-dimensional nanofibrous features into PPy. I have investigated various coating conditions to obtain uniform and conductive nanofibers. Effectiveness of electrical stimulation through the conducting nanofibers was confirmed by in vitro PC12 cell culture. The effects of different conducting nanofiber topographies (random and aligned) on cell adhesion and neurite outgrowth were examined in conjunction with electrical stimulation. The benefits of immobilized-NGF could be combined with electrical stimuli, which could be an ideal platform for neural tissue engineering scaffolds. Thus, I have modified conducting polymers to display neurotrophic activity. Nerve growth factor (NGF) was chemically immobilized on two dimensional and three dimensional PPy substrates. Specific chemical conjugation was achieved and characterized using diverse techniques. Immobilized NGF was as effective as exogenous NGF in medium in inducing neurite development and extension. NGF immobilized on functionalized PPy substrates was stable in a physiological solution and under electrical stimulation, indicating effective prolonged activity. I also investigated another important application of conducting polymer-based materials for neural interfacing - passivating electrodes with a biocompatible polysaccharide, hyaluronic acid (HA). I synthesized electrically polymerizable HA by chemically conjugating amine-functionalized pyrrole derivatives with HA. This coating was stable under physiological conditions for three months and resistant to enzymatic degradation. In vitro studies have shown the minimal adhesion and migration of astrocytes on the HA-coated electrodes. Implantation of HA-coated commercial probes into rat cortices for three weeks revealed attenuated reactive astrocyte responses from the coated wires, and the importance of glial interaction with non-conducting sites was demonstrated. / text

Despite recent advances in surgical technique and the development of numerous therapeutic agents, the formation post surgical adhesions (PSA) continues to cause complications for many patients. In this research, we have employed a rational system to develop a novel treatment to address this clinical need. Based on an understanding of the biochemical events that lead to PSA formation, a series of targeted polymeric biomaterials was designed to interrupt the fibrin gel matrix propagation and suppress PSA formation. Using group transfer polymerization, a series of well controlled block copolymers of polyacrylic acid and poly(ethylene glycol-methacrylate) based materials was synthesized. Subsequent functionalization with the pentapeptide Cys-Arg-Glu-Lys-Ala (CREKA) was employed to target the materials to fibrin as a marker of pro-adhesive sites. While preliminary testing of the untargeted materials verified their ability to suppress non-specific protein adsorption to model surfaces, numerous in vitro tests were conducted to study the ability to inhibit fibrin gel propagation. The ability to inhibit both the rate and quantity of fibrinogen deposition to a fibrin coated surface has been demonstrated. In addition, the rate of fibrin gel propagation and the degree of cellular attachment can modulated. Taking advantage of the systematic variation in structure facilitated by the robust synthetic methodology employed, statistical analysis was used to elucidate the structureproperty relationships governing the performance of these materials. The most important factors that lead to enhanced performance in in vitro tests are the length of PEG chain and number of peptide units conjugated to the polymer: increasing PEG chain length and increasing the number of peptides conjugated to the polymer both improve performance in all tests. The synthetic methods that have been developed, in conjunction with the experimental results, will be used to direct future studies, including cytotoxicity and animal studies.

Cell cultures provide researchers the opportunity to observe cell behavior in response to specific, well-defined environmental cues, leading to insights that enable better engineering design for tissue culture and other biomedical applications. Chemical and electrical stimuli have been successfully applied to cultured cells to approximate aspects of the dynamic conditions experienced in vivo. However, in vitro topographical cues have mostly been limited to static substrates that do not subject cells to the dynamic conditions they experience in vivo when tissue remodels during development and wound healing. Delivering dynamic topographical cues to cultured cells can answer long-standing questions about mechanisms of cell morphology changes. Such capabilities could also facilitate engineering of wound-healing matrices and nerve guidance conduits by promoting migration of cells and providing directional guidance to cellular processes. This dissertation describes the development of approaches for introducing in situ topographical cues to cell cultures and inducing responses such as neurite guidance and cell alignment. Both strategies undertaken in this work make use of multiphoton-promoted photochemistry to print and manipulate three-dimensional microscopic protein hydrogel structures. In one approach, a technique referred to as micro-3D printing, topographical guidance cues are printed in the proximity of cultured cells to guide the growth of cellular processes. By translating a tightly-focused pulsed laser beam through a printing reagent solution flooding cultured cells, features are printed that provide physical guidance to extending neurites from NG108-15 cells, a neuronal model cell type. In another approach, an innovative technique known as micro-3D imprinting is developed for producing micrometer-scale depressions on the surfaces of photoresponsive protein hydrogels. The impact of various experimental parameters on topographical feature dimensions is characterized. Micro-3D imprinting is used to introduce dynamic topographical changes on a cell culture substrate, demonstrating that NIH-3T3 cells, a fibroblast cell model, alter their morphology and alignment in response to the introduction of a grooved surface topography. This set of approaches introduces new tools to the repertoire of cell biologists for exploring the behavior of cells growing in a spatio-temporally dynamic environment, opening possibilities for studies of cellular behavior in conditions that may better reflect environments cells experience in vivo. / text

