Volumetric muscle loss (VML) is a debilitating injury which results in full or partial loss of function. Current clinical options utilize tissue grafts and bracing to restore function. Tissue graft implantation oftentimes leads to serious complications, some of which end in graft rejection and thereby necessitate further surgeries and procedures. Polymeric scaffolds show promise as scaffolding systems due to their mechanical properties and overall degradation profiles. Scaffolds need appropriate mechanical properties, 10-60 kPa modulus, and overall degradation times, five days to two weeks, to initiate tissue regeneration. Poly(β-amino ester)s (PBAE), a class of synthetic polymers, act as a safe biocompatible material with overall degradation times that are suitable for healing; however, due to harmful ultraviolet light (UV) irradiation from common crosslinking methods, these scaffold systems cannot be synthesized in vivo. This research presents the development and characterization of blue light (BL) crosslinked PBAEs. BL PBAEs showed vastly higher swelling ratios, 300-400% increase; decreased mechanical strength, an average decrease of 877 kPa in compressive modulus and 431 kPa in tensile modulus; and prolonged degradation patterns, 22% average mass retention. BL PBAEs show mechanical properties and degradation profiles that could be used as a skeletal muscle scaffolds.

The threat of antibiotic resistance and the global rise of pan-resistant bacteria is a serious concern at present. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is frequently associated with multi and pan-drug resistant infections. This research delves into the mechanism of resistance to a class of drugs known as the β-lactams. AmpC β-lactamase encoded chromosomally in P. aeruginosa is one of the predominant causes of resistance to many β-lactams. Previous research on this pathway identified the AmpC regulatory protein - AmpR and elaborated on its regulon in P. aeruginosa. In this dissertation, further investigation in the mechanisms associated with AmpR regulation of AmpC and its connection with the cell-wall recycling pathway is explored. Cell-wall recycling, a common phenomenon in both Gram-positive and negative bacteria is investigated in some detail in P. aeruginosa for the first time. The identity of the cell-wall recycling products or muropeptides in P. aeruginosa is elucidated. Around 20 distinct muropeptides were identified through liquid chromatography/mass spectrometry analyses of bacterial extracts. Furthermore, iv the muropeptide effector of AmpR that is instrumental in the activation of this transcription factor is identified. The role of two permeases AmpG and AmpP in antibiotic resistance and cell-wall recycling are also investigated by comparing antibiotic susceptibility and muropeptide profile of the isogenic mutants of ampG and ampP with the wild-type PAO1. Along with investigating permeases, the role of a putative N-acetylglucosaminidase FlgJ is also investigated. Finally, keeping in mind the broad role of AmpR in regulating P. aeruginosa virulence and antibiotic resistance, we try to identify small -molecule inhibitors for AmpR. In our effort to identify inhibitors, a novel reporter-based screening assay is developed. In summary, this dissertation increases our understanding of cell-wall recycling and mechanisms of β-lactam resistance and attempts at establishing novel-antibacterial targets and inhibitors.

Drug development research on wound repair is challenging and inefficient due to the complex nature of wound healing and scarring processes and the limitations of available in vitro or in vivo models used for preclinical drug testing. Many patients who undergo elective back surgery develop post-surgical complications resulting from excess peridural scarring in and around the site of operation. We tested the effects of two anti-inflammatory compounds, quercetin and L-2-oxothiazolidine-4-carboxylate (OTC), in ameliorating peridural scar formation following spinal laminectomy surgery in laboratory rats. Western blot and immunocytochemical analyses indicated that the peridural scar tissue contained MyoD-positive myoblast cells and expressed prolyl-4-hydroxylase (P4H), a fibroblast marker. Treatment with 1 mM OTC reduced activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPK) at 21 days post-surgery suggesting potential anti-scarring mechanism. However, large animal to animal variation in the expression levels of collagen biosynthesis markers made it difficult to demonstrate any efficacy of quercetin or OTC in reducing peridural scar formation. The shortcomings of this live animal approach led us to develop a novel three-dimensional (3-D) <i>in vitro</i> wound repair model for evaluating quercetin and OTC effects. High-density micromass co-cultures seeded at a 1:3 ratio of FR 3T3 fibroblast cells and L8 myoblast cells formed 3-D microtissues <i>in vitro</i> that expressed MyoD, P4H, and á-smooth muscle actin. The micromass tissue layer remained adherent to the culture plate when inflicted with a single laceration injury, which allowed monitoring of cell migration into the wound site. Wounded cultures were treated with quercetin, OTC and other agents (TGF- â1, mitomycin, p38 inhibitor SB202190, ERK inhibitor PD184352) to determine their effects on collagen accumulation, wound closure rates, MAPK activation, and gene transcript expression. Both OTC and quercetin treatments reduced collagen biosynthesis in dose-dependent manner. In addition, 1.5 mM OTC accelerated wound closure and significantly reduced p38 MAPK activation without affecting ERK1/2. In contrast, 40 µM quercetin delayed wound closure in micromass co-cultures and reduced ERK1/2 activation. Our in vitro findings suggest that OTC might have potential as an anti-scarring agent. Importantly, our novel micromass co-culture system shows promise as an improved 3-D scaffold-free in vitro model for use in preclinical drug development research.

