Proteases play a key role in the cascade of tumor-associated proteolysis leading to extracellular matrix degradation, stromal invasion and blood vessel recruitment and inroad. Protease systems are widely described as implicated in the formation of new blood vessels. Until now, only few datas are available concerning their role in lymphangiogenesis. We successfully transposed the aorta ring assay to a mouse lymphatic thoracic duct assay. By immunochemistry and transmission electron microscopy, we characterized the outgrowing cells as being lymphatic cells that organize into microvessels containing a lumen and that conserved lymphatic endothelial cell features. This quantifiable model responds to several well-known lymphangiogenic factors as the VEGF-C but not to specific angiogenic factors. This model is so suitable to screen growth factors and inhibitors as well as conditioned media. Plasminogen activator inhibitor-1 is a component of the plasminogen cascade and, though it was critical for angiogenesis, it comes out that it is dispensable for lymphatic outgrowth. In sharp contrast, synthetic and physiological inhibitors of matrix metalloproteases inhibit lymphangiogenesis, and thoracic duct rings derived from MMP-2- but not MMP-9-deficient mice showed an impaired lymphatic cell outgrowth. These data identify MMP2 as an important player in lymphangiogenesis and was confirmed by an in vivo experiments. Proteases are thus also implicated in lymphangiogenesis and the lymphatic ring assay seems to be helpful to discover novel genes and mechanisms that underly the lymphangiogenesis process, including by comparing with angiogenesis in a similar system.

An in vitro model system utilizing de-mineralized periodontium tissues was developed for investigating the molecular controls involved in the spatial deposition of minerals on collagenous tissues. Preferential mineral deposition was observed when the de-mineralized tissue sections were incubated in solutions containing a supersaturation of calcium and phosphate ions with respect to hydroxyapatite (HA). Energy dispersive X-Ray (EDX) analysis demonstrated that these minerals are likely to be octacalcium phosphate (OCP) or dicalcium phosphate dihydrate (DCPD); further characterization with a secondary technique is required to draw a more definitive conclusion. The role of collagen fibrils in mineralization was tested by removing all the non-collagenous components from the tissue sections with proteolytic enzymes and exposing them to similar mineralization conditions as the control samples. A substantially less amount of minerals were formed in these samples; this correlates well with the hypothesis in the literature that collagen fibrils alone cannot initiate mineral formation.

An in vitro model system utilizing de-mineralized periodontium tissues was developed for investigating the molecular controls involved in the spatial deposition of minerals on collagenous tissues. Preferential mineral deposition was observed when the de-mineralized tissue sections were incubated in solutions containing a supersaturation of calcium and phosphate ions with respect to hydroxyapatite (HA). Energy dispersive X-Ray (EDX) analysis demonstrated that these minerals are likely to be octacalcium phosphate (OCP) or dicalcium phosphate dihydrate (DCPD); further characterization with a secondary technique is required to draw a more definitive conclusion. The role of collagen fibrils in mineralization was tested by removing all the non-collagenous components from the tissue sections with proteolytic enzymes and exposing them to similar mineralization conditions as the control samples. A substantially less amount of minerals were formed in these samples; this correlates well with the hypothesis in the literature that collagen fibrils alone cannot initiate mineral formation.

acase@tulane.edu / The ability of humans to regenerate complex tissue structures after amputation is not completely absent. Clinical reports have described random spontaneous cases of digit tip regeneration in young adults. Regeneration of a structure such as a limb or a digit requires tight orchestration of environmental cues and cells that come together and coordinate the regeneration of the missing body part. Studies on animal models have been crucial to have a better understanding on relevant components and mechanisms that are involved in epimorphic regeneration. Mechanistic studies however, are difficult to perform due to the lack of spatial and temporal control of microenvironmental factors. The overall approach of this proposal is to develop a blastema-like in vitro model to conduct comparative studies between connective tissue cells from regeneration-competent (P3) and incompetent (P2) regions of the mouse digit tip, and to control the cellular microenvironment to modulate P2 cells regeneration-incompetent behavior. A 3D spheroid culture model was identified to serve as a 3D biomimetic blastema model that preserves the inherent regenerative properties of regeneration-incompetent and regeneration-competent phenotypes. Relevant factors associated with either wound healing or a regenerative response, were evaluated in both P2 and P3 cells cultured as spheroids. It was found that the expression of wound healing markers, particularly known to be involved in scar tissue formation, were significantly higher in P2 spheroids. Conversely, the expression of markers indicative of a regeneration-permissive microenvironment was significantly higher in P3 spheroids. We evaluated the effects of oxygen modulation in P2 during 2D expansion and/or during 3D spheroid culture and found that preconditioning of P2 cells in 2D increases P2 cell number and promotes spontaneous aggregation. We also found that modulation of oxygen concentration during 3D culture significantly decreases expression of both, wound healing and regenerative markers. This physiologically relevant in vitro model provides a platform to characterize cellular processes involved in the wound healing and regenerative responses. Additionally, it allows for the incorporation of environmental cues, such as oxygen concentration, to better understand the key target mechanisms to shift the default wound response from scar formation to epimorphic regeneration. / 1 / Lina M. Quijano

No description available.

