**2.4.2 Microcapsules affect nociceptive response study**

Rats were injected with 30 µl 0.9% saline or 15 µg CFA and then 24 hours later the rats were injected with either saline or 30 µl of the microcapsule slurry (20% w/v) in 0.9% saline. Before and after injection of the microcapsules the meal duration was recorded.

### **2.4.3 MMP effect on degradation study**

Rats were injected with 30 µl saline or 15 µg CFA or saline with an MMP inhibitor or CFA with an MMP inhibitor added and then 24 hours later these groups of rats were injected with 30 µl microcapsules or microcapsules plus the MMP inhibitor. The MMP inhibitor was a 1 µM solution of the MMP-2,9 inhibitor IV (EMD Biosciences, cataloge # 444274). In the first experiment the rats were sacrificed 0, 5 and 10 days following microcapsule injection, the TMJ was removed, sectioned and the microcapsules counted. In the second experiment the rats nociceptive response was assayed in the pellet feeders, as described below.

### **2.4.4 Drug loaded microcapsules study**

Rats were injected into the upper joint space of the TMJ with a 30 µl slurry of microcapsules containing 15% ibuprofen or 1% morphine in oil or aslurry of microcapsules containing just oil. After twenty-four hours a portion of these animals were subdivided further; one group received bilateral injections of 0.9% saline (30 µl) and the other group received an injection of CFA into the upper joint space. Before and after injection the nociceptive response (i.e., meal duration) was assayed by placing the rats in pellet feeders. Also, on day 7 post-injection the TMJ tissue of a portion of these rats was harvested for quantitation of the cytokine IL-1β.

### **2.5 Nociceptive assay**

The rats were housed individually in sound-attenuated chambers equipped with photobeam computer-activated pellet feeders (Med Assoc. Inc., East Fairfield, VT). The rats were given 45 mg rodent chow pellets (Product No. FO 165, Bioserv, Frenchtown, NJ). When a rat removes a pellet from the feeder trough, a photobeam placed at the bottom of the trough is no longer blocked, signaling the computer to drop another pellet, record the date and time, and keep a running tally of the total daily food consumption. The computer record of pellets dropped over time establishes the meal duration, which is a continuous non-invasive biological marker of TMJ nociception (surface and deep) in undisturbed male and female rats (Kerins, Carlson, Hinton, Grogan, Marr, Kramer, Spears & Bellinger, 2005; Kerins, Carlson, McIntosh & Bellinger, 2003).