<p>In this thesis different aspects of calcium-aluminate (CA) as biomaterial are presented. Calcium aluminate is a chemically bonded ceramic with inherent properties making it suitable for use as biomaterial in some applications. In this thesis the emphasis is put on the basic chemical, physical and mechanical properties that may be achieved using the CA system as well as synthesis of the CA raw material. The basis for using CA in any application is the synthesis of the raw material. Different synthesis routes for producing CA are presented with focus on high temperature routes and the micro-structural and phase development during synthesis. As a base for further understanding of the CA properties a thorough outline of the reaction chemistry for CA is presented also including a description of how the reactions may be controlled and how formulations can be designed. The surface reactions of CA when subjected to simulated body fluid showed that CA is <i>in vitro</i> bioactive. An <i>in vivo</i> study in teeth also indicates that CA produces apatite at the tooth material interface. Dental materials are subjected to a harsh environment in the mouth with high mechanical forces, erosion and thermal changes. Also the demands on precise handling characteristics are high. For these reasons the <i>in vitro</i> evaluation of physical and mechanical properties are important. In this work several mechanical and physical properties of Ca-based formulations for dental applications has been tested using different methods. Some attention is also put on the specific characteristics of CA and the difficulties that arise when new material classes needs to be tested according to consensus standard methods. Finally studies on a CA-based formulation intended for Vertebroplasty is presented. The studies include basic mechanical properties as well as testing the material in an <i>in vitro</i> model utilising synthetic cancellous bone.</p>

Novel net shaping technique Laser Engineered Net shaping™ (LENS) laser based manufacturing solution (Sandia Corp., Albuquerque, NM); Laser can be used to deposit orthopedic implant alloys. Ti-35Nb-7Zr-5Ta (TNZT) alloy system was deposited using LENS. The corrosion resistance being an important prerequisite was tested electrochemically and was found that the LENS deposited TNZT was better than conventionally used Ti-6Al-4V in 0.1N HCl and a simulated body solution. A detailed analysis of the corrosion product exhibited the presence of complex oxides which are responsible for the excellent corrosion resistance. In addition, the in vitro tests done on LENS deposited TNZT showed that they have excellent biocompatibility. In order to improve the wear resistance of the TNZT system boride reinforcements were carried out in the matrix using LENS processing. The tribological response of the metal matrix composites was studied under different conditions and compared with Ti-6Al-4V. Usage of Si3N4 balls as a counterpart in the wear studies showed that there is boride pullout resulting in third body abrasive wear with higher coefficient of friction (COF). Using 440C stainless steel balls drastically improved the COF of as deposited TNZT+2B and seemed to eliminate the effect of “three body abrasive wear,” and also exhibited superior wear resistance than Ti-6Al-4V.

The polymer gels especially hydrogels have a very special structure and useful features such as unusual volume phase transition, compatibility with biological systems, and sensitivity to environmental stimuli (temperature, pH value, electric field, light and more), which lead to many potential applications in physical and biochemical fields. This research includes: (1) the theoretical and experimental studies of polymer gels on swelling kinetics, spinodal decomposition, and solution convection in gel matrix; (2) applications of polymer gels in wound dressing, tissue-simulating optical phantom and gel display. The kinetics of gel swelling has been theoretically analyzed by considering coupled motions of both solvent and polymer network. Analytical solutions of the solvent and the network movement are derived from collective diffusion equations for a long cylindrical and a large disk gel. Kinetics of spinodal decomposition of N-isopropylacrylamide (NIPA) polymer gel is investigated using turbidity and ultrasonic techniques. By probing movement of domains, a possible time-dependent gel structure in the spinodal decomposition region is presented. Theoretical studies of solution convection in gel matrix have been done and more analysis on dimensionless parameters is provided. To enhance the drug uptake and release capacity of silicone rubber (SR), NIPA hydrogel particles have been incorporated into a SR membrane. This SR/NIPA composite gel has promising attributes for wound dressing and other uses. Tissue-simulating optical phantom has been synthesized and studied using NIPA solution trapped inside a hydrogel. Polymer gels with engineered surface patterns were implemented. NIPA gel deposited on the surface of an acrylamide gel can be used as responsive gel display. A dynamically measurement technique of local shear modulus and swelling ratio of gel is presented based on an engineered periodic surface pattern as square array.