**2.2 Detection of Ibuprofen and morphine by HPLC**

Known weights of beads were dissolved in acetonitrile and analyzed via high performance liquid chromatography (HPLC) as follows. A six point calibration curve was prepared by serial dilution and HPLC analysis. The stock solution of 1mg/mL was prepared in 1:1 deionized water (pH 2.5) and acetonitrile. Concentrations of 1, 5, 10, 25, 50 and 100 µg/mL were made in methanol. Samples of 20 µL were injected onto a C-18 column (Supelco Discovery C-18, 4.6 x 15 cm). The isocratic separation was carried out at 30°C using a mobile phase of 1:1 deionized water (pH 2.5) and acetonitrile. The flow rate was 2.0 ml/min with a run time of 10 minutes. Absorbance at 214 nm was taken using a Waters 486 UV detector. The absorbance of the sample was compared to a six-point standard curve from which concentration and percent loading in capsules was determined.

### **2.3 In vitro assay for microcapsule degradation**

In 24 well tissue culture plates approximately 80,000 microcapsules were placed in each well with either 0.9% saline, 15 µg/ml CFA (paraffin oil/15g *Mycobacterium tuberculosis*) mixed in water, or a 50:50 mixture of 0.9% saline and 15 µg/ml CFA. A homogenate of the water and oil was created by passing the solution through a 20 gauge needle 5 times. The plates were placed in a 5% CO2 chamber at 37◦C. The microcapsules were counted in triplicate in a hemacytometer chamber 0, 5 and 10 days following addition to the different solutions.

### **2.4 TMJ Injections**

The Baylor College of Dentistry Institutional Animal Care and Use Committee approved the experimental protocol. Male (250 grams) Sprague-Dawley rats from Harlan Industries, Houston, TX were kept on a 12:12 light/dark cycle with lights on at 08:00 hours. The animals were housed individually in our computerized feeding modules and given food and water ad libitum. They were acclimated to the surroundings for two days before

Cross-Linked Gelatin Microcapsules for Drug Delivery in a Arthritic TMJ 259

and female rats (Kerins, Carlson, Hinton, Grogan, Marr, Kramer, Spears & Bellinger, 2005;

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.

according to the manufacturer's directions (R&D Systems, Minneapolis, MN).

The TMJ tissue was homogenized in buffer (75 mM potassium acetate pH 7.4, 300 mM NaCL, 10 mM EDTA, 0.25% Triton X-100, protease inhibitors). It has previously been shown that TMJ-injected CFA can increase IL-1β (Kerins, Carlson, McIntosh & Bellinger, 2003) . Therefore, the cytokines IL-1 were quantitated from this homogenate using ELISA kits

The data were analyzed using a two-way analysis of variance with repeated measurement. Independent variables were treatment and time. The dependent variable was either the bead count or meal duration. The data found to be significant were further analyzed by

Images of the microcapsules in solution show that the capsules average size was 30 µm (Fig. 1A) and that the inside of the microcapsule is loaded with the dye/mineral oil mixture (Fig. 1B). About 80% of the mass of these microcapsules is the fluid mineral oil and about 20% of

Kerins, Carlson, McIntosh & Bellinger, 2003).

**2.6 Animals and tissue preparation** 

**2.7 Cytokine assay** 

**3. Statistics** 

**4. Results** 

Bonferonni pos-hoc test.

the mass consists of the gelatin shell.

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