The Mechanical Regulation of Biological Mediators of Cartilageand Joint Destruction in Osteoarthritis

Currently in the U.S., musculoskeletal conditions are the leading cause of disability.Joint diseases account for 50% of all chronic conditions in the elderly. Worldwide, OA is only the 6^th leading cause of years of life lost to ill health. The burden of musculoskeletal disease, both in terms of human illness and heath care costs is projected to widen significantly by the year 2030. The aging of the U.S. population is expected to produce an additional 21 million individuals in the 65-and-over age group, representing a 20% increase over current demographics. Surprisingly, despite the wealth of clinical data on OA, surgical treatment which culminates in total joint replacement, remains the most effective therapy for progressive OA. Relatively little is known about the basic biology of OA especially how mechanical wear, the major hallmark of OA, influences fundamental biological control mechanisms in chondrocytes, the cells that populate cartilage.

Recently, through the use of specially-designed mechanical compression devices, we have found that chondrocytes respond to increasing mechanical loading signals much as other tissues respond to increasing concentrations of hormones or growth factors by activating 3 distinct MAPK signaling pathways, including the so-called ‘stress-activated’ protein kinase pathways. This finding not only opens the way for further research into what actions these signaling pathways have on specific genes involved in OA but also provides an opportunity to intervene with pathway-selective MAPK inhibitors in the treatment of OA

The overall goal of these projects is to advance the understanding of the molecular mechanisms of osteoarthritis (OA) progression through the novel finding that mechanical force activates critical cellular-signaling pathways in cartilage. Specific primary goals include:

• Mechanical loading of articular cartilage regulates ECM turnover through specific mechano-sensitive cellular signaling pathways:Physical Loading in the form of mechanical compression regulates both normal and abnormal (osteoarthritis) extracellular matrix (ECM) turnover in articular cartilage. Investigations are underway to elucidate both the specific geneexpression events and the cellular signaling pathways responsible for their regulation under various loading conditions that simulate normal and abnormal loading conditions. Such findings will not only shed light on the basic mechanisms of osteoarthritis but also identify potential therapeutics through the use of small-molecule signaling inhibitors.

Aim1: Continue work in progress to identify matrix metalloproteinases (MMPs) which are regulated by mechanicalcompression in articular cartilage explants.

Aim2: Continue work in progress to identify cellular signaling pathways regulatedby mechanical compression in articular cartilage explants.

Aim3: Integrate mechanically-regulated gene expression events (MMPs) with mechanically-regulated signaling pathways events through the use of pathway-selective inhibitors.

• Differential MMP expression due to differential loading of articular cartilage in the lateral vs. medial compartments of the varus/valgus kneeOur previous work has identified signaling and MMP expression events in compression studies via the use of animal tissues. Human cartilage ex vivo compression studies have been hampered due to the relative thinness of human articular cartilage. Human varus/valgus malformations produce a distortion in the normal weight bearing from one compartment to the other within a single tibio-femoral joint and therefore provide an opportunity to examine differential loading effects.

Aim1: examination of differential MMP expression events by comparing medial vs. lateral human condylar samples.

Aim2: examination of differential mechano-signaling events in medial vs. lateralsamples

• Long-term articular cartilage ex vivo compression methodology optimization:OA is a chronic disease, yet the molecular events that accompany chronic loading have not been assessed in ex vivo tissue culture models of cartilage compression. This is, in part, due to the reliance upon an organ culture environment that includes high amounts of animal serum as a media supplement. This medium likely contributes to the de-differentiation of chondrocytes in longer-term experiments and precludes the ability to sample the medium following experimental conditions due to added high protein concentrations.

Aim1: Evaluate various types of defined media and media supplements incartilage explant cultures for markers of differentiation and de-differentiation and ability to transduce mechanical signals.

Aim2: Compare results to conventional serum-containing media systems using abovecriteria.

Aim3: Introduce hypoxic (low oxygen) environment to mechanically-stimulated cartilage explants

• Tetracycline-family drugs in an ex vivo model of early osteoarthritis development: An important application of this ex vivo articular cartilage mechanobiological system is to test candidate drugs that potentially interfere with MMP production and the cell signaling events that lead to their production and activation.Therefore, tetracyclines, a family of drugs with anecdotal evidence of anti-MMP activities, have been tested. In addition, the nitric oxide (NO) signaling pathway, an important multifunctional messenger in tissue destruction events has not previously been examined under ex vivo mechanical loading situations in articular cartilage. The biochemical relationship between mechanical loading, mitogen-activated protein kinase (MAPK) signaling, nitric oxide (NO) signaling and MMP regulation have been demonstrated through the following aims:

Aim1: Examination of effects of tetracyclines on various cartilage MMP levels andactivation states.

Aim2: Analysis of relative activation states of mechanically-activated MAPK and SAPKsignaling events in the presence and absence of various tetracyclines and dosages.

Aim3: Analysis of the NO signaling pathway under various loading conditions andin combination with various tetracycline treatment regimens.