A multitude of biophysical signals, including chemical, mechanical, and contact guidance cues, are embedded within the extracellular matrix (ECM) to dictate cell behavior and determine cell fate. To understand the complexity of the cell-matrix interaction and how changes to the ECM contribute to the development of tissues or diseases, three-dimensional (3D), culture systems that can decouple the effects of these cues on cell behavior are required. This dissertation describes the development and characterization of approaches based on multiphoton excitation (MPE) to control the chemical, mechanical, and topographical presentation of micro-3D-printed (μ-3DP) protein hydrogels independently. Protein hydrogels were chemically functionalized via the MPE-induced conjugation of benzophenone-biotin without altering the physical properties of the matrix. Complex, immobilized patterns and chemical gradients were generated within protein hydrogels with a high degree of spatial resolution in all axes. Hydrogel surfaces were also labeled with adhesive moieties to promote localized Schwann cell adhesion and polarization. Laser shrinking, a method based on MPE to manipulate the topographical and mechanical presentation of protein hydrogels after fabrication, is also presented. Topographical features on an originally flat substrate are created with depths approaching 6 μm. The Young’s modulus of protein hydrogels can also be increased by 6-fold (~15 – ~90 kPa) using laser shrinking, and parameters can be adjusted to create continuous gradient profiles for studying durotaxis. At determined scan conditions, the two properties can be adjusted independently of each other. Most importantly, the physical properties of the hydrogels can be manipulated in situ to study the effects of dynamic changes to the substrates on cells. As a potential tool to monitor cellular responses to presented cues, fluorescent probes that detect nitric oxide are characterized. Collectively, these technologies represent a key advance in hydrogel tunability, as the platforms presented offer independent, dynamic, and spatiotemporal control of the chemical, mechanical, and topographical features of protein hydrogels. The introduced technologies expand the possibilities of protein hydrogels to clarify underlying factors of cell-matrix interactions that drive morphogenesis and pathogenesis, and are broadly applicable to a multitude of physiological systems. / text

In vivo, adipose tissue demonstrates only a limited capacity for self-repair, and the long-term treatment of subcutaneous defects remains an unresolved clinical problem. With the goal of regenerating healthy tissues, many tissue-engineering strategies have pointed to the potential of implementing three-dimensional (3-D), cell-seeded scaffolds for soft tissue augmentation and wound healing. In particular, microcarriers have shown promise as both cell expansion substrates and injectable cell-delivery vehicles for these applications. However, limited research has investigated the engineering of tissue-specific microcarriers, designed to closely mimic the native extracellular matrix (ECM) composition. In this work, methods were developed to fabricate microcarriers from decellularized adipose tissue (DAT) via non-cytotoxic protocols. Characterization by microscopy confirmed the efficacy of the fabrication protocols in producing stable beads, as well as the production of a microporous surface topography. The mean bead diameter was 934 ± 51 μm, while the porosity was measured to be 29 ± 4 % using liquid displacement. Stability and swelling behavior over 4 weeks indicated that the DAT-based microcarriers were effectively stabilized with the non-cytotoxic photochemical crosslinking agent rose bengal, with only low levels of protein release measured within a simulated physiological environment. In cell-based studies, the DAT-based microcarriers successfully supported the proliferation and adipogenic differentiation of human adipose-derived stem cells (hASCs) in a dynamic spinner flask system, with a more favorable response observed in terms of adhesion, proliferation, and adipogenesis on the DAT-based microcarriers relative to gelatin control beads. More specifically, dynamically-cultured hASCs on DAT-based microcarriers demonstrated greater lipid loading, as well as higher glycerol-3-phosphate dehydrogenase (GPDH) activity, a key enzyme involved in triacylglycerol biosynthesis, at 7 days and 14 days in culture in an inductive medium. Overall, the results indicated that the DAT-based microcarriers provided a uniquely supportive environment for adipogenesis. Established microcarrier sterility and injectability further support the broad potential of these tissue-specific microcarriers as a novel, adipogenic, clinically-translatable strategy for soft tissue engineering. / Thesis (Master, Chemical Engineering) -- Queen's University, 2010-12-01 14:28:14.628

In recent years, respiratory diseases have emerged as a leading cause of mortality across the globe. In the United States alone respiratory diseases are the fourth leading cause of deaths annually. Moreover, with the rapid increase of industrialization and urbanization, the occurrences of respiratory diseases are expected to remain high with strong chances of increasing in the future. To ameliorate the epidemic of respiratory disease, it is first important to understand its underlying mechanisms. Respiratory research studies in animals have elucidated the chronological order of the pathological events and systemic responses inside the lung, but understanding the response of individual cell types inside the lung is necessary to outline the initiators and mediators of the pathological events. Many research studies have aimed to understand the behavior of individual cell types, from the lung, under different pathological conditions specific to the respiratory system. However, the cell culture systems used in most of these studies were limited by the absence of the dynamic cell growth environment present in actual lung tissues. The lung exists in a mechanically active environment, where different amounts of circumferential and longitudinal expansion and contraction occur during breathing movements. Thus, simulating the biomechanical environment in in vitro cell culture models may improve the cellular functionality and the outcome of the research studies. Moreover, the stimulation of biomechanical forces in in vitro cell cultures provides the advantage of mimicking the mechanical environment, related to different pathological conditions. In our study we used a dynamic in vitro cell culture system capable of implementing cyclic equibiaxial deformation in cell monolayers to stimulate different biomechanical environments similar to conditions inside the lung. The dynamic cell growth condition was used to determine the effects of ventilator-induced lung injury and nano-material/pollutant exposure in A549 cell cultures. Examples of such pollutants are diesel particulate matter, multi-walled carbon nanotubes, and single-walled carbon nanotubes. Our results indicated that the dynamic cell growth condition specific to ventilator induced lung injury facilitated an increase in inflammatory and tissue remodeling activities in A549 cells. Under the nano-material/pollutant exposure assessment studies, the dynamic cell growth condition induced changes in inflammation and oxidative stress level which closely resembled those in in vivo studies.

