Scholarship & Research
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/1
Browse
2 results
Search Results
Item Microgels for single-cell culturing of neurons and chondrocytes(Montana State University - Bozeman, College of Engineering, 2023) Fredrikson, Jacob Preston; Chairperson, Graduate Committee: Abigail Richards; This is a manuscript style paper that includes co-authored chapters.Tissue engineering is a multidisciplinary field that combines engineering and life sciences to restore, improve, or generate biological substitutes to replace damaged tissues or organs. This is often performed using hydrogels that serve as scaffolds for the growth and maintenance of target tissues. Hydrogels, crosslinked polymer networks composed primarily of water, are excellent tissue mimics with highly tunable mechanical and biochemical properties. Hydrogels can be fabricated at the microscale, termed microgels, using drop-based microfluidics, which enables the precise control of cell density within the microgels down to a single cell. Encapsulating cells in microgels allows for the manipulation of microgels after production for single cell analyses. In this dissertation, human articular cartilage (HAC) cells and neurons are cultured within and upon microgel particles that serve as microscale tissue models for the study of chondrocyte matrix production and Herpes Simplex Virus type -1 (HSV-1) infection studies. HAC is the load-bearing tissue that lines the interfaces of joints and is responsible for shock and wear resistance. Chondrocytes, the cells in HAC, are responsible for producing and maintaining HAC. The chondrocyte pericellular matrix (PCM) regulates the metabolism and mechanical strain of the cells, which is critical to cellular function and cartilage homeostasis. However, the PCM is challenging to produce in vitro. The first half of this work applies microgels for PCM formation in chondrocytes. Immunofluorescence and high-performance liquid chromatography-mass spectrometry data demonstrate that chondrocytes grown in alginate microgels form a collagen VI-rich PCM, significantly altering the cells' metabolic response to dynamic compression. Atomic force microscopy data demonstrates that when chondrocytes are grown in alginate microgels for ten days, the elastic modulus of the PCM increases an order of magnitude. HSV-1 is a human pathogen that invades the peripheral nervous system. Understanding the complexities of HSV-1 infection at the single-cell level could lead to better therapeutics and reduced disease outcomes. Drop-based microfluidics (DBM) has recently been adapted for studying single-cell viral infection but has not been applied to neurons and HSV-1. The second half of this work develops a method for growing individual neurons in microgels. These microgel-embedded neurons are isolated, encapsulated with precise inoculating doses of HSV-1 using DBM, and the kinetics of viral gene expression are tracked in individual neurons using a fluorescent-recombinant HSV-1 virus. The data demonstrate that microgels provide a solid scaffold for neuronal development that supports single-cell productive HSV-1 infection within droplets.Item Development and validation of a system for studying chondrocyte mechanotransduction with preliminary metabolomic results(Montana State University - Bozeman, College of Engineering, 2013) Jutila, Aaron Arthur; Chairperson, Graduate Committee: Ronald K. June IIOsteoarthritis (OA) is a degenerative disease currently affecting over 46 million Americans. OA is most commonly characterized by breakdown of articular cartilage within the joint resulting in abnormal loading, loss of motion, and pain. Articular cartilage is the tough, flexible, load-bearing material that allows for joints to articulate smoothly i.e. running with relative ease. Currently there is no cure for this disease and the exact causes remain relatively unknown. Chondrocytes are the only cell type found in cartilage and are responsible for all biological maintenance and repair. Previous studies have shown that chondrocytes respond to mechanical load by cellular mechanotransduction, the process by which cells convert mechanical stimuli into biochemical activity. The aim of this thesis is to study the effects that mechanical loads have on human chondrocyte metabolism to better understand OA. To study chondrocyte mechanotransduction it was vital to develop a machine that could simulate in vivo loading of chondrocytes within the human knee joint. A bioreactor was designed, built, and validated that can simulate physiological loading in a tissue culture environment. This bioreactor was then used to characterize the mechanical properties of a viscoelastic material (agarose) capable of maintaining viable 3-Dimensional cell cultures. Inside the body chondrocytes are surrounded by a pericellular matrix (PCM), which provides a unique stiffness much less than the stiffness of cartilage. The mechanical property tests performed on agarose allowed for an accurate representation of this cellular microenvironment. Agarose gel concentrations were found that can model both healthy PCM and OA PCM stiffness. Methods were then developed to encapsulate living chondrocytes within these physiologically stiff gels. Utilizing these newly developed gel constructs and custom built bioreactor, 3-Dimensional chondrocyte suspensions were subjected to dynamic compression simulating normal physiological loading i.e. walking. It was hypothesized that moderate dynamic loading would promote changing in the central metabolism pathways, such as glycolysis. To study this hypothesis, mass-spectrometry techniques were utilized to identify metabolites present in each sample, and if the amount of each metabolite changed due to dynamic compression. These results provide a robust foundation for understanding cellular mechanotransduction.