**5. Discussion**

Gelatin microcapsules have the capability for sustained drug release after intra-articular injection (Inoue, Takahashi, Arai, Tonomura, Sakao, Saito, Fujioka, Fujiwara, Tabata & Kubo, 2006; Lu, Zhang, Sun & Zhong, 2007; Saito, Takahashi, Arai, Inoue, Sakao, Tonomura, Honjo, Nakagawa, Inoue, Tabata & Kubo, 2009). Sustained drug delivery into a diseased TMJ has the potential to improve the treatment of TMJ pain and inflammation. In this report a rat model of TMJ inflammatory arthritis was used and we tested the effect of intra-

Ibuprofen drug loaded microcapsules did not significantly affect the nociceptive response (Fig. 8A) or the immune response (Fig. 8B) of CFA injected rats. Morphine loaded microcapsules did significantly decrease the nociceptive response (p<0.05, days 4 and 5) and post-hoc testing showed a significant decrease on the fourth day post-CFA injection

Fig. 8. Nociceptive and inflammatory response after injecting microcapsules containing ibuprofen or morphine. Rats TMJs were injected with a 30 µl solution of microcapsules (bead inj). The microcapsules contained oil (Blank microcapsules) or oil with 15% ibuprofen (Ibuprofen microcapsules) or oil containing 1% morphine (Morphine microcapsules). 24 hours after microcapsule injection the rats were subdivided further for TMJ injection of either saline or 15 µg CFA (CFA inj). A) Before and 8 days after injection of the saline/CFA the daily meal duration was recorded. There were 8 animals in each of the four treatment groups. B) The amount of IL-1β in the TMJ retrodiscal, synovial and disc tissue 7 days after injection with microcapsules. There were 3-4 animals in each of the four treatment groups

Gelatin microcapsules have the capability for sustained drug release after intra-articular injection (Inoue, Takahashi, Arai, Tonomura, Sakao, Saito, Fujioka, Fujiwara, Tabata & Kubo, 2006; Lu, Zhang, Sun & Zhong, 2007; Saito, Takahashi, Arai, Inoue, Sakao, Tonomura, Honjo, Nakagawa, Inoue, Tabata & Kubo, 2009). Sustained drug delivery into a diseased TMJ has the potential to improve the treatment of TMJ pain and inflammation. In this report a rat model of TMJ inflammatory arthritis was used and we tested the effect of intra-

(Fig. 8A).

for the cytokine analysis.

**5. Discussion** 

articularly injected gelatin microcapsules on the nociceptive response as well as on the inflammatory response. Loading the gelatin microcapsules with the ibuprofen or morphine was accomplished because we hypothesized that intra-articular injection of drug loaded beads would reduce the nociceptive response. We determined that gelatin microcapsules do not increase IL-1β levels in the disc and synovial tissues after injection into the TMJ. Nor do the capsules increase the nociceptive response in an arthritic joint. Importantly, injection of morphine but not ibuprofen loaded microcapsules decreased the nociceptive response of a rat with an arthritic TMJ. Because injection of microcapsules loaded with morphine decreased the nociceptive response of a rat with inflammatory arthritis we expect that the microcapsules will amealorate the pain response in patients with TMJ disease.

