In Vitro Culture Conditions and Mechanical Stimulation on Inorganic Polyphosphate Production in Chondrocytes

Abstract

Osteoarthritis (OA) is degenerative joint disease that is characterized by the progressive loss and destruction of articular cartilage, the connective tissue that provides a smooth and lubricated surface for articulation and the transmission of mechanical forces. OA affects almost 5 million Canadians and can cause individuals to experience pain, joint stiffness, and a decreased range of motion, all of which have a significant impact on their quality of life. There is currently no cure to stop the progression of OA; hence, current treatments are solely focused on symptom management, while the disease progresses.

The lack of effective treatments is largely attributed to our limited knowledge of cartilage biology and of the mechanisms involved in the pathogenesis of the disease. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphates that are linked together by high energy phosphoanhydride bonds. Previous studies have shown that the exogenous administration of polyP to cultures of chondrocytes stimulates matrix accumulation, while intra-articular injection in an OA animal model protected the cartilage from degenerative changes. Polyphosphate has also been observed in native articular cartilage. Therefore, polyP demonstrates the potential to be integrated into therapeutics to address the degenerative nature of OA. Despite this potential as a chondroprotective agent, polyP remains understudied in cartilage. In this work, we investigated the impact of culture conditions, exogenous polyP administration, and static compression on the levels of polyP and other key metabolites in cartilage tissue.

Our work demonstrated that the polyP found in cultures of chondrocytes can be synthesized by the cells, as opposed to accumulated from media supplements. Cultures grown in the combination of DMEM 4.5 g/L glucose and fetal bovine serum (FBS) yielded the greatest accumulation of polyP in the tissue constructs. PolyP levels were influenced by media composition, as greater levels of polyP per cell were quantified in DMEM compared to Ham’s F12. Glucose, lactate, and polyP content were monitored over a 72-hour period between media changes, with a downward trend observed in the glucose content and an upward trend in lactate and polyP accumulation. Exogenous polyP administration elicited minimal differences in glucose consumption and lactate build-up. Static compression of tissue constructs resulted in an increase in cell proliferation and depletion of collagen and proteoglycans, the two major components of the extracellular matrix. The metabolic response to static loading appeared to be minimal due to the stable levels of glucose, lactate and polyP quantified, suggesting the chondrocytes may reach a state of equilibrium similar to the controls within the compression session. Results from this thesis provide insight into the factors that influence polyP metabolism, furthering our understanding of this biomolecule’s role in cartilage tissue. Further work needs to be done to identify the key factors involved in modulating polyP levels, the effect of exogenous polyP administration on metabolic pathways, and short-term effects of static compression on metabolism.