Alginate is a popular biomaterial in tissue engineering. When crosslinked with calcium ions (Ca2+), alginate forms a hydrogel which provides necessary mechanical support as a scaffold. The material properties as well as the biological properties of alginate scaffold are of great importance. In this thesis, the aim is to use traditional methods, such as scanning electron microscopy (SEM) and light microscopy, and emerging X-ray imaging techniques, such as micro-computed tomography (micro-CT) and synchrotron radiation (SR) X-ray imaging, to characterize the alginate scaffolds. Firstly, the material properties of freeze-dried alginate scaffolds were evaluated using micro-CT, as it is a non-destructive and non-invasive imaging method, and can provide three-dimensional information. Alginate scaffolds made with different sodium alginate concentrations and frozen to different temperatures were scanned and analyzed in micro-CT. Results indicated that lower freezing temperature and higher sodium alginate concentration lead to smaller pore size and porosity. Secondly, cell culture experiments were carried out to study the biological properties and the interactions of alginate hydrogel with cells. A Schwann cell line was either blended with alginate solution before crosslinking with calcium chloride (CaCl2) or put around alginate gel in the culture dish. Light microscopy of sectioned slices showed that cells surrounding the alginate gel could not grow into the gel, while cells blended with alginate solution before crosslinking could proliferate inside the hydrogel. Cells grown inside a thin slice of alginate gels appeared to be in better condition and were larger in size and also grew in clusters. Thirdly, in order to image soft tissue buried inside alginate gels, such as brain slices, novel imaging methods based on synchrotron radiation (SR) were applied, such as absorption and phase contrast imaging, diffraction-enhanced imaging (DEI) and also combined with computed tomography (CT). Synchrotron-based monochromatic X-ray imaging proved to be good at distinguish objects of similar density, especially biological soft tissue samples, even without any staining material, such as osmium tetroxide (OsO4). These three pieces of research work show the potential in applying the emerging X-ray imaging in soft tissue engineering.

Tissue-engineering (TE) is one of the most innovative approaches for tackling many diseases and body parts that need to be replaced, by developing artificial tissues and organs. For this, tissue scaffolds play an important role in various TE applications. A tissue scaffold is a 3D (3D) structure with interconnected pore networks and used to facilitate cell growth and transport of nutrients and wastes while degrading gradually itself. Many fabrication techniques have been developed recently for incorporating living cells into the scaffold fabrication process and among them; dispensing-based rapid prototyping techniques have been drawn considerable attention due to its fast and efficient material processing. This research is aimed at conducting a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. Dispensing-based polymer deposition system was used to fabricate 3D porous hydrogel scaffolds. Sodium alginate was chosen and used as a scaffolding biomaterial. The influences of fabrication process parameters were studied. With knowledge and information gained from this study, 3D hydrogel scaffolds were successfully fabricated. Calcium chloride was employed as crosslinker in order to form hydrogels from alginate solution. The mechanical properties of formed hydrogels were characterized and examined by means of compressive tests. The influences of reagent concentrations, gelation time, and gelation type were studied. A post-fabrication treatment was used and characterized in terms of strengthening the hydrogels formed. In addition, the influence of calcium ions used as crosslinker on cell viability and proliferation during and after the dispensing fabrication process was examined and so was the influence of concentration of calcium solutions and exposing time in both media and alginate hydrogel. The study also showed that the density of encapsulated cells could affect the viscosity of alginate solution. In summary, this thesis presents a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. The results obtained regarding the influence of various factors on the cell viability and scaffold fabrication would form the basis and rational to continue research on fabricating 3D cell-encapsulated scaffolds for specific applications.

