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Abstract #66 — The immunocytochemical distribution of IL-2 and IL-2R in dental pulp
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AAE Abstracts of Papers
Journal of Endodontics
(Abstract #63, continued) controlling moderate to severe pain for the first 24 hours. Eryc(500 mg) and penicillin were the most effective medications in controlling moderate and severe pain compared to placebo and other medications for the next48 hours. In addition, we foundpostoperative complications were positively correlated with preoperative symptoms.
Research Seminar V Saturday, May 1, 8:30 - 11:30 a.m. Abstract # 6 4 - Effects of r e c o m b i n a n t BMP-2 and Vitamin D on RPC C2A. S.R. Srinivasan*, J. Zhang, R. Croft, W. Lorenz, A.C. Karim,
M.Z. Hussain University of California, San Francisco, California and University of Manitoba, Winnipeg, Canada RPC C2A is a clone line of adult rat dental pulp cells with high Alkaline Phosphatase (ALP) activity, indicating dentinogenic potential. Recombinant Bone Morphogenetic Protein 2 (BMP-2) and Vitamin D3 (Vit D3) have regulatory effects on osteogenic cells both in vivo and in vitro. The effects of BMP-2 on non-skeletal tissue, including pulpal cells is unknown. The purpose of our studies was to examine the effects of BMP-2 and Vitamin D3 on RPC C2A cells in vitro. Confluent cultures (treated with 100-500ng/ml of BMP-2 and 100ug/ml Vit D3, alone and in combination, and untreated controls) were assayed for ALP activity and synthesis of collagenous and non-collagenous proteins. ALP activity was assayed using the method of Lowry et al. (1954), while collagen synthesis was quantitated with the bacterial collagenase digestion technique. BMP-2 and Vit D3 elevated ALP levels ten and eight-fold respectively. BMP-2 and Vit D3 stimulated synthesis of both collagenous and noncollagenous proteins, with a consistently greater increase in extracellular(EC) collagen. TheseresultssuggestthatALP activity and dentinal collagen synthesis maybe co-regulated. Further, RPC C2A may be a suitable in vitro-model for studying dentinogenesis. This study was supported by a Grant from the Research and Education Foundation of the AAE.
Abstract #65 - Effect of IL-1 on collagen synthesis in human dental pulp fibroblasts.
R.A. Barkhordar*, L.W. Lim, J.C. Zhang, M.Z. Hussain University of California, San Francisco, California Immunopathologic reactions play a significant role in inflammatory disease ofdentalpulp. In addition, interleukin (IL-1) stimulates collagen synthesis in cultured fibroblasts. The purpose of this study was to examine the effect of IL-1 on collagen synthesis in fibroblasts grown out of two healthy and two diseased human pulpal samples. Cultures maintained in DM E-H 16 containing antibiotics were seru m-starved for 48 hours. Quiescent fibroblasts were then treated with 10-50 pg/ml of IL-1 in the presence of 10 Ci/ml 3H-proline and 50 g/mr ascorbate for 20 hours. Collagen synthesis was measured by following the incorporation oPH-proline into collagenase-sensitive protein in both cell and medium layers. Our results showed that collagen synthesis was significantly stimulated in healthy fibroblasts while it was lowered in the diseased cells, compared to untreated controls (healthy:untreated = 1.09 _+0.12, 50 pg/ml IL-1 = 2.25 + 0.28; diseased:untreated = 1.83 + 0.22, 50 pg/ ml IL-1 = 1.63 + 0.21 x 104 dpm/106 cells). Further analysis using ILl ELISA kit revealed that diseased pulp fibroblasts contained nearly 2.5-fold IL-1 than healthy pulp fibroblasts (healthy 754.6_+152;diseased 1898 +_315 pg/mg protein). These results suggest that although IL-1 is produced and released in inflammatory pulpal fibroblasts, they respond poorly or adversely to this cytokine with respect to collagen synthesis. Supported partly by NIDR Summer Research Fellowship.
