Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.

Craniofacial defects resulting from trauma and resection present many challenges to reconstruction due to the complex structure, combinations of tissues, and environment, with exposure to the oral, skin and nasal mucosal pathogens. Tissue engineering seeks to regenerate the tissues lost in these defects; however, the composite nature and proximity to colonizing bacteria remain difficult to overcome. Additionally, many tissue engineering approaches have further hurdles to overcome in the regulatory process to clinical translation. As such these studies investigated a two stage strategy employing an antibiotic-releasing porous polymethylmethacrylate space maintainer fabricated with materials currently part of products approved or cleared by the United States Food and Drug Administration, expediting the translation to the clinic. This porous space maintainer holds the bone defect open allowing soft tissue to heal around the defect. The space maintainer can then be removed and one regenerated in the defect. These studies investigated the individual components of this strategy. The porous space maintainer showed similar soft tissue healing and response to non-porous space maintainers in a rabbit composite tissue defect. In humans, the porous space maintainers were well tolerated and maintained a soft tissue envelope for closure after implantation of a bone regeneration technology. The antibiotic-releasing space maintainers showed release of antibiotics from 1-5 weeks, which could be controlled by loading and fabrication parameters. In vivo, space maintainers releasing a high dose of antibiotics for an extended period of time increased soft tissue healing over burst release space maintainers in an infected composite tissue defect model in a rabbit mandible. Finally, stabilization of bone defects and regeneration could be improved through scaffold structures and delivery of a bone forming growth factor. These studies illustrate the possibility of the two stage strategy for repair of composite tissue defects of the craniofacial complex.

Tissue engineering is a multidisciplinary subject aimed at producing the immunologically tolerant artificial tissues/organs to repair or replace damaged ones. In this field, tissue scaffold plays a key role to support cell growth and new tissue regeneration. For fabrication of tissue scaffolds with individual external geometry and predefined inner structure, rapid prototyping (RP) systems based on fluid dispensing techniques have proved to be very promising. The present research conducted a comprehensive study on the dispensing-based fabrication process. <p>First of all, the scaffold materials are characterized in terms of their biocompatibility and flow behaviour. The biocompatibility of biomaterials of PLLA, PCL, collagen, chitosan, and gelatine is evaluated in terms of supporting neuron cells adhesion and outgrowth. Chitosan solution (2% w/v) in acetic acid is shown to be the most promising among the examined biomaterials for the fabrication of nerve tissue scaffolds. Its non-Newtonian flow behaviour is identified by using a commercial rheometer. <p>In the fabrication process, the flow rate of biomaterials dispensed, the profile of strand cross-sections, and the scaffold porosity are very important and must be precisely controlled. A model is developed to represent the flow rate of biomaterials dispensed under the assumptions that the flow is incompressible, steady, laminar, and axisymmetric. Also, the profile and size of line strands at different layers and portions are modeled based on the Young-Laplace equation. Thus the dispensing-based fabrication process can be predicted in terms of the flow rate and the scaffold porosity. <p>The effects of operation conditions on the fabrication result are identified theoretically and experimentally. Simulation result shows that a higher driving pressure, a higher temperature, and a larger needle diameter will result in a larger size of the strand cross-sections and lower scaffold porosity. The change pattern, however, is nonlinear, which is affected by the fluid surface tension and non-Newtonian flow behaviour of scaffold biomaterials. <p>To verify the effectiveness of the developed models, experiments were carried out on a commercial dispensing system (C-720, Asymtek, USA). To avoid the possible error derived from the temperature difference between the dispensing system and the rheometer, a new method is presented to characterize the fluid properties used for model predictions. Experimental results illustrate that the developed models, combined with the new identification method, are very promising to predict the dispensing-based fabrication process.

Biologically-inspired materials have emerged as promising substrates for enhanced repair in various therapeutic and regenerative medicine applications, including nervous and vascular tissues, bone, and cartilage. These strategies focus on the development of materials that integrate well-characterized domains from biomacromolecules to mimic individual functions of the extracellular matrix (ECM), including cell adhesive motifs, growth factor binding sites, and protease sensitivity. A vital property of the ECM is the fibrillar architecture arising from supramolecular assembly. For example, the fibrillar structure of fibronectin (FN) matrices modulates cell cycle progression, migration, gene expression, cell differentiation, and the assembly of other matrix proteins. Current biomaterials do not actively promote deposition and assembly of ECM. In this research, we describe the rational design and investigation of non-fouling biomimetic surfaces in which an oligopeptide sequence (FN13) from the self-assembly domain of FN is tethered to non-fouling substrates. This surface modification directs cell-mediated co-assembly of robust fibrillar FN and type I collagen (COL) matrices reminiscent of ECM, and increases in cell proliferation rates. Furthermore, the effect of this peptide is surface-directed, as addition of the soluble peptide has no effect on matrix assembly. We have also identified a critical surface density of the immobilized peptide to elicit the full activity. These results contribute to the development and design of biomimetic surface modifications that direct cell function for biomedical and biotechnology applications.