### **5.1 TMJ intra-articular model for testing microcapsules**

The premise of our animal (i.e., meal duration) model is that the CFA induced TMJ pain would affect the rat, such that, when a hungry animal initiated a meal the animal would eat slowly due to the TMJ pain associated with the movement of the mandible during the chewing process. This is exactly what we observed following bilateral TMJ CFA injections in males and females, i.e., the rats had longer meal durations (Harper, Kerins, Talwar, Spears, Hutchins, Carlson, McIntosh & Bellinger, 2000; Kerins, Carlson, Hinton, Grogan, Marr, Kramer, Spears & Bellinger, 2005; Kerins, Carlson, McIntosh & Bellinger, 2003). Support that meal duration is a measure for orofacial pain it that when ibuprofen is administered directly into a inflamed, CFA-injected TMJ meal duration was normal in both male and female rats (Kerins, Carlson, McIntosh & Bellinger, 2003). Our selection of CFA was made because it produces a persistent arthritic pain response that last for over two weeks (Hill, Bellinger, Spears, Hutchins, Kerins & Kramer, 2007; Ren, 1999). Thus, of all the agents, CFA was best when trying to establish long duration pain. Injection of CFA into the TMJ significantly lengthened meal duration in rats, while the same amount of CFA in the knee did not affect meal duration (Kerins, Carlson, Hinton, Grogan, Marr, Kramer, Spears & Bellinger, 2005) indicating meal duration is a specific measure of orofacial pain. Interestingly IL-1β remained significantly elevated in the TMJ of the ibuprofen treated animals injected with CFA (Kerins, Carlson, McIntosh & Bellinger, 2003), suggesting that some inflammation from the CFA injection remained. In another study, cyclooxgenase-II (COX-2) inhibitors normalized meal duration in rats after CFA injection (Kerins, Carlson, McIntosh & Bellinger, 2004). In this study the COX-2 inhibitor also attenuated the inflammation, i.e., TMJ tissue IL-1 normalized (Kerins, Carlson, McIntosh & Bellinger, 2004). In still another study, rats were given capsaicin or vehicle at 2 and 10 days of age; capsaicin permanently destroyed afferent nociceptive fibers in these animals (Bellinger, Spears, King, Dahm, Hutchins, Kerins & Kramer, 2007). When these male rats reached adulthood saline or CFA was injected into the TMJ and their meal duration was measured. Capsaicin treatment alone had no effect on meal duration, because saline injected, non-capsaicin treated rats had the same meal duration as saline injected, capsaicin treated rats. Non-capsaicin treated rats injected with CFA had longer meal durations than rats that were pre-treated with capsaicin, which demonstrated meal duration after CFA injection was normalized due to a capsaicin-induced loss of afferent nociceptive neuronal fibers (Bellinger, Spears, King, Dahm, Hutchins, Kerins & Kramer, 2007). The lack of change in meal duration in these capsaicin treated male rats occurred despite CFA inducing greater TMJ swelling, which demonstrated that the physical and mechanical changes in the inflamed TMJ synovial joint did not affect meal duration measurements. Another rationale for suggesting that meal duration is a measure of

Cross-Linked Gelatin Microcapsules for Drug Delivery in a Arthritic TMJ 269

2004) but in this intra-articular paradigm we did not want intracellular drug release thus

In conclusion drug loaded gelatin microcapsules reduce the nociceptive response of an arthritic TMJ. These microcapsules are expected to be useful not only for the treatment of pain but also to modify the joint environment prior to implantation by delivering proregenerative signals in a spatially and temporally controlled fashion. The limitations of current therapeutic strategies for TMJ disorders have led to increased interest in tissue engineering strategies, which combine cells, bioactive factors, and implantable scaffolds to trigger joint regeneration (Detamore & Athanasiou, 2003). In addition to the added benefit of sustained drug release local administration of drug has the added benefit of avoiding

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**6. References** 

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nociception stems from the finding that eating is impaired in patients with TMD (Haketa, Kino, Sugisaki, Amemori, Ishikawa, Shibuya, Sato & Yoshida, 2006) and from a clinical study of juvenile rheumatoid arthritic children (Harper, Brown, Triplett, Villasenor & Gatchel, 2000) that examined chewing performance as an objective measure of masticatory function. It showed that the juvenile rheumatoid arthritic children with TMD symptoms changed their chewing habits presumably to "guard" against pain. Most recently, meal duration in the rat was shown to be increased over the course of a week following pulp exposures demonstrating meal duration can also be used as a measure of tooth nociception (Bellinger, He & Kramer, 2010).
