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The primary objective of this study was to identify, develop and characterise a novel bioactive surface capable of binding hepatocytes and enabling the retention of hepatocyte-specific cell function during in-vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) found on hepatocytes. Hydrogels were created by modifying a commercially available block co-polymer of polyethylene glycol (PEG) and acrylamide, (PEGA) with galactose moieties contained within lactobionic acid (LA), producing a unique bioactive sugar-based gel. A control sugar, D-glucuronic acid (GA), was used as a non-ASGP-R binding control. Monomers used were mono- and bis-acryloamido PEG (Mw=1900 gmol-1), and dimethylacrylamide. The pendant PEGA amine groups were used as ligands to bind to the sugars. The resultant gels were characterised using Fourier Transform Infrared Spectroscopy (FT-IR), protein adsorption, Fmoc-Phe and dansyl chloride labelling. The biocompatibility of the gel surfaces was evaluated using a hepatocyte cell line and the degree of attachment, proliferation, and morphology was characterised using light microscopy, live/dead assays, DNA assays, immunochemical staining, flow cytometry and reverse-transcription polymerase chain reaction (RT-PCR).FT-IR analysis of LA revealed a distinctive band at approximately 1740cm-1 corresponding to carbonyl stretching (C=O) of carboxylic acid. This unique peak disappeared as the galactose moieties within the LA were incorporated into the PEGA gel. A similar trend was also observed with the control GA sugar within the PEGA gel, confirming that the sugars had been integrated into the material. Protein adsorption assays confirmed the non-fouling nature of PEGA. Cell culture experiments showed that hepatocytes attached preferentially to the sugar surfaces, with few cells seen on the PEGA surfaces. It was observed that cells on the PEGA with LA surface were more metabolically active, than the controls and proliferated to a monolayer by day 7 in culture. Immunocytochemical staining of the cells for actin, vinculin and phosphorylated focal adhesion kinase illustrated differences in cell morphology between cells grown on different surfaces. It was determined that the sugar PEGA surfaces maintained some characteristics of hepatocyte functionality e.g. urea synthesis over the course of 7 days. To improve the reproducibility of the surfaces generated, a preliminary investigation of two-dimensional PEG monolayer surfaces as a well defined platform for surface reactions was conducted. These were chemically functionalised in a stepwise manner with the sugars. The number of coupling steps and the choice of solvent were shown to affect the efficiency of the reaction. Further more, the need for careful sample preparation was highlighted as contamination could potentially inhibit the interpretation of the surface chemistry.The overall conclusion of this work is that saccharides within non-fouling surfaces composed of thin layers of PEG-acrylamide hydrogels are able to support hepatocyte attachment and the retention of cell type specific functions in culture. However, this preliminary work has shown that much further research is necessary to elucidate the role that the surface chemistry plays in the attachment of hepatocytes.

Cells are complex active materials that display fascinating phenomena in response to changes in their physical environments. It is well established that the physical environment dictates cell fate and function; nevertheless, the standard method of culturing and studying cells is on stiff 2- dimensional Petri dishes and glass cover slips. The difference in the magnitude of the stiffness of the substrate in addition to the 2-dimensional character, leads to an incomplete and perhaps misleading picture of the cellular process under scrutiny. As such, an entire field has been dedicated to developing materials that more closely match the characteristics of the natural cellular milieu: biomaterials. Despite significant progress in the field, we are still far from fully recapturing the native environment. Importantly, many of the current strategies for engineering 3-dimensional biomaterials have specific applications yet lack flexibility to be adapted to a wide variety of functions. Our approach is to repurpose existing complex, readily available materials to create a platform for biomaterial production; our biomaterials are derived from plant tissue. Plants have evolved over millions of years to attain structures with intricate geometries for specialized functions. Due to the wide variety of plant structures, one can easily select a plant-based material with analogous features to the tissue of interest. A series of investigations are presented on these novel biomaterials to demonstrate this