|Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1995: ¥1,900,000 (Direct Cost: ¥1,900,000)
The purpose of this study was to investigate the role of matrix metalloproteianses (MMPs) on extracellular matrix (ECM) degradation in the destruction of human periapical connective tissue. We examined the effects of several inflammatory cytokines on the production of MMPs and their specific inhibitors (Tissue inhibitors of metalloproteinases ; TIMPs) by human dental pulp (DP) cells and human periodontal ligament (PL) cells. DP and PL cells were obtained from dental pulp tissue and periodontal ligament tissue of freshly extracted human teeth and used for experiments at passage levels 5-9. After the cells had reached confluence, they were further incubated for 48 hours in the medium with inflammatory cytokines, Interleukin-1alpha, beta (IL-1alpha, beta), Tumor necrosis factor alpha (TNFalpha), Interleukin-6(IL-6) and Interleukin-8 (IL-8). And we determined MMPs (collagenase : MMP-1, gelatinase : MMP-2) activities and TIMPs (TIMP-1 and TIMP-2) concentrations in culture medium. Furthermor
e, zymogram analysis was performed to identified to the type of gelatinase and northern blot analysis was performed to confirme the expession of mRNA of MMP-2 and TIMP-1.
The results were as follows :
1. Concerning the effects of inflammatory cytokines on DP cells, the production of MMP-1 was significantly accelerated by IL-1alpha, beta and TNF alpha, however, it was scarcely accelerated by IL-6 and IL-8. On the other hand, the production of TIMP-1 was slightly accelerated by IL-1alpha, beta and TNF alpha, however, it was not accelerated by IL-6 and IL-8. Therefore, IL-1alpha, beta and TNF alpha were considered to have the effect on the emphasis of the collagenolytic activity of DP cells by changing the balance between MMP-1 and TIMP-1.
2. Concerning the effects of inflammatory cytokines on PL cells, both the total collagenase and gelatinase activities were markedly enhanced by both IL-1alpha and beta in dose dependent manner. On the other hand, the production of both TIMP-1 and TIMP-2 was slightly accelerated by IL-1alpha and beta in dose dependent manner. Therefore, IL-1alpha and beta were considered to have the effect on the emphasis of the collagenolytic activity of PL cells by changing the balance between MMP-1 and TIMP-1.
3. Gelatinase activity was identified to be Gelatinase A (72kDa Gelatinase) by zymogram analysis in the culture media from PL cells.
4. The expression of MMP-2 and TIMP-1 mRNA in both DP cells and PL cells was confirmed by northern blot analysis, however, there were no differences between the IL-1 treated group and the control group.
Taken together, each inflammatory cytokines appeared to show different effects on the induction of MMPs and TIMPs produced by DP and PL cells. Present findings suggest MMPs and TIMPs may be involved in the pathogenesis of periapical disease. Less