This work describes the use of microfluidic technology and biomaterials in cancer research by mimicking the extracellular matrix (ECM) and development of drug delivery system. Initially, biomaterials such as Gelatin methacryloyl (GelMA) and collagen type I were combined to create a hydrogel composite able to mimic both healthy and cancerous ECM. The impact of the tumor microenvironment was analyzed by using the hydrogel inside of a pressurized microfluidic device and by tracking the movement of gold nanoparticles (GNPs). The GNPs showed a decrease in diffusion coefficient of 77% when analyzed in cancerous conditions. This investigation was further explored by analyzing the diffusion of charged GNPs in the same system, while also tracking cellular uptake. An inverse correlation between diffusion and cellular uptake was obtained for charged GNPs in breast cancer cells. Due to the tunable properties and biocompatibility of GelMA, this hydrogel was also employed in the development of pH-responsive drug delivery systems. Since GelMA contains a gelatin backbone, two responsive polymers (Polymers A and B) were synthesized. Microspheres of ~40 μm were fabricated in flow focusing microfluidic devices. Polymer A microspheres displayed a swelling increase of 167% in pH 6.0, while polymer B spheres showed a 296% swelling in pH 10. Considering the unique properties of the tumor microenvironment such as leaky vasculature and acid pH environment, polymer A was selected to be used in the production of nanocarriers. The behavior of this polymer in acidic environment illustrated its potential applicability as drug delivery systems to the tumor area. Polymer A nanogels displayed a uniform size of 74 ± 7 nm. Lastly, GNPs were added to the solution of polymer A, leading to the fabrication of GNPs-loaded nanogels, presenting a homogenous distribution of gold particles inside nanogels. / Graduate / 2023-07-05

Nasal high flow (NHF) cannulae are used to deliver heated and humidified air to patients at steady flows ranging from 5-50 l/min. Knowledge of the airflow characteristics within the nasal cavity with NHF and during natural breathing is essential to understand the treatment's efficacy. In this thesis, the distribution and velocity of the airflow in the human nasal cavity have been mapped during natural and NHF assisted breathing with planar- and stereo-PIV in both steady and oscillatory flow conditions. Anatomically accurate transparent silicone models of the human nasal cavity were constructed using CT scan data and rapid prototyping. Breathing flowrates and waveforms were measured in vivo and dimensionally scaled by Reynolds and Womersley number matching to reproduce physiological conditions in vitro. Velocities of 2.8 and 3.8 m/s occurred in the nasal valve during natural breathing at peak expiration and inspiration, respectively; however on expiration the maximum velocity of 4.2 m/s occurred in the nasopharynx. Velocity magnitudes differed appreciably between the left and right sides of the nasal cavity, which were asymmetric. NHF modifies nasal cavity flow patterns significantly, altering the proportion of inspiration and expiration through each passageway and producing jets with in vivo velocities up to 20.8 m/s for 40 l/min cannula flow. The main flow stream passed through the middle airway and along the septal wall during both natural inspiration and expiration, whereas NHF inspired and expired flows remained high through the nasal cavity. Strong recirculating features are created above and below the cannula jet. Results are presented that suggest the quasi-steady flow assumption is invalid in the nasal cavity during both natural and NHF assisted breathing. The importance of using a three-component measurement technique when investigating nasal flows has been highlighted. Cannula flow has been found to continuously flush the nasopharyngeal dead space, which may enhance carbon dioxide removal and increase oxygen fraction. Close agreement was found between numerical and experimental results performed in identical conditions and geometries.

Accessible in vitro models recapitulating the human airway that are amenable to study whole cannabis smoke exposure are needed for immunological and toxicological studies that inform public health policy as well as medicinal and recreational cannabis use. In the present study, we developed and validated a novel three-dimensional (3D)-printed in vitro exposure system (IVES) that can be directly applied to study the effect of cannabis smoke exposure on primary human bronchial epithelial cells (HBECs). Using commercially available design software and a 3D printer, we designed a four-chamber Transwell insert holder for exposures to whole smoke. COMSOL Multiphysics software was used to model gas distribution, concentration gradients, velocity profile, and shear stress within IVES. Following simulations, primary HBECs cultured at the air–liquid interface on Transwell inserts were exposed to whole cannabis smoke using a modified version of the Foltin puff procedure. Following 24 h, outcome measurements included cell morphology, epithelial barrier function, lactate dehydrogenase (LDH) levels, cytokine expression and gene expression. HBECs exposed to cannabis smoke using IVES showed changes in cell morphology and disruption of barrier function without significant cytotoxicity. Cannabis smoke elevated interleukin-1 (IL-1) family cytokines and elevated CYP1A1 and CYP1B1 expression relative to control. These findings validate IVES to have an effect in HBECs at a molecular level following cannabis smoke exposure. In addition, HBECs stimulated with a viral mimetic, Poly I:C, challenge following cannabis smoke exposure showed a suppression of key antiviral cytokines. The growing legalization of cannabis on a global scale must be paired with research related to potential health impacts on lung exposures. IVES represents an accessible, open-source, exposure system that can be used to model varying types of cannabis smoke exposures with HBECs grown under air–liquid interface culture conditions. / Thesis / Master of Science (MSc) / Despite its recent legalization in Canada, cannabis smoke has been understudied and a lack of evidence exists to inform legislative policies, medicinal and recreational usage. Due to a lack of relevant ways to study cannabis smoke in a lab setting, it is difficult to accumulate literature around its impacts in the lungs. Here, we addressed this gap by engineering and validating a novel model to expose lung cultures to cannabis smoke. In addition, we investigated its impact on the immune response. Our findings suggest exposure to cannabis smoke alters the immune functions of these cells. We also found that in response to a viral mimetic stimulus, cell cultures pre-exposed to cannabis smoke exhibited impaired immune responses. Our novel model to expose cell cultures to cannabis smoke creates a foundation for future researchers to investigate environmental insults, such as cannabis smoke, in the context of respiratory health and infectious disease.