approach, quantify the mechanical properties, and study the cellular responses. First, we developed a method of processing plant materials to yield decellularized, cellulose-based, biocompatible scaffolds that can be repopulated with mammalian cells. We then created composite materials by casting hydrogels around the cellulose-based scaffolds, which allowed us to incorporate distinct temporal and spatial cues to the local cell populations. Spatial organization of tissues and tissue interfaces remains a primary challenge in biomedical engineering, as tissue interfaces mark complex transitional zones between distinct cell populations. Replicating and repairing this intricate delineation of cell types and mechanical profiles has proven to be a major concern in regenerative medicine. As such, we sought to develop a platform for engineered tissue interfaces, wherein components are combined in a modular fashion into a functional unit. The mechanical cues of the microenvironment affect a plethora of cellular processes, namely cell migration, proliferation, and differentiation. Consequently, the rheological properties of our decellularized, plant-based scaffolds were thoroughly investigated. An in-depth knowledge of the mechanics of the underlying substrate is required to guide future applications and refinements of this technology. The potential applications of these 3-dimensional constructs, as demonstrated through our findings, include designing in vitro models of tissue interactions, new biomaterials for in vivo applications, and studies on fundamental cellular processes. We highlight the significance of our results in a collection of scientific articles, which are presented in the body of this thesis (Chapters 2-5). This work is focused on the use of plant- derived cellulose materials, which forms a subsection of the cellulose biomaterial field. A review article centered on the use of cellulose materials for tissue engineering serves as an introductory chapter.

Indiana University-Purdue University Indianapolis (IUPUI) / A novel antimicrobial dental composite system has been developed and evaluated. Both alumina and zirconia filler particles were covalently coated with an antibacterial resin and blended into a composite formulation, respectively. Surface hardness and bacterial viability were used to evaluate the coated alumina fi ller-modif ed composite. Compressive strength and bacterial viability were used to evaluate the coated zirconia ller-modi ed composite. Commercial composite Kerr was used as control. The specimens were conditioned in distilled water at 37 °C for 24 h prior to testing. Four bacterial species Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were used to assess the bacterial viability. Effects of antibacterial moiety content, modif ed particle size and loading, and total fi ller content was investigated. Chapter 2 describes how we studied and evaluated the composite modi fed with antibacterial resin-coated alumina llers. The results showed that almost all the modi ed composites exhibited higher antibacterial activity along with improved surface hardness, as compared to unmodi fed one. Increasing antibacterial moiety content, particle size and loading, and total fi ller content generally increased surface hardness. Increasing antibacterial moiety, fi ller loading, and total fi ller content increased antibacterial activity. On the other hand, increasing particle size showed a negative impact on antibacterial activity. The leaching tests indicate that the modiChapter 3 describes how we studied and evaluated the composite modif ed with antibacterial resin-coated zirconia fi ller. The results showed that almost all the modif ed composites exhibited higher antibacterial activity along with decreased compressive strength, as compared to the unmodif ed control. It was found that with increasing antibacterial moiety content and modi fedfi ller loading, yield strength, modulus and compressive strength of the composite were decreased. In addition, the strengths of the composite were increased with increasing powder/liquid ratio. On the other hand, with increasing antibacterial moiety content, fi ller loading and powder/liquid ratio, antibacterial activity was enhanced. In summary, we have developed a novel antibacterial dental composite system for improved dental restoratives. Both composites modif ed with the antibacterial resin-coated alumina and zirconia fi ller have demonstrated signi cant antibacterial activities. The composite modi fed with the alumina fi ller showed improved hardness values, but the composite modif ed with the zirconia fi ller showed decreased compressive strength values. It appears that the developed system is a non-leaching antibacterial dental composite. ed experimental composite showed no leachable antibacterial component to bacteria.

