Clear aligners are becoming a popular mode of treatment in orthodontics. Despite their increase in use and demand, there is a general lack of literature regarding the fundamental intrinsic mechanical properties of clear aligners. Therefore, it becomes difficult for patients and clinicians to compare the performances of different aligner products. Existing studies have investigated the biomechanical properties of raw plastic sheets used to make aligners, and sheets thermoformed over simple geometries (i.e. standard blocks). However, the results obtained from standard blocks have limited clinical significance. Instead, aligner samples with actual dental morphology need to be tested to yield clinically meaningful data. Therefore, the aim of this study is twofold. First, we set forth a standardized protocol to characterize significant properties of both raw thermoplastic sheets and fabricated aligners. Second, we evaluated the effects of processing and intraoral components on the aligner materials. These components include heat treatment during aligner manufacturing processes, water soaking, and pH alternations.To characterize the mechanical properties of the thermoplastic sheets and fabricated aligners, we first characterized the structural compositions of aligners by measuring the degree of crystallinity of each material. Second, we used TA Instruments� Dynamic Mechanical Analyzer (DMA) to quantify critical properties such as stress relaxation and creep of aligner samples with high accuracy and precision. Third, we compared strength and resistance from macroscopic and microscopic levels to analyze crack resistance and microhardness. We also measured Arrhenius activation energy and glass transition temperature to study physical phase transition and molecular interactions after thermomechanical changes. Moreover, we mapped out thickness distributions and quantified the light transmission of different aligner materials. Lastly, we examined the effects of heat treatment and water-polymer interactions on the aforementioned mechanical properties. Our findings suggest that thermoforming processes reduced aligner thickness and stiffness, increased hydrophilicity and water absorption, modified crack resistance, transparency, glass transitioning temperature, and microhardness in some materials. Water soaking resulted in reduced initial stress and Arrhenius energy, but increased stress relaxation and creep compliance compared to the unsoaked controls. Aligners with different chemical compositions exhibited distinct mechanical characteristics and responded differently to the changes in heat treatment and water soaking. In summary, we found that heat treatment and water immersion can lead to significant degradation of various important mechanical and thermal properties of aligners. Thus, the clinical performances of aligners may be affected by thermoprocessing and intraoral use. The long-term goal is to share our protocols with other independent institutions so that we can create collaborative monitoring and analysis of all aligner products in the market. The clinical impact is to improve patient care by allowing clinicians to make evidence-based decisions in material selection.

Chronic wounds affect approximate 6.5 million individuals in the United States alone. These types of wounds are usually locked in a permanent cycle between tissue inflammation, remodeling, and breakdown without appreciable progress towards complete healing. Current methods of treatment include consistent delivery of growth factors in hopes of easing the soft tissue through the proper stages of repair. While there is evidence that dose delivery of growth factors is more desirable than continuous dosing, there are no current on-demand delivery platforms for such growth factors. Our studies show the feasibility of a duplex oligonucleotide tethering system to create a heat-sensitive linker that can release a growth factor on-demand. Our system has the ability to last for a minimum of five days and has the potential to be incorporated into other biodegradable implants for delivery.