In the US alone, more than 500,000 coronary artery bypass procedures are performed annually. Tissue engineering shows the potential to construct functional grafts to overcome the limited availability of autologous saphenous veins, relatively poor elasticity and low compliance of synthetic materials (mainly Dacron and polytetrafluoroethylene). In order to meet the low modulus associate with myocyte differentiation, the high suture retention and an ultimate tensile strength (UTS) sufficient to withstand implantation and peak physiological stresses, we designed and characterized a multi-component scaffold comprised of polyurethane electrospun mesh layers bonded together by a fibrin hydrogel matrix. We have demonstrated this composite construct retains the high tensile strength and suture retention strength but displays a "J-shaped" mechanical response similar to that of native coronary artery. To improve our design, poly(ethylene glycol) diacrylate based hydrogel system was utilized as a blank slate to study the phenotypic regulation by cell material interactions. Fibrinogen, fibronectin, laminin and collagen type IV were incorporated into the hydrogel to mimic the stimuli from extracellular matrix (ECM) proteins. Surprisingly, no significant effect was detected on induction of smooth muscle cell (SMC) differentiation marker expression, activation of mitogen-activated protein (MAP) kinases pathway, or alteration of surface integrin expression profile. However, fibronectin showed repression of undesired phenotypes in SMC differentiation. In contrast to ECM proteins, glycosaminoglycans (GAGs) showed more influence on regulating SMC phenotype. By using a scaffold environment intended to be mimetic of early atherosclerosis, the impact of GAG identity on SMC foam cell formation was explored. We focused on chondroitin sulfate C (CSC), dermatan sulfate (DS), and an intermediate molecular weight hyaluronan (HA_IMW, ~400 kDa), the levels and/or distribution of which are significantly altered in atherosclerosis. CSC and DS hydrogels were associated with greater SMC phagocytosis of apolipoprotein B than HA_IMW gels. However, only SMCs in DS constructs maintained increased expression of adipocyte marker A-FABP relative to HA_IMW gels over 35 days of culture. Combined, our results suggested interesting roles for fibronectin and HA_IMW in repression of undesired phenotypes in SMC differentiation, which could give insights into rational design of novel biomaterials for vascular tissue engineering applications.

This dissertation describes the development and application of an approach for creating multiphoton crosslinked protein microchambers to characterize bacterial group behaviors in small populations (~10¹ - 10⁵ cells). Porous protein cavities of desired size and geometry are made with sub-micrometer three-dimensional (3D) resolution using a dynamic mask-based multiphoton lithography (MPL) technique previously developed in the Shear Group. One aspect of this dissertation focuses on basic characterizations of properties of these materials key to their utility in studying entrapped bacteria. Studies are presented on the mass transport across microcavity walls (important for growth and signaling), and the temperature- and light-induced volume response (used to open/close microchamber apertures for cell entry/exit). Fabrication parameters are optimized to trap and manipulate small populations under in vitro conditions that are relevant to in vivo environments. The ability to culture bacteria at physiologic growth rates within protein microstructures has provided a unique platform to study the group behaviors of quorum sensing (QS) and antibiotic resistance in biologically relevant population sizes, a platform I have exploited to study group behaviors in the opportunistic pathogen, Pseudomonas aeruginosa. This work presents the first experimental evidence supporting the efficiency sensing QS model by showing that QS-dependent gene expression is affected by both the population size and density, as well the external flow rate in the surrounding environment. The onset of antibiotic resistance is observed in as few as ~150 P. aeruginosa cells, and is shown to increase with cell density. Lastly, the development of a gelatin-based MPL approach that is demonstrated in situ to create confined populations of non-motile cells, free-floating 3D cultures, nested colonies, and spatially patterned polymicrobial communities of P. aeruginosa and Staphylococcus aureus. / text