Towards the creation of the next generation of biomedical implants that effectively integrate in tissues, understanding cell behaviour at the material-host interface to control and optimize the biological outcome is a crucial endeavour. It is now well known that the nanoscale surface properties of biomaterials play a significant role in directing the activity of adherent cells at the implant-host tissue interface. A variety of cellular functions, ranging from adhesion and proliferation to differentiation along specific lineages, are guided by the nanoscale topographical and physicochemical features of the substrate. This evidence reaffirms the role of surface features on eliciting an enhanced response of cells towards improved biological outcomes (e.g., bone integration) of implanted biomaterials. In this context, Titanium (Ti) and its alloys are popular biomaterials widely used in orthopedic, dental, and cardiovascular applications. In particular, in the field of osseointegrated devices, chemical treatments of titanium, specifically oxidative nanopatterning (i.e., a simple yet effective treatment with a H₂SO₄/H₂O₂ solution), have shown to be a promising strategy for guiding and controlling the fate of relevant cells (e.g., osteoblasts, stem cells), thereby achieving the ability to direct the biological response towards the desired outcome. In this context, the sponge-like nanoporous surface resulting from oxidative nanopatterning of titanium allows direct surface cueing to bone cells. It also has the capacity to selectively regulate cell behaviour, modulate the expression of crucial determinants of cell activity, and offers the potential to harness the power of stem cells. However, the mechanisms that control how cells sense and respond to these nanometric cues are still elusive. A novel strategy to elucidate them takes inspiration from in-vivo protocols, where "knock-out" animal models are used to determine the role of a specific gene. Based on this, I propose an original approach aimed at investigating cell response under conditions known to affect specific cellular processes, thereby determining whether these activities can be rescued by direct cueing by the substrate, ultimately elucidating their implication in responding to a given nanostructured substrate. In particular, hyperglycemic culturing conditions often used to mimic diabetes in-vitro are known to exert detrimental effects on the proliferation and differentiation of osteoblasts, and thereby could be an excellent opportunity to test whether the nanometric surface features resulting from oxidative nanopatterning of titanium also possess the ability to compensate to the cell-level changes caused by higher levels of glucose. This would ultimately demonstrate a direct effect of the substrate on these events and help us understand the mechanisms involved in cell-biomaterial interactions. To address this challenge, I propose to investigate the response of human MG-63 osteoblastic cells to nanoporous titanium under hyperglycemic conditions. The goal is, therefore, to understand whether direct nanotopographical cueing at the nanoscale can rescue MG-63 cells from the effects of hyperglycemia, thereby casting new light on the mechanisms underlying the interactions between this widely used cell line and nanoporous titanium. In parallel, results from my work aim at providing new fundamental evidence to interpret results from that body of literature that uses high glucose content as a way to mimic the osseointegration of biomaterials in diabetic conditions.

Nanoindentation techniques have not been standardized for use on bone tissues, making comparison of bone material properties obtained via nanoindentation across studies difficult and unreliable. This study determined a set of optimal parameter values for thermal drift correction time, dwell time, and loading rate that can be used to obtain accurate and repeatable material properties from human femoral trabecular bone through experimentation and statistical analysis. All testing was conducted using a single nanoindenter on a single trabeculae, with the assumption that material properties within the individual trabeculae were internally consistent. Parameters not of interest during this study, such as ambient temperature, maximum load, and maximum indentation depth were held constant throughout all experiments. Elastic modulus and hardness data were calculated using the Oliver-Pharr technique. The optimal values for these parameters are as follows: 150 seconds for thermal drift correction time, 30 to 60 seconds for dwell time, and 0.4 to 0.8 mN/s for loading rate.

Coronary heart disease is the leading cause of death in the United States and occurs when plaque occludes coronary arteries. Coronary stents, which may be used to treat coronary occlusions, are small metal tubes that are implanted in coronary arteries to restore blood flow. After stent implantation, endothelial cells grow over the stent so that blood contacts the endothelial cells instead of the stent surface; this event is known as re-endothelialization. Re-endothelialization prevents blood from clotting on the stent surface and is a good predictor of stent success. Blood vessel mimics (BVMs) are in vitro tissue engineered models of human blood vessels that may be used to preclinically test coronary stents for re-endothelialization. BVMs have been developed in straight geometries, but the FDA has recommended that coronary devices be preclinically tested in complex-shaped simulated vessels when the complex geometries of coronary arteries may negatively affect device performance. Coronary geometries may negatively affect the tissue response to coronary stents, therefore BVMs should be developed in complex geometries. The goal of this thesis research was to fully develop complex-shaped scaffolds and bioreactors, to develop complex-shaped BVMs with cells located throughout all regions of the BVMs, and to develop a complex-shaped BVM with a confluent region of cells. First, bioreactors that can house complex-shaped scaffolds were designed, constructed, and validated. Complex-shaped BVMs were then developed by depositing cells throughout the entire inner surface of complex-shaped scaffolds, and the average and median cell densities throughout all regions of the BVMs were shown to be approximately the same order of magnitude as endothelial cell densities in native blood vessels. A stent was then successfully deployed in a complex-shaped BVM. The complex-shaped BVM straightened out to conform to the stent, which also occurs in native blood vessels. Finally, a confluent region of cells was developed on a complex-shaped scaffold. Complex-shaped BVMs could eventually be used to preclinically test coronary stents, coronary drug-delivery systems, coronary imaging modalities, and other intravascular technologies.