Head and neck squamous cell carcinomas (HNSCC) are among the leading causes of cancer related deaths throughout the world. The survival rate for this type of cancer is extremely low and has not changed significantly in recent decades. There is an imperative need to study tumor progression in a representative model in order to generate more knowledge about this disease as well as develop more effective treatment options. Multiple methods already exist for studying HNSCC and other types of cancers, including in vitro and in vivo models. Although in vivo models are more representative of the human carcinomas in terms of complexity of the microenvironment the tumor cells experience, they are difficult to manipulate and many experiments cannot be performed easily in whole organisms; therefore, in vitro models are used. Current in vitro models are typically two-dimensional (2D) monolayer cultures that are easily manipulated for a controlled environment, but these fail to mimic the native microenvironment in terms of three-dimensional (3D) interactions present in vivo. The literature documents that several 3D organotypic models of HNSCC have been created, showing significant differences in tumor response to drugs between these models and traditional 2D culture systems, perhaps suggesting a closer representation of human HNSCC. However, these models were not rigorously validated, with little comparison to in vivo tumor behavior. We developed a 3D HNSCC in vitro model using electrospun scaffolds to mimic the extracellular matrix as well as using a HNSCC-derived tumor cell line, HN12, in co-culture with a supporting fibroblast cell line. We compared the model to the same tumor cell line grown in vivo in immunodeficient mice. We also investigated drug sensitivity of tumor cells in our model compared to conventional monocultures to determine whether differences exist. Finally, we investigated pro-angiogenic properties of tumor cells in this model. The long-term goal is to develop a model that can be manipulated easily to study tumorigenic mechanisms and potential treatments.

Many congenital skeletal muscle disorders including muscular dystrophies are caused by genetic mutations that lead to a dysfunction in myocytes effectively binding to the extracellular matrix. This leads to a chronic and continuous cycle of breakdown and regeneration of muscle tissue, ultimately resulting in loss of muscle function and patient mortality. Such disorders lack effective clinical treatments and challenge researchers to develop new therapeutics. The current drug development process often yields ineffective therapeutics due to the lack of genetic homology between pre-clinical animal models and humans. In addition current engineered tissue models using human cells fail to properly emulate native muscle morphology and function due to necrotic tissue cores and an abundance undigested ECM protein. Thus, a more precise benchtop model of 3D engineered human muscle tissue could serve as a better platform for translation to a disease model and could better predict candidate drug efficacy during pre- clinical development. This work presents the methodology for generating a high-content system of contiguous skeletal muscle tissue constructs produced entirely from human cells by using a non-adhesive hydrogel micro-molding technique. Subsequent culture and mold modifications confirmed by morphological and contractile protein analysis improve tissue longevity and myocyte maturation. Finally, mechanical strength and contractile force measurements confirmed that such modulations resulted in skeletal muscle microtissues that were more mimetic of human muscle tissue. This cell self-assembly technique yielded tissues approximately 150um in diameter with cell densities approaching that of native muscle. Modifications including seeding pre-differentiated myoblasts and the addition of ECM producing fibroblasts improved both tissue formation efficiency and cell alignment. Further culture modifications including supplementation of the culture medium with 50ug/ml ascorbic acid and 100ng/ml Insulin-like growth factor-1 coupled with a mold redesign that allowed tissue to passively contract during maturation while still remaining anchored under tension further improved ECM production, myogenic differentiation, and long-term longevity in culture. Further confirmation of the culture improvements were demonstrated by increases in mechanical strength and contractile force production. In conclusion, this approach overcomes cell density limitations with exogenous ECM-based methods and provides a platform for producing 3D models of human skeletal muscle by making tissue entirely using cells. Future work will attempt to translate the methodology used for tissue generation and long-term culture to create benchtop models of disease models of skeletal muscle, streamlining pre- clinical benchtop testing to better predict candidate drug efficacy for skeletal muscle diseases and disorders along with elucidating side effects of non-target drugs.