**2.6 Animals and tissue preparation**

258 Recent Advances in Arthroplasty

receiving injections. Prior to the TMJ injections, the rats were removed from their cages and anesthetized with a 5% gas flow of isoflurane. Following the TMJ injections and removal

Saline or 15 µg of CFA in 15 µl was injected into the upper joint space of the TMJ followed by a second injection 24 hours later of 30 µl of dye loaded microcapsules (90 mg microcapsules [dry weight]/1 mlTris buffer or 0.9% saline) into the same location. The animals were sacrificed 5 hours (day 0), 5 and 10 days after injection of microcapsules and TMJ was removed en bloc, fixed in paraformaldehyde, sectioned and stained as described

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.

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

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

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

Before and after injection of the microcapsules the meal duration was recorded.

the rats nociceptive response was assayed in the pellet feeders, as described below.

from anesthesia, the rats began freely moving within two minutes.

**2.4.2 Microcapsules affect nociceptive response study** 

**2.4.3 MMP effect on degradation study** 

**2.4.4 Drug loaded microcapsules study** 

harvested for quantitation of the cytokine IL-1β.

**2.5 Nociceptive assay** 

**2.4.1 Bead degradation study** 

below.

Rats were sacrificed within 20 sec to minimize stress. For the cytokine study the TMJ anterior, disc, retrodiscal and synovium were dissected from one side by performing a superficial, horizontal skin incision parallel and just inferior to the zygomatic arch. The masseter and temporalis muscles were cut away from the arch and the ramus of the mandible so that the arch could be removed with ronjeurs. The neck of the condyle was grasped with hemostats and the condylar neck fractured to allow removal of any remaining musculature and access to the anterior, disc, retrodiscal and synovial tissues. Dissected TMJ tissue was excised from the posterior neck of the condyle, and all anterior tissue, articular disc, retrodiscal tissue and synovium were removed, placed in liquid nitrogen and stored at –80°C. To count the number of microcapsules in the TMJ a 0.5 cm cube of TMJ tissue centered on the condyle from the other side was removed and placed in 4% paraformaldehyde at 4°C overnight. The tissue was then decalcified in 0.5 M EDTA in a Pelco microwave (Ted Pella Inc., Redding CA) for 2 weeks, frozen and 20 m sections were cut and mounted on Superfrost glass slides (Fisher Scientific, Pittsburgh, PA). Every 5th section was stained with hematoxylin and eosin for counting and a small number of the slides were mounted with fluormount and DAPI. Microcapsules were counted on every 5th section to obtain a representative sample through the entire TMJ region (West & Slomanka, 2001). The fluorescent signal was imaged using MetaMorph Imaging System software (Molecular Devices Corporation, Sunnyvale, CA), a Photometrics CoolSnap K4 CCD camera (Roper Scientific, Inc, Duluth, GA) and a fluorescent microscope using a DAPI filter with excitation between 395-410 nm and an emission between 450-470 nm, as well as, a FITC filter with excitation between 490-505 nm and an emission between 515-545 nm.