BACKGROUND: Magnesium-based biomaterials might provide an innovative therapeutic potential to substantially enhance regeneration of dental tissues. Magnesium (Mg2+) has been considered for its potential ability to accelerate proliferation and differentiation of human osteoblasts. However, to date, the dentinogenic effect of magnesium chloride (MgCl2) and magnesium oxide (MgO) on human dental pulp cells (HDPCs) has not been investigated. PURPOSE: This study was designed to compare the stimulatory effect of different concentrations of MgCl2 and MgO on dentinogenesis of HDPCs and to explore associated cellular signaling pathways. METHODS: HDPCs were cultured with 0.5mM,1mM, 2mM, 4mM, 8mM concentrations of supplemental MgCl2 and MgO, 0 mM as negative control group, lignin sulfonic acid sodium salt and xanthan gum as vehicle control groups. Crystal violet staining was used to determine cell attachment and proliferation rate. Cell viability was investigated by MTT assay. Odontogenic differentiation was assessed by evaluating alkaline phosphatase (ALP) activity, expression of dentin sialoprotein (DSP), dentin matrix protein1(DMP-1), dentin sialophosphoprotein (DSPP), type I collagen (COL-I), and mineralization. Expression of bone morphogenic protein (BMP-2), phosphorylated SMADs 1/5/9, p-p38, p38, p-JNK, JNK, p-ERK1/2, ERK1/2 mitogen activated protein kinase (MAPK) were also investigated. Statistical analysis was applied using multi-way ANOVA with Wilks’ lambda test. RESULTS: 0.5mM-2mM MgCl2 elicited the highest stimulatory effect on attachment, proliferation rate, ALP activity, expression of dentinogenic proteins (DSP, DMP-1, DSPP, COL-I), expression of cellular signaling proteins (BMP-2, phosphorylated SMADs1/5/9, p-p38, p-JNK) mineralization, and down regulation of p-ERK 1/2 compared to negative control (P< 0.0001). 0.5mM supplemental MgO showed higher attachment, proliferation, cell viability, ALP activity, expression of dentinogenic proteins (DSP, DMP-1, DSPP, COL-I), cellular signaling proteins (BMP-2, phosphorylated SMADs1/5/9) and mineralization, compared to negative control (P<0.001). CONCLUSION: This study demonstrated the significant benefit imparted by optimum concentrations of MgCl2 and MgO on HDPCs evidenced by upregulated cell attachment, proliferation, cell viability, ALP activity, mineralization, expression of odontogenic proteins and cellular signaling proteins. Compared with MgO, MgCl2 yielded a wider range of effective concentrations (0.5mM-2mM MgCl2 vs. 0.5mM MgO) for upregulating the dentinogenic effect of HDPCs. MgCl2 at optimal concentrations could be a potential novel material for dentin repair in regenerative endodontics.