Abstract #66 - The i m m u n o c y t o c h e m i c a l distribution of IL-2 and IL-2R in dental pulp. C. Rauschenberger*, C. Cootauco University of Maryland, Baltimore, Maryland IL-2, agrowth factor responsible forT-cell proliferation and maturation, activates a variety of other cells which destroy connective tissues and may contribute to the pathogenesis of irreversible pulpal disease. Cox etal., 1992, suggested that increases in IL-2 receptor (IL-2R) associated with inflammation may bind released IL-2 and therefore block the detection of this product in extracellular tissue fluid. The purpose of this study was to evaluate the distribution of IL-2 and IL-2R in healthy and inflamed dental pulps using ultracryoimmunocytochemical techniques. Pulp tissue samples were obtained from 12 impacted third molars and 12 carious molars with irreversible pulpitis. Immediately following extraction, the pulp tissue was fixed in 4% paraformaldehyde at 4°C, immersed into graded concentrations of sucrose with polyvinyl pyrrolidone, frozen in liquid nitrogen, cryo-sectioned, and immunostained for IL-2 and IL-2R. Twenty tissue sections per sample were evaluated for the presence of gold labelled IL-2 and IL-2R by transmission electron microscopy. Inflamed pulpal tissue sections exhibited intense IL-2 labelling when compared to the healthy pulpal tissue sections. Immunogold labelling for IL-2 in the inflamed tissues was localized intracellulafly subjacent to the cell membrane of pulpal lymphocytes and released extracellularly to the pulpal collagen matrix. Healthy tissues displayed mild intracellular IL-2 labelling and no extracellular labelling. Both healthy and inflamed pulpal tissues exhibited moderate i mmunogold IL2R labelling on the surface membrane of inflammatory cells as well as in small aggregates in the extracellular connective tissue matrix. TheobservedresultssuggestthatlL-2isreleasedfrom pulpal T-cell associated with irreversible pulpitis. No difference in intensity or localization of immunogold l L-2R could be determined when irreversibly inflamed tissue sections were compared to normal samples. Therefore, IL-2 may be suitable as a marker for inflammatory pulpal disease and IL-2R should not substantially interfere with released IL2 associated with irreversible pulpitis.
Abstract #67 - The effects of three retrofilling materials on selected oral bacteria. C.U. Hong*, M. Torabinejad, J.D. Kettering Loma Linda University, Loma Linda, California An antibacterial effect of filling materials has been considered as an ideal property. The purpose of this study was to compare the effects of amalgam (AM) Super EBA, and a mineraltrioxide (MT) aggregate on five facultative oral bacteria. Lactobacillus sp (Ls), Streptococcus fecaelis (SF), Streptococcus mitis (Smi), Streptococcus mutans (Smu), and Streptococcus safivanus (Ss) were grown overnight in a broth culture. The bacteria were standardized (O.D. = 0.02 at 590 mm). 0.1 ml aliquots of each bacteria were inoculated onto separate blood agar plates (BAP) and spread evenly over the surface. The three materials were mixed and placed in a mold to form a plug 1 cm in diameter x 5 mm high. Materials were tested using freshly mixed samples. The materials were placed in the center of the BAP and incubated at 37 ° for 24 hours. Antibacterial effect was demonstrated by a zone of inhibition (no bacterial growth around the materials). The diameter of the zones were measured using a ruler and analyzed statistically (ANOVA). Fresh EBA had no antibacterial effect on Ls, Sf and Smu, but slight effect on Smi (1 mm) and Ss (1 mm). AM had no antibacterial effect on any organism tested. MT aggregate, however, had an anti-bacterial effect on all microorganisms used (Ls = 4.58 + .20 mm, Sf = 1.38 + .14 mm, Smi = 3.17 + .20 mm, Smu = 3.5 + .32 mm and Ss = 4.84 + .19 mm). In conclusion, AM and EBA demonstrated no antimicrobial properties against five facultative oral microorganisms, whereas MT aggregate had more effect with Ls, SmL Smu and Ss being more sensitive than Sf (p < O.05).
AAE Abstracts of Papers
Journal of Endodontics
(Abstract #63, continued) controlling moderate to severe pain for the first 24 hours. Eryc(500 mg) and penicillin were the most effective medications in controlling moderate and severe pain compared to placebo and other medications for the next48 hours. In addition, we foundpostoperative complications were positively correlated with preoperative symptoms.