Cartilage regeneration is a promising field with many attempts at creating a tissue-engineered cartilage substitute. While many tissue-engineered skin and bone products exist and continue to enter the market, there remains no FDA-approved solution to regenerate the avascular, aneural cartilage tissue other than autologous chondrocyte implantation, which is both costly and requires weeks of cell culture time, not to mention healthy cartilage from which to harvest the chondrocytes. Many attempts to create biomaterial scaffolds for implantation are being made, yet these technologies often require seeded cells, and the resulting cartilage (if there is proper cartilage regeneration and not fibrocartilage production) often lacks the zonal architecture that gives native cartilage its unique structural and functional properties. Lots of recent focus has been given to growth factor delivery from within scaffolds to assist cartilage regeneration, specifically to signal ECM production by chondrocytes. However, most diffusion-based growth factory delivery systems suffer from a large burst release and do not provide a sustained release over the course of many weeks, which is required for cartilage production. Materials with growth factors chemically conjugated to their surface offer some advantage of a gradual release as the bulk material degrades and permanent presentation of the bioactive molecule, but certain growth factors require internalization for activation of their signaling cascades and conjugation chemistry to surfaces can be inefficient or render the protein biologically inactive. This project aims to develop an acellular scaffold implant with biomimetically designed micro- and macro-architecture that also delivers a chondrogenic growth factor via a sustained release mechanism to promote cartilage regeneration. This scaffold would be implanted into the joint during resurfacing and would facilitate the instant uptake of underlying bone marrow and residing mesenchymal stem cells, which would then be guided to differentiate into chondrocytes once in contact with the slowly diffusing chondrogenic growth factor. This system would address the many challenges facing cartilage tissue engineers today and has the potential to become an off-the-shelf solution for cartilage regeneration.

Three-dimensional printing (3DP) uses inkjet printheads to selectively deposit liquid binder to adjoin powder particles in a layer-by-layer fashion to create a computer-modeled 3D object. Two general approaches for 3DP have been described for biomedical applications-- direct and indirect 3DP. The two approaches offer competing advantages, and both are limited by print resolution. This study describes materials processing strategies to enhance both indirect and direct 3DP resolution by shrinking.

For indirect 3DP, 3DP resolution was improved by controlled shrinking of net-shape scaffolds. Briefly, porogen preforms are printed and infused with the desired monomer or polymer solution. After solidification or polymerization, the porogen is leached and the polymer is allowed to shrink by controlled drying. Heat treatment is performed to retain the dimensions against swelling forces. The main objectives are to determine the effects of polymer content and post-processing on dimension, microstructure, and thermomechanical properties of the scaffold. For polyethylene glycol diacrylate, reducing polymer content corresponded with greater shrinkage, with maximum shrinkage of ~80 vol% at polymer content of 20%. The secondary heat treatment retains the microarchitecture and new dimensions of the scaffolds, even when the scaffolds are immersed into water. This material processing strategy provides an alternative method to enhance the resolution of 3D scaffolds, for a wide range of polymers, without optimizing the binder-powder interaction physics for each material combination.

For direct 3DP, a novel fabrication method was developed to create prints without organic solvent in the binder and shrinking was induced by a solvent plasticizer. PLGA was first made into microparticles and then mixed with sucrose particles to create the printing powder. A water-based binder was deposited onto the powder layers to create a print and then the polymer microparticles fused together with solvent vapor fusion. The sucrose was then removed by leaching and PLGA scaffolds permanently shrunken ~80% volumetrically in a solution of methanol to a final resolution of ~400 μm. The methanol was acting as a plasticizer by decreasing the Tg below room temperature to allow for the polymer network to collapse upon itself. This approach to increasing the resolution allows for material flexibility of 3DP by increasing the range of materials used in this process since polymer microparticles are fused after printing and polymer-binder printing conditions do not need to be optimized.

This thesis presents novel materials processing strategies to improve the resolution of indirect and direct 3DP resolution for biomaterials.

Current methods for gene delivery utilize nanocarriers such as liposomes and viral vectors that may produce in vivo toxicity, immunogenicity, or mutagenesis. Moreover, these common high-cost systems have a low efficacy of gene-vehicle transport across the cell plasma membrane followed by inadequate release and weak intracellular stability of the genetic sequence. Thus, this study aims to maximize gene transfection while minimizing cytotoxicity by utilizing supersaturated blood-plasma ions derived from simulated body fluids (SBF).

