Osteoarthritis (OA) is the degeneration of articular cartilage that lines and protects the ends of your long bones to cushion and dissipate the forces applied to the body during physical activity. Articular cartilage can withstand millions of cyclic loads but eventually wears down, afflicting 50% of the population 65 and over. Those afflicted with OA suffer from activity limitations, pain, and increased risk of comorbidities, significantly decreasing their quality of life and incurring an economic burden of almost $100 billion annually. The homeostasis of cartilage is a refined balance between anabolic and catabolic factor production. Alterations to this delicate balance, specifically insensitivity to anabolic factors and increases in production of catabolic factors, stimulates degradation of the tissue. Degeneration of articular cartilage modulates the biomechanical properties of cartilage and elicits aberrant function of the resident cells, chondrocytes. Therefore, determining the influence of anabolic factors and degradation of the tissue on chondrocyte function and phenotype will provide valuable targets for the potential treatment of OA. Overall, the purpose of this dissertation was to determine how an anabolic growth factor, insulin-like growth factor-1 (IGF-1), and degradation of the extracellular matrix participate in the response of chondrocytes to physical cues through regulating the mechanosensitive non-selective ion channel Transient Receptor Potential Vanilloid 4 (TRPV4).

TRPV4 is a non-selective channel that is ubiquitously expressed throughout the body and can be activated by biomechanical stimuli, temperature, noxious nociceptive stimuli, natural and synthetic ligands, and endogenous phospholipids. Activation of this channel leads to an influx of calcium to transiently increase intracellular calcium ([Ca²⁺]_i). The spatiotemporal effect of TRPV4-mediated increases in [Ca²⁺]_i regulates the response of chondrocytes to compression and osmotic stimuli to enhance biosynthesis of two extracellular matrix protein of articular cartilage, proteoglycans and type II collagen (Col2). Additionally, activation of TRPV4 stimulates expression of the anabolic growth factor transforming growth factor-β3 while inhibiting the effect of the inflammatory cytokine interleukin-1. Knockout of TRPV4 in mice leads to spontaneous age-related OA and mutations of the channel cause aberrant [Ca²⁺]_i dynamics, impairing chondrogenesis and leading to skeletal dysplasia. While TRPV4 integrates the response of chondrocytes to mechanical stimuli and stimulate growth factor production, how anabolic factors and decreasing compressive modulus of the tissue during OA influence TRPV4 gating and chondrocyte function remain poorly defined.

During the development of articular cartilage, chondrocytes are solely responsible for the synthesis of the ECM responsible for the biomechanical and biophysical properties of the tissue. IGF-1 is a polypeptide growth factor that uniquely modulates chondrocyte function by acting as a mitogen and providing anti-catabolic effects. IGF-1 enhances the production of proteoglycans and Col2, promotes reparative cellular functions, and suppresses endogenous inflammatory cytokine induced matrix degeneration. In addition, synergism between dynamic mechanical loading and IGF-1 enhances proteoglycan and Col2 biosynthesis in neocartilage constructs. Following administration of physiologic concentrations to IGF-1, chondrocytes reorganize the actin cytoskeleton to resist and respond to mechanical deformations applied to the cells during physiologic loading of the tissue. The actin cytoskeleton interacts with TRPV4 to modulate gating of the channel and we have previously shown that growth factors can regulate mechanosensitive channels in the musculoskeletal system. Thus, we postulated that IGF-1 may regulate TRPV4 activation via the remodeling of the actin cytoskeleton during cartilage homeostasis.

In Chapter 2, we demonstrate that physiologic levels of IGF-1 suppress hypotonic-induced TRPV4 currents and intracellular calcium flux by increasing apparent cell stiffness that correlates with actin stress fiber formation. Disruption of F-actin following IGF-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Using point mutations of the TRPV4 channel at the microtubule-associated protein 7 (MAP-7) site shows that regulation of TRPV4 by actin is mediated via the interaction of actin with the MAP-7 domain of TRPV4. We further highlight that ATP release, a down-stream response to mechanical stimulation in chondrocytes, is mediated by TRPV4 during hypotonic challenge. This response is significantly abrogated with IGF-1 treatment.

During the initiation and progression of OA, alterations to the biomechanical and biophysical increase the magnitude of the compression, osmotic tension, fluid flow, and deformations applied to chondrocytes. This is evident as early hallmarks of OA are tissue and chondrocyte swelling and chondrocyte apoptosis near the articulating surface of the tissue. Regulatory volume decrease is an integral part of chondrocyte homeostasis as the cell swell during the dynamic mechanical loads they experience during normal physical activity. Impairment of this process is observed in OA chondrocytes and when chondrocytes are challenged with inflammatory cytokines. Previous research highlights the importance of TRPV4 activation in mediating the response of chondrocytes to hypotonic swelling and the subsequent regulatory volume decrease. Furthermore, substrate elasticity, or the compressive modulus of the tissue influences chondrocyte homeostasis and cell morphology. Normal cartilage has compressive moduli between 0.2 MPa-2 MPa, which can decrease up to an order of magnitude during the early stages of pathogenesis. While previous studies implicate this decrease in causing a catabolic phenotype in chondrocytes, how degradation of the ECM influences TRPV4-mediated mechanotransduction and chondrocyte function requires further study.

In Chapter 3, to elucidate how the chondrogenic ATDC5 cells alter TRPV4- mediated calcium signaling and cell phenotype in response to softer substrates, we created PEGDA-RGDS hydrogels with Young’s moduli that simulated healthy (~350 kPa), OA (~175 kPa) and severe OA (~35 kPa) tissue. Using these substrates, we found that softer substrates reduced the influx of calcium through TRPV4 when challenging chondrocytes with hypotonic swelling (HTS). Chondrocyte apoptosis also increased on the OA and severe OA gels due to elevated basal [Ca²⁺]_i which is attenuated with pharmacological stimulation of TRPV4. Pharmacological activation of TRPV4 rescued the expression of the anabolic markers of aggrecan and TRPV4 in chondrocytes cultured on OA gels and enhanced the type II collagen (col2) expression in cells on the normal and OA gels. These data suggest that the biomechanical properties of degraded cartilage alter TRPV4-mediated mechanotransduction in chondrocytes. Given that TRPV4 reduced apoptosis and improved the chondrogenic capacity of cells, TRPV4 stimulation could provide a potential therapeutic target in patients with early to moderate OA.