Although medical devices and biomaterial implants are used clinically in a variety of applications, the process of implanting them damages local tissue and initiates a localized non-specific inflammatory response that is detrimental to device performance. Extensive research efforts have focused on developing material surface treatments and systems to deliver anti-inflammatory agents to abrogate such biomaterial-mediated inflammation, yet long-term use of these traditional materials in vivo is limited due to continued inflammation and fibrous encapsulation. This work aims to address these limitations by developing a versatile implant coating with non-fouling properties using a system based on hydrogel microparticles (i.e. microgels). The overall objective of this project was to evaluate host responses to these microgel coatings. Microgel particles were synthesized from poly(N-isopropyl acrylamide) cross-linked with poly(ethylene glycol)-diacrylate and were successfully deposited onto polymeric substrates using a simple and reproducible spin coating technique. We determined that microgel-coated samples adsorbed significantly lower levels of human fibrinogen than controls. Further characterization using an in vitro culture system demonstrated that microgel coatings significantly reduced the adhesion and spreading of murine macrophages and primary human blood-derived monocytes compared to controls. Materials were then evaluated for early cellular responses following implantation in the intraperitoneal cavity of mice to model acute inflammation. Analyses of explanted biomaterials using immunofluorescence staining techniques revealed that microgel-coated samples significantly reduced the density of surface-adherent cells. Additional analysis using flow cytometry revealed that microgel-coated samples exhibited significantly lower levels of pro-inflammatory cytokines in adherent leukocytes compared to controls, indicating that these coatings modulate cellular pro-inflammatory activities. Finally, we implanted samples subcutaneously in rats to determine the efficacy of microgel coatings at longer time points using an established model of chronic inflammation. Explants were processed histologically and stained for various markers. Importantly, staining demonstrated that the microgel coatings significantly reduced fibrous capsule thickness, the capsules appeared less compact and structurally ordered than controls, and also contained significantly fewer cells. Collectively, these results demonstrate that microgel particles can be applied as polymeric coatings to modulate inflammation and achieve more desirable host responses in vivo, with the potential to extend implant lifetime.

A comprehensive understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is essential in the design of advanced biomaterials that better promote bone growth and osseointegration of implants. Dental implants with roughened surfaces and high surface energy are well known to promote osteoblast differentiation in vitro and promote increased bone-to-implant contact in vivo. In addition, increased surface roughness increases osteoblasts response to the vitamin D metabolite 1α,25(OH)2D3. However, the exact mechanisms mediating cell response to surface properties and 1α,25(OH)2D3 are still being elucidated. The central aim of the thesis is to investigate whether integrin signaling in response to rough surface microtopography enhances osteoblast differentiation and responsiveness to 1α,25(OH)2D3. The hypothesis is that the integrin α5β1 plays a role in osteoblast response to surface microtopography and that 1α,25(OH)2D3 acts through VDR-independent pathways involving caveolae to synergistically enhance osteoblast response to surface roughness and 1α,25(OH)2D3. To test this hypothesis the objectives of the studies performed in this thesis were: 1) to determine if α5β1 signaling is required for osteoblast response to surface microstructure; 2) to determine if increased responsiveness to 1α,25(OH)2D3 requires the vitamin D receptor, 3) to determine if rough titanium surfaces functionalized with the peptides targeting integrins (RGD) and transmembrane proteoglycans (KRSR) will enhance both osteoblast proliferation and differentiation, and 4) to determine whether caveolae, which are associated with integrin and 1α,25(OH)2D3 signaling, are required for enhance osteogenic response to surface microstructure and 1α,25(OH)2D3. The results demonstrate that integrins, VDR, and caveolae play important roles in mediating osteoblast response to surface properties and 1α,25(OH)2D3. Silencing of the β1 integrin in osteoblast-like MG63 cells significantly reduced osteogenic response to surface topography and 1α,25(OH)2D3. Silencing of the α5 subunit did not alter the response of MG63 cells to changing surface roughness or chemistry, although future work must confirm these results given similar cell surface α5 integrin expression observed in control and α5-silenced cells. Multifunctional RGD, KRSR, and KSSR coated surfaces show that RGD increased osteoblast proliferation and reduced differentiation, KRSR had no affect on osteoblast phenotype, and KSSR increased osteoblast differentiation. These results suggest that titanium surfaces can be modified to manipulate proliferation and differentiation and that RGD/KSSR functionalized surfaces could be further investigated for use as osteointegrative surfaces. The results using VDR deficient osteoblasts demonstrate that 1α,25(OH)2D3 acts via VDR-dependent mechanisms in cells cultured on titanium surfaces that support terminal differentiation. In caveolae deficient osteoblasts, 1α,25(OH)2D3 affected cell number, alkaline phosphatase activity, and TGF-β1 levels, although levels of osteocalcin and PGE2 were not affected. These results are consistent with the hypothesis that VDR is required for the actions of 1α,25(OH)2D3, but that caveolae-dependent membrane 1α,25(OH)2D3 signaling modulates traditional VDR signaling. The exact mechanisms for this interaction remain to be shown. Overall, these results are important in better understanding the role of β1 integrin partners in mediating osteoblast response to implant surfaces and in understanding how integrin signaling can alter osteoblast differentiation and responsiveness to 1α,25(OH)2D3 via genomic and non-genomic pathways.