With favorable electrostatic interactions to create biocompatible calcium-phosphate nanoparticles (NPs) derived from biomimetic apatite (BA), results suggest that the SBF system, though naturally sensitive to reaction conditions, after optimization can serve as a tunable and versatile platform for the delivery of various types of nucleic acids. From a systematic exploration of the effects of nucleation pH, incubation temperature, and time on transfection efficiency, the study proposes distinct characteristic trends in SBF BA-NP morphology, cellular uptake, cell viability, and gene modulation. Specifically, with aggressive nucleation and growth of BA-NPs in solution (observed via scanning electron microscopy), the ensuing microenvironment imposes a more toxic cellular interaction (indicated by alamarBlue and BCA assays), limiting particle uptake (fluorescence experiments) and subsequent gene knockdown (quantitative loss of function assays). Controlled precipitation of BA-NPs function to increase particle accessibility by surrounding cells, and subsequently enhance uptake and transfection efficiency. By closely examining such trends, an optimal fabrication condition of pH 6.5-37C can be observed where particle growth is more tamed and less chaotic, providing improved, favorable cellular interactions that increase cell uptake and consequently maximize gene transfection, without compromising cellular viability.

Despite significant improvements in cardiac care over the past 50 years, ischemic heart disease continues to be the leading cause of morbidity and mortality worldwide. While therapies such as percutaneous coronary intervention have greatly improved reperfusion rates in acute myocardial infarction, effective therapies for the prevention and treatment of ischemia-reperfusion injury remain lacking. Some of the causes for this may include certain technical limitations of drug therapy, such as poor bioavailability, which can potentially be addressed through use of nanoparticle-based targeted drug delivery. This thesis evaluates the use of a nanoparticle-based induced Cre system in the development of nanoparticles for ischemia targeting and drug delivery. PLGA/4-Hydroxytamoxifen nanoparticles were fabricated using an emulsion-solvent evaporation method. These nanoparticles were then demonstrated to successfully induce gene recombination in Rosa26-CreER x tdTomato and Rosa26-CreER x Rainbow transgenic mouse models both in vitro and in vivo, through direct intracellular delivery of 4-Hydroxytamoxifen to specific tissues. In addition, tissue specificity of gene recombination was enhanced through attachment of targeting antibodies such as CD31 and CD11b to the nanoparticles. Using this system, the uptake and drug delivery of nanoparticles to cardiac tissue subjected to ischemia-reperfusion injury was studied. Nanoparticle uptake and resultant drug delivery was found to be increased in areas of injury, and this uptake could be further increased by nanoparticle targeting of macrophages in the injury region. Control of vascular permeability through the administration of histamine was also found to influence nanoparticle uptake in certain organs. This research provides a framework for further studies to develop a nanoparticle-based system for delivery of targeted therapeutics for ischemia-reperfusion injury.

Bioactive ceramics, such as calcium phosphate-based materials, have been studied extensively for the regeneration of bone tissue. Accelerated apatite coatings prepared from biomimetic methods is one approach that has had a history of success in both in vitro and in vivo studies for bone regeneration [1]-[4]. However, how cells interact within the apatite microenvironment remains largely unclear, despite the vast literature available today. In response, this thesis evaluates the in vitro interactions of a well-characterized osteoblast cell line with the apatite microenvironment. For this, the cellular response to several aspects of the apatite microenvironment was separately examined in order to piece together a more simplified picture of a complex and dynamic system: (1) the influence of accelerated apatite on local calcium and phosphate concentration, (2) the role of protein adsorption onto apatite surfaces, and (3) apatite surface charge. Furthermore, the immunopotentiating properties of apatite were also characterized by examining monocyte response to 2D and 3D apatite-coated model culture systems in vitro.