The primary goal of this research is to engineer solutions facilitating the utilization of bacteriophages as naturally occurring bactericidal agents for combatting multidrug-resistant (MDR) bacterial infections. Bacteriophages, which are bacterial viruses, represent self-replicating antibacterial agents known for their remarkable specificity in targeting bacterial cells. This specificity stands in sharp contrast to the indiscriminate and broad-spectrum actions of many currently employed antimicrobials across various sectors. Specificity of bacteriophages is a double-sided sword, often requiring large-scale phage hunting and phage biobank screening. This, combined with the lack of a global phage biobank can significantly limit access to phage therapeutics. I have developed a rapid, high-throughput platform focused on the detection of phage-mediated adenosine triphosphate (ATP) release via enzymatic ATP bioluminescence assay to identify highly lytic phages targeting MDR bacterial pathogens. I also used pullulan-trehalose sugar mixture to stabilize the ATP bioluminescence assay components at physiological temperatures. The sugar mixture also enhanced the desiccation tolerance of the ATP assay components along with phage, enabling the creation of all-inclusive shelf-stable tablets. The resulting tablets proved effectiveness and reliability in tracking phage-mediated bacterial cell lysis, and the pullulan-trehalose encapsulation significantly enhanced both the signal and desiccation tolerance of the phage and assay components. Next, I developed a bi-functional phage delivering nanoclay-based injectable hydrogel that can serve as both antibacterial and osteoinductive therapeutic hydrogel for treating bone and implant associated infections. The in vitro results for phage-loaded injectable hydrogels confirmed strong antimicrobial action against bacterial biofilms, in both biofilm prevention and biofilm dispersion challenges. Continuing the phage biomaterials research, I also co-developed a combination of phage-collagen conjugated liquid infused coating on titanium implant that enhanced osteointegration and was remarkably effective against implant-associated infections as a prophylactic measure in vivo. Lastly, and as a proof of the utility of phage biocontrol beyond biomedical applications, I demonstrated biofilm removal and full signal regeneration for dissolved oxygen (DO) sensors using a phage cocktail. / Thesis / Doctor of Philosophy (PhD) / Antibiotic resistance is rapidly spreading worldwide, leading to a substantial loss of lives each year and imposing a significant economic burden. Bacteriophages, natural bactericidal viruses, are emerging as a promising solution due to their unique properties. This thesis focuses on the practical implementation of bacteriophages to address real-world challenges linked to antibiotic resistance. I worked on facilitating the process of selecting phages for personalized phage therapy through detecting phage-mediated release of bacteria encoded biomolecules. I also developed phage-loaded injectable hydrogels and phage-conjugated liquid infused coatings to combat bone and implant-related infections. Moreover, I have shown the promise of phage biocontrol beyond biomedical application by demonstrating its effectiveness in restoring a heavily biofouled sensor used for measuring dissolved oxygen, a critical water quality indicator.

To date, protein adsorption is an unavoidable response to implanted biomaterials. When proteins interact with materials, adverse biological events such as thrombus formation and inflammation can occur and challenge device efficacy. Protein adsorption is influenced by various material and surface properties which can be modified in efforts to alter the protein-material interactions and the subsequent cellular response. There is a need for simple modifications of commonly used biomaterials and the effect of these modifications on (1) material properties (2) proteins and (3) cells is important to study. In this work, the effect of modifying polydimethylsiloxane (PDMS) and its interactions with fetuin-A are studied for potential immunomodulatory properties. PDMS modifications are achieved by altering the ratio of PDMS formulations to simply and effectively control elastic modulus, and by coating PDMS with polydopamine (PDA), a molecule commonly used as a bioglue. Surface characterization confirmed that altering the PDMS formulation changed the elastic modulus without affecting surface wetting properties. Minor changes in surface roughness via atomic force microscopy and surface chemistry via x-ray photoelectron spectroscopy were detected on some samples, and the deposition of PDA was confirmed. Protein adsorption studies provided quantitative and qualitative data on fetuin-A interactions. It was determined that fetuin-A adsorption was influenced by the PDMS formulations, and that the preferential adsorption changed when adsorbed from a competitive environment. Following modification of samples with adsorbed fetuin-A, the inflammatory effects of fetuin-A were investigated by measuring the concentration of pro- and anti-inflammatory cytokines in response to modified and unmodified samples. Data suggest that elastic modulus influences cytokine secretion at certain timepoints, a result of varied protein adsorption amounts and orientations in response to material stiffness. The addition of a PDA layer demonstrated the potentially cytokine mitigating effect of PDA cell interactions and protein immobilization when compared to unmodified PDMS samples. / Thesis / Master of Applied Science (MASc)