Many important biomaterials are composed of multiple layers of networked fibers. A prime example is in the field of tissue engineering, in which damaged or diseased native tissues are replaced by artificial tissues that are grown on fibrous polymer networks. For load bearing tissues, it is critical that the mechanical behavior of the engineered tissue be similar to the behavior of the native tissue that it will replace. In the case of soft tissues such as heart valves, the macroscale mechanical behavior is highly anisotropic and nonlinear. This behavior is a result of complex deformations of the collagen and elastin fibers that form the extracellular matrix (ECM). The microstructure of engineered tissues must be properly designed to reproduce this unique macroscopic behavior. While there is a growing interest in modeling and simulation of the mechanical response of this class of biomaterials, a theoretical foundation for such simulations has yet to be firmly established. This work introduces a method for modeling materials that have a layered, fibrous network microstructure. Methods for characterizing the complex network geometry are first established. Then an algorithm is developed for generating realistic network geometry that is a good representation of electrospun tissue scaffolds, which serve as the primary synthetic structure on which engineered tissues are grown. The level of fidelity to the real geometry is a significant improvement on previous representations. This improvement is important, since the scaffold geometry has a strong influence over the macroscopic mechanical behavior of the tissue, cell proliferation and attachment, nutrient and waste flows, and extracellular matrix (ECM) generation. Because of the importance of scaffolds in tissue formation and function, this work focuses on characterizing scaffold network geometry and elucidating the impact of geometry on macroscale mechanics. Simulation plays an important role in developing a detailed understanding of scaffold mechanics. In this work, Cosserat rod theory is used to model individual fibers, which are connected to form a network that is treated as a representative volume element (RVE) of the material. The continuum theory is the basis for a finite element discretization. The nonlinear equations are solved using Newton's method in a parallel implementation that is capable of accurately capturing the large, three-dimensional fiber rotations and large fiber stretches that result from the large macroscopic deformations experienced by these biomaterials in their natural environment. Comparisons of simulation results with existing analytical models of soft tissues show that these models can predict the behavior of scaffold networks with reasonable accuracy, despite the significant differences between soft tissue and scaffold network microstructural geometry. The simulations also reveal how macroscale loading is related to the microscale fiber deformations and the load distribution among the fibers. The effects of different characteristics of the microstructural geometry on macroscopic behavior are explored, and the implications for the design of scaffolds that produce the desired macroscopic behavior are discussed. Overall, the improved modeling of electrospun scaffolds presented in this work is an important step toward designing more functional engineered tissues.

Lung cancer has the highest mortality rate relative to all types of cancers, and unfortunately there still exist numerous challenges towards decreasing this rate. One of these challenges is gaining a clear understanding of why metastatic lung cancer cells attach and detach to colonize other areas of the body. Reports suggest that the attachment of cells to secondary tumor sites does not occur randomly. It is theorized that both the physical and chemical properties of the tissue are able to create a suitable environment for their adhesion. Therefore, the motivation for the work presented herein is to use dynamic thermoresponsive polymer surfaces as a tool towards unraveling this seemingly mysterious behavior of metastatic cancer cells. This type of polymer is able to swell and deswell as a function of temperature. As such, spin-cast thin films of this polymer provide for topographies that have been used to investigate how highly metastatic lung cancer cells are able to rearrange their structure, specifically the cytoskeleton. Changes in cell to surface anchorage as a function of thin film structure can also monitored. One of the most studied reversibly binding surfaces is poly(Nisopropylacrylamide) (PNIPAAm), which has been considered for the past two decades as a non-destructive method for the harvest of confluent cell sheets. As a result of this property, a series of photocrosslinkable copolymers based on PNIPAAm and methacroyloxybenzophenone (MaBP) have been developed. Coatings are created by spin-casting the polymer followed by ultraviolet (UV) radiation, which triggers the transition in the benzophenone groups. This leads to the formation of a biradicaloid triplet that abstracts a hydrogen from a neighboring aliphatic C-H group, leading to a stable C-C bond. The characteristics of the polymer film, i.e. thickness, pattern, and topography, can be tuned during the spin casting and subsequent exposure/ developing process.The ease of tunability of this polymer allows for the investigation of the aforementioned parameters and their possible effects on bioadhesion.