A rapid "pull-down" of extracellular Ca2+and PO43- ions onto the apatite surface could be measured upon the incubation of apatites in cell culture medium, suggesting that cells may be subject to changing levels of Ca2+and PO43- within their microenvironment. Changing levels of Ca2+and PO43- are likely to have large implications for the biological response to apatites as increasing concentrations above a certain threshold were confirmed to be cytotoxic. Proteins were found to be critical in the mediation of cell-apatite interactions, as adherence of MC3T3-E1 cells to apatite surfaces without protein coatings resulted in significant levels of cell death within 24 hours in serum-free media. In the absence of protein-apatite interaction, cell viability could be restored upon treatment of the cells with inhibitors to PO43- transport, suggesting that PO43- uptake may play a role in viability. In contrast, rescue was not observed upon treatment with calcium channel inhibitors. The apatite surface charge could be modulated by treating the apatite surface with biomolecular coatings (proteins, polyamino acids), or with non-biological coatings of carbon or gold. In general, surface treatments that resulted in a more negatively-charged apatite surface, relative to that of bare apatite, promoted cell survival in a dose-dependent manner. A potential immunomodulatory role for apatite may contribute to its overall pro-osteogenic capacity, as apatite coatings could enhance monocyte adhesion in the absence of activation factors. Moreover, the presence of monocytes or monocyte conditioned media was shown to promote osteoblastic differentiation on apatite-coated substrates in vitro. Taken together, this investigation provides an initial understanding of the cellular response to various elements within the apatite microenvironment, and may provide the foundation for furthering the development of apatite materials for bone tissue engineering.

Soft tissue loss can be the result of many clinical issues. An engineered injectable material to stimulate soft tissue regeneration would be an improvement for many clinical applications within tissue augmentation including dermal augmentation, periurethral bulking, and gingival augmentation.

Implanted biomaterials are usually held to a standard of biocompatibility meaning that the overall goal for a biomaterial is to have it as inert as possible and to act stealthily when it comes in contact with the body. This is to avoid a chronic foreign body response and ultimately rejection of the biomaterial. The goal of this project was to utilize a mild, transient immune response within the soft tissue of the body to accelerate regeneration of the extracellular matrix proteins and therefore tissue augmentation.

An injectable biomaterial was utilized to increase the volume of the soft tissue, then it was combined with a bioactive coating that elicited an immune response. The injectable material was fabricated out of macroporous poly ε-caprolactone (PCL) microparticles and coated in the bioactive polymer, poly lactic co-glycolic acid (PLGA). The host-material interaction of the bioactive PLGA with biological cells was characterized in vitro by inflammatory cytokine production using a multiplex system. It was found that the co-culture of human fibroblasts and monocytes produced elevated levels of IL-1β, IL-6, GM-CSF, and TNF-α in response to the PLGA biomaterial. Subsequently, the cell infiltration, inflammatory response, blood vessel infiltration, and collagen deposition were analyzed to determine tissue regeneration and healing in vivo. The murine model showed more thorough tissue integration and healing with added porosity and bioactive PLGA coating through blood vessel infiltration and extracellular matrix deposition. The accelerated healing response seen with the bioactive coating is shown to be important to prevent migration of the particles within tissue. Additionally the PLGA coating did enhance the immune response early on in the in vivo studies corroborating with the in vitro findings.

The overall goal of this research was to engineer an injectable material that can be used to induce an acute immune response which will lead to the accelerated production of extracellular matrix proteins to augment tissue and eventually create a soft tissue network with the ultimate clinical application being dermal augmentation, periurethral bulking, or gingival augmentation.

Clinical parameters for dental whitening such as peroxide concentration and treatment time have been empirically derived. However, limited quantitative analyses examine reactivity of hydrogen peroxide on in vivo tooth stains under various catalytic settings. The wide range of possible activators and stains are challenging in creating a standardized tooth model to isolate various effects for clinical applications. This study uses three model systems to determine the effects of heat, light, metal catalysts, and pH on peroxide bleaching. By first using a model chemical stain, alkaline pH levels (7.4), 35°C heat treatment, and 490nm light activation were optimal for increasing overall bleaching efficacy. When the same conditions were applied to bleaching stained bovine teeth using liquid peroxide, only 35°C heat treatment showed catalytic effect, increasing overall luminosity and decreasing yellowness. A final evaluation of the activation parameters on teeth treated with commercially used gel peroxides revealed positive catalytic activity for heat alone. These results, in conjunction with future clinical studies, can provide the basis for optimizing clinical whitening parameters and ultimately control peroxide reactivity to enhance bleaching efficacy while minimizing undesired side-effects.