A study on application of combinatorial methods (CM) and high-throughput methods (HTM) to biomaterials design, characterization, and screening are reported in this thesis - focusing on screening the effects of biomaterial surface features on adherent bone cell cultures. Polymeric biomaterials were prepared on two-dimensional combinatorial libraries that systematically varied the size and shape of chemically-distinct microstructural patterns - generated from blends of biodegradable polyurethanes and polyesters. Characterization and screening were performed with high-throughput optical and fluorescence microscopy. A unique advance of this work is the application of data mining techniques to identify the controlling structural features that affect cell behavior from among the myriad variety of metrics from the microscope images. The results from this study demonstrated the potentials of CM/HTS to be applied to exploratory studies involving complex systems in life sciences. This study accomplishes the goal to demonstrate the efficient screening and exploration of vast and complex dataset, extracting important and meaningful information to narrow down the future path of study in this field. Further study aimed to tuning cellular responses via signals from surface cues will be necessary to examine the causal relationships beyond the observed correlations shown in this exploratory study. It is recommended for further studies to narrow down the range for surface patterning around each of the three 'activation' ranges found in this study: apoptotic, viable, and one unknown state to be studied further. Different cellular-function staining methods will be necessary to be used in cellular imaging techniques in order to explore this unknown state further.

Commercial organic dyes are widely used for cellular staining due to their small size, high brightness, and chemical functionality. However, their blinking and photobleaching are not ideal for studying dynamics inside live cells. An improvement over organics and much larger quantum dots, silver nanodots (Ag NDs) exhibit low cytotoxicity and excellent brightness and photostability, while retaining small size. We have utilized ssDNA hairpin structures to encapsulate Ag NDs with excellent spectral purity, high concentration, and good chemical and photophysical stability in a variety of biological media. Multi-color staining of fixed and live cells has been achieved, suggesting the promise of Ag NDs as good fluorophores for intracellular imaging. The great brightness and photostability of Ag nanodots indicate that they might be outstanding imaging agents for in vivo studies when encapsulated in delivery vehicles. In addition, Ag NDs can be optically modulated, resulting in increased sensitivity within high backgrounds. These good characteristics are combined with delivery vehicles such as PLGA and nanogels. After encapsulation, Ag nanodots still retain their good photophysical properties and modulation. It might be useful for in vivo applications in the near future

Novel surgical strategies for spinal disc repair are currently being developed that require materials that (1) possess the appropriate mechanical properties to mimic the tissue the material is replacing or repairing and (2) maintain their mechanical function for long durations without negatively affecting the tissue response of adjacent tissue (i.e. bone). Polymers formed through photopolymerization have emerged as candidate biomaterials for many biomedical applications, but these materials possess limited toughness in vivo due to the presence of water inherent in most tissues. Therefore, the overall objective of this research was to develop photopolymerizable (meth)acrylate networks that are both mechanically and biologically compatible under physiological conditions to be implemented in spinal repair procedures. The fundamental approach was to determine structure-property relationships between toughness and network structure in the presence of phosphate buffered saline (PBS) using several model copolymer networks in order to facilitate the design of photopolymerizable networks that are tough in physiological solution. It was demonstrated that networks toughness could be optimized in PBS by tailoring the Tg of the copolymer network close to body temperature and incorporating the appropriate "tough" chemical structures. The ability to maintain toughness up to 9 months in PBS was dependent upon the viscoelastic state and overall hydrophobicity of the network. In tandem, the effect of network chemistry and stiffness on the response of MG63 pre-osteoblast cells was assessed in vitro. The ability of MG63 cells to differentiate on (meth)acrylate network surfaces was found to be primarily dependent on surface chemistry with PEG-based materials promoting a more mature osteoblast phenotype than 2HEMA surfaces. Amongst each copolymer group, copolymer stiffness was found to regulate osteoblast differentiation in a manner dependent upon the surface chemistry. In general, photopolymerizable (meth)acrylate networks that were deemed "tough" were able to promote osteoblast differentiation in a manner comparable if not exceeding that on tissue culture polystyrene (TCPS). This research will impact the field of biomaterials by elucidating the interrelationships between materials science, mechanics, and biology.