In 2015, American Heart Association (AHA) reported that 1 in 9 deaths are attributed to Heart failure (HF), the number one killer in the world. While advancements in interventional cardiology in conjunction with pharmacotherapies have significantly reduced the rate of mortality following MI, there has been a corresponding rise in chronic heart failure (CHF) in surviving patients, largely attributed to the limited regenerative capacity of the heart and the inadequate healing response. Myocardial ischemic injury triggers an exuberant local and systemic inflammation, and the extent and quality of the cardiac wound healing process is intricately tied to the delicate equilibrium of this inflammatory response. While cardiac regeneration is an important goal, it is imperative in the meantime to explore therapeutic strategies that target these inflammatory mediators of early cardiac repair. These interventions to influence and improve cardiac wound healing can represent a new therapeutic window to halt the progression of heart failure between the few hours that may be used to limit infarct size by reperfusion and an irreversible non-contractile cardiac scar. This dissertation examines three therapeutic delivery strategies aimed at modulating the immune response to enhance cardiac repair in rodent models MI: 1) Polyketal nanoparticles as siRNA delivery vehicles for antioxidant therapy; 2) Spherical nucleic acid particles for anti-inflammatory therapy and; 3) Bioactive PEG (polyethyleneglycol)-based hydrogel for immunomodulation. The work presented here applies novel nucleic acid delivery strategies for cardiac gene silencing and has contributed to new knowledge with regard to modulating the immune response following MI.

Dendritic cells (DCs) are regulators of the immune system and express a class of pattern recognition receptors known as C-type lectin receptors (CLRs) to recognize and respond to carbohydrates (glycans). Dendritic cells are hypothesized to be key mediators in the immune response to implanted materials and ligation of CLRs has been shown to have diverse effects on DC phenotype ranging from tolerogenic to pro-inflammatory. Thus, designing future biomaterials and combination products that harness the potential of CLR ligation on DCs has great promise. Additionally, many of the proteins which adsorb to biomaterials when implanted are glycosylated and thus understanding this interaction would provide further insight into the host response to currently implanted materials. However, DC responses to glycans presented from non-phagocytosable surfaces has not been well characterized and optimal factors for DC phenotype modulation by surface presented glycans are unknown. Additionally, studies relating DC response to glycan structures from soluble and phagocytosable displays to that of non-phagocytosable display have not been performed. This is of critical importance to the field because of the extremely limited supply of complex glycan structures that are able to be obtained. Because of this limited supply of glycans the trend in glycomics has been toward creation of glycan microarrays to assess initial candidates of interest for further study. However, the assumption that cell response to these glycoconjugate microarrays is equivalent to soluble or phagocytosable conjugates has not been validated. Therefore, the purpose of this study was to 1) determine the optimal molecular contextual variables of glycoconjugate presentation from a non-phagocytosable surface, namely, charge, density, and glycan structure for modulating DC phenotype; and 2) determine if modality of glycoconjugate presentation, i.e. soluble, phagocytosable, or non-phagocytosable will modulate DC phenotype differentially. To determine the effect of the molecular contextual variables primary human immature DCs (iDCs) were exposed to a range of adsorbed glycoconjugates in a 384 well plate and their subsequent phenotype assessed via a novel in house produced high throughput (HTP) assay. Bovine serum albumin (BSA) was modified to have a range of glycan densities and isoelectric points to determine which of these were optimal for DC phenotype modulation. Next, several poly-mannose structures were presented to DCs to determine if DC response was structure specific. Finally, contextual variables were modeled in a multivariate general linear model to determine underlying trends in DC behavior and optimal factors for glycan presentation from non-phagocytosable surfaces. To determine the effect of the modality of glycoconjugate display on DCs, optimized glycoconjugates from 1) were adsorbed to the wells of a 384 flat well plate, delivered at varying soluble concentrations, or adsorbed to phagocytosable 1 µm beads and subsequent DC phenotype assessed via the HTP assay. The cell response to the glycoconjugates was then validated to be CLR mediated and the DC response to glycan modality was modeled in another general linear model. Results from these studies show that highly cationized high density glycoconjugates presented from non-phagocytosable flat well display modulate DC phenotype toward a pro-inflammatory phenotype to the greatest extent. Additionally, significant impacts on DC phenotype in response to adsorbed conjugates can be seen when grouping glycan structure by terminal glycan motif. Finally, DC response to glycoconjugates were found to be CLR mediated and that each modality of glycan display is significantly different, in terms of DC phenotype, from the others. These results provide indications for the future design of glycan microarray systems, biomaterials and combination products. Furthermore, this work indicates that different mechanisms are involved in binding and processing of surface bound versus soluble glycoconjugates. With further study these differences could be harnessed for use in the next generation of biomaterials.