A cerebral aneurysm is a weakened area of an artery in the brain, creating an abnormal expansion. Recent research for treatment is utilizing a photopolymerizable hydrogel as a possible operation for injection in situ. This paper aimed to achieve bioconjugation of peptides on a PEGDMA polymer network (using the photoinitiator PEG-BAPO) to form a biocompatible photopolymerizable hydrogel, without compromise to any of its mechanical attributes. Achieving cell adhesion to the hydrogel surface is a critical requirement as that could drive the growth of endothelium between aneurysm and artery, to considerably enhance its sustainability and decrease the risk of inflammation. The hydrogel was synthesized by functionalizing RGD with a PEG-spacer and co-polymerize it with PEGDMA using UV-radiation to create an intertwined cross-linking network. Samples of various peptide concentrations were studied in cell culture to analyze cell adhesion, followed by mechanical tests to identify possible deviations. A subsequent study was established to create a dynamic prototype as a quantifiable replication of a hydrogel inside an aneurysm in vivo. The model was designed in SolidWorks and connected with an Ibidi sticky-Slide to roughly replicate a cerebral aneurysm connected to an artery with space to introduce a hydrogel sample.

L’organisme humain présente de nombreuses constantes de régénération tissulaires et c’est cette caractéristique essentielle qui maintient l’équilibre physiologique. Toutefois, l’existence de lésions importantes provoquée par un déséquilibre interne ou externe peut empêcher l’organisme de s’auto-régénerer. Dans ce cas, l’application des biomatériaux développés pour des applications biomédicales peuvent améliorer le processus de guérison. Pour les applications en tissus durs, les biomatériaux doivent posséder des propriétés similaires aux matrices naturelles tant sur le plan biologique que physico-mécanique. Dans les applications en bioingénierie osseuse, les composites à base de collagène (Col) et d’hydroxiapatite (HA) sont devenus tellement performant qu’ils peuvent être classifiés comme des matériaux biomimétiques. Cette thèse propose la production d’une matrice 3-D poreuse à base d’HA et de Col (50:50wt%). Ce composite réticulé par le glutaraldéhyde a été caractérisé par des différentes techniques et servira de support pour la culture cellulaire. Des cellules estromales ostéoprogénitrices ont été cultivées dans un environnement statique et dynamique (deux vitesse de flux) et leurs capacités de colonisation ainsi que leurs comportements d’adhésion, de prolifération, de différentiation seront observés. A travers les résultats de diffraction de rayons X et de spectroscopie infrarouge, il est possible d’affirmer la présence dans la matrice collagène d’une phase minérale peu cristalline constituée par de l’hydroxiapatite carbonatée du type-B déficiente en calcium. La viabilité cellulaire a été fortement influencée par les systèmes de culture au cours des 21 jours. Les résultats du système dynamique en haute vitesse montrent une excellente capacité du composite à supporter les processus cellulaires. Les cellules sont capables d’adhérer, de proliférer et de coloniser la matrice tridimensionnelle. / The progress in Tissue Engineering area allows the development of biomaterials that mimic the properties of natural tissues. For biomedical applications in mineralized tissues, composites based on hydroxyapatite (HA) and collagen (Col) have presented good results when implanted in vivo. The aim of this work was to produce a 3-D matrix and to observe the cell behaviour when stromal cells are cultured in contact with HA-Col scaffold under static and dynamic conditions. For in vitro biological evaluation, osteoprogenitor human cells (Stro+1A cells) were grown and their colonization capacity and adhesion, proliferation and differentiation behaviour were quantified. Two perfusion flow rates (0,03ml/min and 0,3ml/min) were proposed for dynamic culture. The HA-Col composite was prepared by reorganization of Col fibrils simultaneously with HA crystal nucleation and precipitation from calcium and phosphate rich solutions. Afterwards, the composites were crosslinked and sterilized by gamma radiation. Stro+1A cells were inoculated (5x105 cells/sample) into the scaffolds and cultured over 21 days in a humid incubator at 37°C and 5% CO2. Infrared spectroscopy and X-ray diffraction results suggested a calcium-deficient hydroxyapatite as mineral phase. About cell culture, the cell number increased under higher flow rate dynamic culture. By scanning electron microscopy and histological sections, we observed cells adhered and spread inside colonized scaffolds.