This thesis is concerned with the design of a novel system for inserting porogen particles within internal structure of the bone scaffold. The proposed system would be integrated with a 3D printing machine to create macro-pores based on the conventional porogen leaching method. The system is capable of inserting porogens on pre-designed locations within the scaffold structure to realize the generation of macro-porosity within scaffolds. Several alternatives for such a porogen insertion mechanism are proposed based on employing a mechanical actuator for opening and closing the path of porogen particles from a porogen reservoir to the build chamber. Another possible design that offers significant advantages over its actuator-based alternatives is a pneumatic-based mechanism that picks up porogens from a porogen reservoir and places them at pre-designed locations. Among all the presented alternatives, the pneumatic-based system is selected by utilizing the value matrix method, and detail design of the different parts of this system is presented. The required pilot test setups for performing the feasibility study of the proposed method have been designed and successfully developed, and the practicality of the designed porogen insertion mechanism is proven through experiment.

The conventional treatments for craniofacial bone defects currently are unsatisfactory due to several drawbacks. Replacement of lost bone by autografts typically causes donor site morbidity while allografts, xenografts, and demineralized bone matrix all have a chance of disease transmission. Current synthetic implants placed within the defect site generally lack osseointegration and biodegradability. There are several methods of generating a hybrid extracellular matrix (ECM) and synthetic material construct. These include coating the synthetic material scaffold with collagen and calcium phosphate, incorporating acellular biological tissue within the scaffold material, and using cells to generate an ECM coating on the synthetic material scaffold. The research performed for this thesis developed and characterized mesenchymal stem cell (MSC)-generated extracellular matrix poly(ε-caprolactone) constructs (PCL/ECM) for the replacement of bone tissue. The osteogenic potential of the PCL/ECM constructs was explored by culturing i) MSCs and ii) whole marrow cells combined with MSCs onto the construct with or without the osteogenic differentiation supplement, dexamethasone. It was established that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs was maintained even in the absence of dexamethasone and that the co-culture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow. The osteogenicity of the constructs encouraged the characterization of the protein and mineral composition of the ECM coating on the PCL/ECM constructs. Characterization revealed that at short culture durations the MSCs used to generate the ECM deposited cellular adhesion proteins that are a prerequisite protein network for further bone formation. At the later culture durations, it was determined that the ECM was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. The prior studies on the PCL/ECM constructs persuaded exploration of the effect of various devitalization and demineralization processes on the retention of the ECM components within and the osteogenicity of the PCL/ECM constructs. Analysis demonstrated that the freeze-thaw technique is a milder method of devitalization of cell-generated ECM constructs as compared to other methods, but it reduced the osteogenicity of the constructs. In addition, it was elucidated that void spaces in the surface of the constructs are important for allowing access of MSCs into the interior of the constructs.

This thesis is concerned with the design of a novel system for inserting porogen particles within internal structure of the bone scaffold. The proposed system would be integrated with a 3D printing machine to create macro-pores based on the conventional porogen leaching method. The system is capable of inserting porogens on pre-designed locations within the scaffold structure to realize the generation of macro-porosity within scaffolds. Several alternatives for such a porogen insertion mechanism are proposed based on employing a mechanical actuator for opening and closing the path of porogen particles from a porogen reservoir to the build chamber. Another possible design that offers significant advantages over its actuator-based alternatives is a pneumatic-based mechanism that picks up porogens from a porogen reservoir and places them at pre-designed locations. Among all the presented alternatives, the pneumatic-based system is selected by utilizing the value matrix method, and detail design of the different parts of this system is presented. The required pilot test setups for performing the feasibility study of the proposed method have been designed and successfully developed, and the practicality of the designed porogen insertion mechanism is proven through experiment.