Ongoing advances in tissue engineering with the goal to address the clinical shortage of donor organs have encouraged the design and development of biomaterials to be used in tissue-engineered scaffolds. Furthermore, biomaterials have been used as delivery vehicles for vaccines that aim to enhance the protective immunity against pathogenic agents. These tissue-engineered constructs or vaccines are usually combination products that combine biomaterial and biological (e.g. cells, proteins, and/or DNA) components. Upon introduction into the body, the host response towards these products will be a combination of both a non-specific inflammatory response towards the biomaterial and an antigen-specific immune response towards the biological component(s). Recently, the biomaterial component was shown to influence the immune response towards a co-delivered antigen. Specifically, poly(lactic-co-glycolic acid) (PLGA), but not agarose, scaffolds or microparticles (MPs) enhanced the humoral response to a model antigen, ovalbumin. This in vivo result echoed with the in vitro study that PLGA, but not agarose, supported a mature phenotype of dendritic cells (DCs), the most potent antigen-presenting cells. Therefore, it is hypothesized that the effect of biomaterials on DC phenotype may influence the adaptive immunity against a co-delivered antigen. Understanding how biomaterials affect DC response will facilitate the selection and design of biomaterials that direct a desired immune response for tissue engineering or vaccine delivery applications. The objectives of this research were to elucidate the correlations between material properties and DC phenotype, develop predictive models for DC response based on material properties, and uncover the molecular basis for DC response to biomaterials. Well-defined biomaterial systems, including clinical titanium (Ti) substrates and two polymer libraries, were chosen to study induced DC phenotype. Due to the time-consuming nature of conventional methods for assessing DC phenotype, a high-throughput (HTP) method was first developed to screen for DC maturation based on surface marker expression (CHAPTER 4). A 96-well filter plate-based HTP methodology was developed and validated for the assessment of DC response to biomaterials. A "maturation factor", defined as CD86/DC-SIGN and measured by immunostaining, was found to be a cell number-independent metric for DC maturation and could be adapted to screen for DC maturation in a microplate format. This methodology was shown to reproducibly yield similar results of DC maturation in response to biomaterial treatment as compared to the conventional flow cytometric method upon DC treatment in 6-well plates. In addition, the supernatants from each treatment could easily be collected for cytotoxicity assessment using glucose-6-phosphate dehydrogenase (G6PD)-based assay and cytokine profiling using multiplex technology. In other words, the 96-well filter plate-based methodology can generate three outcomes from one single cell culture: 1) maturation marker expression, 2) cytotoxicity, and 3) cytokine profile. To examine which material properties were critical in determining DC phenotype, a set of three clinical titanium (Ti) substrates with well-defined surfaces was used to treat DCs (CHAPTER 5). These Ti substrates included pretreatment (PT), sand-blasted and acid-etched (SLA), and modified SLA (modSLA), with different roughness and surface energy. DCs responded differentially to these substrates. Specifically, PT and SLA induced a mature DC (mDC) phenotype, while modSLA-treated DCs remained immature based on surface marker expression, cytokine production profiles and cell morphology. Both PT and SLA induced higher CD86 expression as compared to iDC control, while modSLA maintained CD86 expression at a level similar to iDC. PT- or SLA-treated DCs exhibited dendritic processes associated with a mDC phenotype, while modSLA-treated DCs were rounded, a morphology associated with an iDC phenotype. Furthermore, PT induced increased secretion of MCP-1 by DCs compared to iDCs, indicating that PT promoted a pro-inflammatory environment. SLA induced higher IL-16 production, which is a pleiotropic cytokine, by DCs, most likely as a pro-inflammatory response due to the enhanced maturation of DCs induced by SLA. In contrast, modSLA did not induced enhanced production of any cytokines examined. Principal component analysis (PCA) were used to reduce the multi-dimensional data space and confirmed these experimental results, and it also indicated that the non-stimulating property of modSLA co-varied with certain surface