David Mecham and David Kooyman, Faculty Mentor

Introduction

Osteoarthritis is a degenerative disease of the cartilage that affects more than 80% of Americans above the age of 65 (1). Many adults also suffer from Temporomandibular joint dysfunction (TMD), and 80% of such individuals eventually develop Temporomandibular joint associated Osteoarthritis (TMJ OA) (2). Current pharmaceutical options target symptoms of OA, but there are no known treatments available. Research is being undertaken to elucidate the root causes of the disease at a cellular level. Our lab has previously shown that mice lacking the Receptor for Advanced Glycation End Products are protected from OA (3). Other researchers have shown that blocking Toll-like receptor 4 (TLR-4) also slows disease progression (4). The aim of this project was to determine the additive effects of blocking RAGE and TLR-4 on the progression of TMJ OA. The data presented in this report, while preliminary, provides new and exciting data in the study of OA.

Materials

Bonding of a stainless steel wire to the maxillary molars was performed on RAGE KO and wild type (WT) mice matched in age and gender to induce TMJ OA. This procedure was performed on mice that were 8-10 weeks old, a period where they are considered fully mature. CLI-085, a TLR-4 blocker, was suspended in DMSO and injected into the TMJ capsules of WT and RAGE KO mice at a concentration of 1.0 μM. Mice were injected immediately following and at seven weeks following the procedure. As a control, DMSO without CLI-085 was injected into WT and RAGE KO mice at similar time points. At 2 and 8 weeks following the procedure, mice were sacrificed and joint tissues harvested. Our lab has previously demonstrated this is an appropriate span of time to monitor TMJ OA onset and progression. Tissues were decalcified and embedded in paraffin wax to be sectioned for further analysis. Tissues were stained with Safranin O/Fast Green and scored using the Mankin scoring system, which determines the severity of OA on a scale of 0-15, with higher numbers corresponding to more severe disease.

Results

Preliminary analysis revealed that treatment with CLI-095 does not prevent the onset and progression of TMJ OA in either RAGE KO or WT mice. As expected, WT mice with the TLR-4 blocker do have a slightly higher Mankin score average than RAGE KO mice, indicating that blocking both pathways may slightly attenuate OA progression (Figure 1). However, RAGE KO mice showed significant hypocellularity, indicating that blocking both pathways may also interfere with normal chondrocytic development and proliferation.

Discussion

The results obtained up to this point are demonstrate a trend but are insufficient to obtain statistical significance. We will proceed with our experimental design to increase sample size and complete analysis and comparison of control samples.

References

1. R.C. Lawrence, D.T. Felson, C.G. Helmick, L.M. Arnold, H. Choi, R.A. Deyo, S. Gabriel, R. Hirsch, M.C. Hochberg, G.G. Hunder, J.M. Jordan, J.N. Katz, H.M. Kremers, F. Wolfe. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum, 58 (2008), pp. 26–35.
2. Wadhwa, S. and S. Kapila, TMJ disorders: Future innovations in diagnostics and therapeutics. Journal of Dental Education, 2008. 72(8): p. 930-947.
3. J. Larkin, J.Z. Kartchner, D.M. Peterson, A.S. Doxey, W.R. Hollis, J.L. Rees, C. Ingersoll, G.G. Jackson, S.K. Wilhelm, S.S. Haynie, E. Chavez, P. Reynolds, D.L. Kooyman. The temporal expression of biomarkers associated with osteoarthritis after knee destabilizing surgery in mice with and without Receptor for Advanced Glycation End products (RAGE).
4. P.L.E.M. van Lent, A.B. Blom, L. Grevers, A. Sloetjes, W.B. van den Berg. Toll-like receptor 4 induced FcgammaR expression potentiates early onset of joint inflammation and cartilage destruction during immune complex arthritis: Toll-like receptor 4 largely regulates FcgammaR expression by interleukin 10. Ann Rheum Dis, 66 (2007), pp. 334–340.