The technique of DNA origami utilizes the specific and limited bonding properties of DNA to fold single stranded DNA sequences of various lengths to form a predesigned structure. One longer sequence is used as a scaffold and numerous shorter sequences called staples, which are all complementary to the scaffold sequence, are used to fold the scaffold into intricate shapes. The most commonly used scaffold is derived by extracting the genome of the M13 phage and the staples are usually chemically synthesized oligonucleotides. Longer single stranded sequences are difficult to synthesize with high specificity, which limits the choices of scaffold sequences available. In this project two main methods of single stranded amplification, Rolling Circle Amplification (RCA) and the usage of helper phages, were explored with the goal to produce both a 378 nt scaffold and staple sequences needed for folding a DNA origami structure. To facilitate imaging by Transmission Electron Microscopy (TEM) of this small structure, the DNA origami structure was created to form a polymer structure. Production of the scaffold sequence in high yield was unsuccessful and no well-defined polymers were found in the folded samples, though a few results showed promise for further studies and optimizations. Due to time constraints of this project, only production of the scaffold sequence was tested. Unfortunately the scaffold produced by the helper phages was of the complementary strand to that used to design the DNA origami structure, and could therefore not be used for folding. The correct strand was produced by the RCA where the yield was too low when using Phi29 DNA polymerase for proper folding to take place, though small scale RCA by Bst DNA polymerase on the other hand showed promising results. These results indicate that the scaffold production may not be far off but still more experience in producing intermediate size oligonucleotides may be necessary before succeeding in high yield production of this 378 nt long sequence. The promise given by this production is to enable high yield, high purity, low cost and also an easily scalable process set-up. This would be an important step in future DNA nanotechnology research when moving from small scale laboratory research to large scale applications such as targeted drug delivery systems.

Drug development research on wound repair is challenging and inefficient due to the complex nature of wound healing and scarring processes and the limitations of available in vitro or in vivo models used for preclinical drug testing. Many patients who undergo elective back surgery develop post-surgical complications resulting from excess peridural scarring in and around the site of operation. We tested the effects of two anti-inflammatory compounds, quercetin and L-2-oxothiazolidine-4-carboxylate (OTC), in ameliorating peridural scar formation following spinal laminectomy surgery in laboratory rats. Western blot and immunocytochemical analyses indicated that the peridural scar tissue contained MyoD-positive myoblast cells and expressed prolyl-4-hydroxylase (P4H), a fibroblast marker. Treatment with 1 mM OTC reduced activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPK) at 21 days post-surgery suggesting potential anti-scarring mechanism. However, large animal to animal variation in the expression levels of collagen biosynthesis markers made it difficult to demonstrate any efficacy of quercetin or OTC in reducing peridural scar formation. The shortcomings of this live animal approach led us to develop a novel three-dimensional (3-D) <i>in vitro</i> wound repair model for evaluating quercetin and OTC effects. High-density micromass co-cultures seeded at a 1:3 ratio of FR 3T3 fibroblast cells and L8 myoblast cells formed 3-D microtissues <i>in vitro</i> that expressed MyoD, P4H, and á-smooth muscle actin. The micromass tissue layer remained adherent to the culture plate when inflicted with a single laceration injury, which allowed monitoring of cell migration into the wound site. Wounded cultures were treated with quercetin, OTC and other agents (TGF- â1, mitomycin, p38 inhibitor SB202190, ERK inhibitor PD184352) to determine their effects on collagen accumulation, wound closure rates, MAPK activation, and gene transcript expression. Both OTC and quercetin treatments reduced collagen biosynthesis in dose-dependent manner. In addition, 1.5 mM OTC accelerated wound closure and significantly reduced p38 MAPK activation without affecting ERK1/2. In contrast, 40 µM quercetin delayed wound closure in micromass co-cultures and reduced ERK1/2 activation. Our in vitro findings suggest that OTC might have potential as an anti-scarring agent. Importantly, our novel micromass co-culture system shows promise as an improved 3-D scaffold-free in vitro model for use in preclinical drug development research.