properties, such as high surface hydrophilicity, % oxygen and % titanium of the substrates. In contrast, high surface % carbon and % nitrogen were more associated with a mDC phenotype. Furthermore, PCA also suggested that surface line roughness (Ra) did not contribute to the expression of CD86, an important maturation marker, suggesting that roughness had little impact on DC response (CHAPTER 5). DC response-material property relationships were also derived using more complex materials from a combinatorial library of polymethacrylates (pMAs) (CHAPTER 6). Twelve pMAs were selected and were found to induce differential DC response using the HTP method described in CHAPTER 4. These pMAs resulted in a trend of increasing DC maturation represented by the metric CD86/DC-SIGN, which was consistent with the trends of the production of pro-inflammatory cytokine, TNF-α, and chemokine, IL-8. Interestingly, this set of pMAs induced an opposite trend of IL-16 production, which is most likely released as an anti-inflammatory cytokine in this situation. These polymers were characterized extensively for a number of material properties, including surface chemical composition, glass transition temperature (Tg), air-water contact angle, line roughness (Ra), surface roughness (Sa), and surface area. Similar to the results from the Ti study, PCA determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an iDC phenotype. In addition, Tg, Ra, and surface area were unimportant in determining DC response. Partial square linear regression (PLSR), a multivariate modeling approach, was implemented using the pMAs as the training set and a separate polymer library, which contained methacrylate- and acrylate-based terpolymers, as the prediction set. This model successfully predicted DC phenotype in terms of surface marker expression with R2prediction = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with R2prediction = 0.80 (CHAPTER 6). Nonetheless, one should note that a predictive model can be only as good as what it is trained on and cannot be used to predict the DC response induced by a type of materials different from the training set. Also, this model might not contain all the important material properties such as polymer swelling and cannot predict specific types of immune responses. However, these results demonstrated that a generalized immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection (CHAPTER 6). From the pMA library, pMAs that induced the two extremes of DC phenotype (mature or immature) were identified for elucidating the mechanistic basis of biomaterial-induced DC responses (CHAPTER 7). Two pMAs, polyhydroxyethylmethacrylate (pHEMA) and poly(isobutyl-co-benzyl-co-terahydrofurfuryl)methacrylate (pIBTMA), were selected because they induced the least and the most mature DC phenotype, respectively. These pMAs were used to elucidate the activation profiles of transcription factors in DCs after biomaterial treatment and were compared to the iDC and mDC controls. In addition, a combined treatment of pHEMA and LPS was also included to determine if pHEMA could maintain an iDC phenotype in the presence of LPS. Interestingly, pIBTMA induced DC maturation primarily through the activation of NF-κB, while pHEMA mediated suppression of DC maturation through multiple TFs, including the activation of ISRE, E2F-1, GR-PR, NFAT, and HSF. GR-PR and E2F-1 have been shown to be associated with the suppression of DC maturation; ISRE, E2F-1, and NFAT are linked to apoptosis induction; HSF regulates the production of heat shock proteins (HSPs) that induce DC maturation and inhibit apoptosis. The activation of HSF by pHEMA was most likely a natural defensive mechanism against the other apoptotic signals. Therefore, pHEMA suppressed DC maturation through the induction of apoptosis. Surprisingly, in the presence of pHEMA, the effect of LPS was completely eliminated, suggesting that biomaterials can override the effect of soluble factors. The morphology and surface marker expression of DCs treated with these different biomaterials or controls were consistent with TF activation profiles (CHAPTER 7). Overall, this research illustrates that biomaterial properties, within the chosen biomaterial space, can be correlated to DC phenotype and more importantly, can be used as predictors for relative levels of DC phenotype. Furthermore, the differential responses induced by different biomaterials were mediated through the distinct activation profiles of transcription factors. Together, these findings are expected to facilitate the design and selection of biomaterials that direct desired immune responses.

