Effects of the Extracellular Osmotic Environment on Chondrocyte and Precursor Cell Physical and Metabolic Properties.

摘要

Osteoarthritis (OA) is a degenerative disease that afflicts cartilage of the articulating joints, including the knee joint. Due to the poor inherent healing capacity of this tissue, various strategies for repair have been investigated. These include development of tissue-engineered constructs from differentiated cartilage cells or precursor cells that could be implanted into OA defect sites in the knee. Previous work in our laboratory has explored the application of stimuli inspired by the native joint environment in attempts to increase the biochemical and mechanical properties of engineered constructs to levels that can restore function to the heavily-loaded knee.;The overarching goal of the work detailed in this dissertation was therefore to provide novel insight on the influence of the extracellular osmotic environment on chondrocyte and clinically-relevant cartilage precursor cell physical and metabolic properties over timescales (days, weeks) more relevant to the baseline osmotic environment, as described above. Use of various cell sources and 2D and 3D culture models allowed us to refine measurements of osmolarity-related phenomena, to provide more-sophisticated interpretation of these measurements, and to expand the impact of work previously performed on the extracellular osmotic environment of chondrocytes to clinically-relevant tissue engineering applications.;The major conclusions drawn from this work were multiple and contribute to several research areas, including effects of extracellular osmotic environment on cells in 3D and 2D culture, effects of chondrocyte tissue zone-of-origin on cell phenotype, and use of extracellular osmolarity as a physiologic stimulus for cartilage tissue engineering.;These conclusions were (1) The osmolarity of the 3D culture extracellular environment can have a significant effect on chondrocyte physical properties; (2) The osmolarity and/or ion concentration of the 3D culture extracellular environment can have a significant effect on chondrocyte metabolic properties; (3) These effects can persist over timescales relevant to osteoarthritis disease progression and de novo tissue growth; (4) Choice of osmolyte can affect chondrocyte response to alterations in 3D extracellular osmotic environment; (5) Zonal chondrocytes respond similarly to alterations in extracellular osmotic environment; (6) Tissue zone-of-origin is a greater regulator of cell metabolic activity than is the extracellular osmotic environment; (7) The osmolarity in which cells are expanded prior to transfer into 3D culture can affect expanded cell number in 2D and 3D culture and cell-seeded construct properties in 3D culture; (8) Application of higher-osmolarity (400 mOsM NaCl/KCl) 3D culture media increases the biochemical properties of 3D culture constructs over application of lower-osmolarity (300 mOsM) culture media, potentially recommending its use in tissue engineering protocols; (9) Application of higher-osmolarity (400 mOsM NaCl) media during 2D cell expansion increases the biochemical and mechanical properties of expanded cell-seeded 3D culture constructs over application of lower-osmolarity (300 mOsM) culture media, potentially recommending its use in tissue engineering protocols. (Abstract shortened by UMI.).;In the native articular cartilage tissue, the presence of highly negative-charged proteoglycans in the extracellular matrix gives rise to the extracellular osmotic environment, due to the attraction of positive counter-ions and associated water movement into the tissue. A gradient in proteoglycan content through the depth of the tissue gives rise to increasing extracellular osmotic environment from the articular surface of the tissue to the cartilage-bone interface. It is unclear how the metabolically-active chondrocytes that reside within this gradient regulate its concentration under healthy tissue conditions and under the onset of osteoarthritis, when traumatic loss of tissue proteoglycans leads to decreases in extracellular osmolarity.