M. Tech. Metallurgical Engineering. / StudIes the tribocorrosion behaviour of copper-nickel-titanium shape memory composite reinforced by zirconia,synthesized through powder metallurgy process. The research aims to achieve the following objectives: 1. Study the tribocorrosion mechanisms of the composites in NaCl solution (typical human body fluid). 2. Investigate the tribocorrosion mechanisms of the composites in other environments typical of some engineering applications.The proposed study on incorporating zirconia into the matrix NiTiCu through powder metallurgical process and investigations of the phenomenon of joint wear-corrosion synergism occurring in sodium chloride considered typical of human body system and sulphuric acid environment typical of wide range engineering applications is therefore very novel. It is therefore aimed that information on the tribocorrosion behaviour of NiTiCu as well as with zirconia incorporation will form basis for typical compositional formulation approaches for improved bio-tribocorrosion improvement in biomedical applications and actuators used in other engineering applications.

This dissertation aims to further the understanding of the complex communication that occurs as cells interact with topographical and chemical patterns on a biomaterial interface. The research accomplishes this through two aims fabricating cell substrate surface topography and chemical patterns independently using non-cleanroom approaches, and analyzing higher order cellular response to surface features. The work will impact biomaterial surface modification and fabrication which will apply to biomedical implanted devices, tissue engineering scaffolds, and biological analysis devices. The first aim seeks to apply non-traditional topographical and chemical patterning methods in order to create independent topographical and chemical patterns on cell culture substrates. Experiments use the resulting patterned substrates to quantify cellular alignment to surface topography and compare the relative influence of topographical and chemical patterns on cellular response. The combined patterning methods of imprint lithography and micro-contact printing result in a high-throughput technique applicable to a variety of materials and a range of feature sizes from nanoscale through microscale, thereby enabling future analysis of cell response to surface features. The second aim evaluates the impact of topographical and chemical features on cellular differentiation. Experiments use patterned topography overlaid with a characterized chemical model layer to evaluate the effects of topography on myoblast differentiation and alignment. Chemical patterns that independently control available cell spreading area and modulate cell-cell contact are used to investigate the impact of cell-cell contact on differentiation.

Peripheral nerve injury in adults often leads to permanent functional loss with or without pain. Traumatic injury or surgery, metabolic injury (diabetic neuropathy), and drug toxicity can lead to neuropathies and all negatively impact the quality of life1-8. Damage to the nervous system is often permanent since neurons in the brain and periphery are post-mitotic and have limited regenerative capacity. Nerve repair involves axon regeneration, a complex and incompletely understood process with repair potential declining with age9-15. The research and design discussed involves the induction of endogenous repair mechanisms of the peripheral nerve using embryonic stem cells, alginate hydrogel, and the guided support of a biomaterial scaffold composed of PGS. Three different populations of cells are discussed: human embryonic stem cells, neural progenitor cells derived from human embryonic stem cells16, and primary rat bone marrow stromal cells. This study was innovative in that it was the first attempt for use of an elastomeric biomaterial scaffold in an injury model for the purpose of clinical application. This research is significant as it has direct clinical relevance in that it incorporates both functional and neuropathic recovery of patients affected by peripheral nerve damage.

The overall objective of this work was to assess the suitability of poly(3-octylthiophene) (P3OT) to sustain MC3T3-E1 osteoblast attachment and growth. The central hypothesis was that specific P3OT film properties (i.e., thickness, film preparation conditions, and level of doping) are able to regulate osteoblast functions (i.e., attachment and proliferation). Discrete and combinatorial techniques were utilized to prepare and characterize thin films of P3OT, a semiconductor in its undoped state, and to study its interaction with MC3T3-E1 osteoblasts. In this work we demonstrate that P3OT is a suitable surface to sustain MC3T3-E1 attachment and proliferation with no observed cytotoxicity. We show that P3OT has an effect on MC3T3-E1 attachment and proliferation as area, circularity, and proliferation ratio are significantly different for P3OT compared to control surfaces. We also demonstrate that P3OT doping and film preparation conditions have an effect on osteoblast attachment and proliferation but that thickness over a low and high range does not affect osteoblast functions. This work is significant because it contributes to the growing area of conducting polymers in biomedical applications and establishes P3OT as a potential cell substrate that sustains MC3T3-E1 attachment and promotes high levels of cell proliferation.