Research Seminar V Saturday, May 1, 8:30 - 11:30 a.m. Abstract # 6 4 - Effects of r e c o m b i n a n t BMP-2 and Vitamin D on RPC C2A. S.R. Srinivasan*, J. Zhang, R. Croft, W. Lorenz, A.C. Karim,
M.Z. Hussain University of California, San Francisco, California and University of Manitoba, Winnipeg, Canada RPC C2A is a clone line of adult rat dental pulp cells with high Alkaline Phosphatase (ALP) activity, indicating dentinogenic potential. Recombinant Bone Morphogenetic Protein 2 (BMP-2) and Vitamin D3 (Vit D3) have regulatory effects on osteogenic cells both in vivo and in vitro. The effects of BMP-2 on non-skeletal tissue, including pulpal cells is unknown. The purpose of our studies was to examine the effects of BMP-2 and Vitamin D3 on RPC C2A cells in vitro. Confluent cultures (treated with 100-500ng/ml of BMP-2 and 100ug/ml Vit D3, alone and in combination, and untreated controls) were assayed for ALP activity and synthesis of collagenous and non-collagenous proteins. ALP activity was assayed using the method of Lowry et al. (1954), while collagen synthesis was quantitated with the bacterial collagenase digestion technique. BMP-2 and Vit D3 elevated ALP levels ten and eight-fold respectively. BMP-2 and Vit D3 stimulated synthesis of both collagenous and noncollagenous proteins, with a consistently greater increase in extracellular(EC) collagen. TheseresultssuggestthatALP activity and dentinal collagen synthesis maybe co-regulated. Further, RPC C2A may be a suitable in vitro-model for studying dentinogenesis. This study was supported by a Grant from the Research and Education Foundation of the AAE.
Abstract #65 - Effect of IL-1 on collagen synthesis in human dental pulp fibroblasts.
R.A. Barkhordar*, L.W. Lim, J.C. Zhang, M.Z. Hussain University of California, San Francisco, California Immunopathologic reactions play a significant role in inflammatory disease ofdentalpulp. In addition, interleukin (IL-1) stimulates collagen synthesis in cultured fibroblasts. The purpose of this study was to examine the effect of IL-1 on collagen synthesis in fibroblasts grown out of two healthy and two diseased human pulpal samples. Cultures maintained in DM E-H 16 containing antibiotics were seru m-starved for 48 hours. Quiescent fibroblasts were then treated with 10-50 pg/ml of IL-1 in the presence of 10 Ci/ml 3H-proline and 50 g/mr ascorbate for 20 hours. Collagen synthesis was measured by following the incorporation oPH-proline into collagenase-sensitive protein in both cell and medium layers. Our results showed that collagen synthesis was significantly stimulated in healthy fibroblasts while it was lowered in the diseased cells, compared to untreated controls (healthy:untreated = 1.09 _+0.12, 50 pg/ml IL-1 = 2.25 + 0.28; diseased:untreated = 1.83 + 0.22, 50 pg/ ml IL-1 = 1.63 + 0.21 x 104 dpm/106 cells). Further analysis using ILl ELISA kit revealed that diseased pulp fibroblasts contained nearly 2.5-fold IL-1 than healthy pulp fibroblasts (healthy 754.6_+152;diseased 1898 +_315 pg/mg protein). These results suggest that although IL-1 is produced and released in inflammatory pulpal fibroblasts, they respond poorly or adversely to this cytokine with respect to collagen synthesis. Supported partly by NIDR Summer Research Fellowship.
Abstract #66 - The i m m u n o c y t o c h e m i c a l distribution of IL-2 and IL-2R in dental pulp. C. Rauschenberger*, C. Cootauco University of Maryland, Baltimore, Maryland IL-2, agrowth factor responsible forT-cell proliferation and maturation, activates a variety of other cells which destroy connective tissues and may contribute to the pathogenesis of irreversible pulpal disease. Cox etal., 1992, suggested that increases in IL-2 receptor (IL-2R) associated with inflammation may bind released IL-2 and therefore block the detection of this product in extracellular tissue fluid. The purpose of this study was to evaluate the distribution of IL-2 and IL-2R in healthy and inflamed dental pulps using ultracryoimmunocytochemical techniques. Pulp tissue samples were obtained from 12 impacted third molars and 12 carious molars with irreversible pulpitis. Immediately following extraction, the pulp tissue was fixed in 4% paraformaldehyde at 4°C, immersed into graded concentrations of sucrose with polyvinyl pyrrolidone, frozen in liquid nitrogen, cryo-sectioned, and immunostained for IL-2 and IL-2R. Twenty tissue sections per sample were evaluated for the presence of gold labelled IL-2 and IL-2R by transmission electron microscopy. Inflamed pulpal tissue sections exhibited intense IL-2 labelling when compared to the healthy pulpal tissue sections. Immunogold labelling for IL-2 in the inflamed tissues was localized intracellulafly subjacent to the cell membrane of pulpal lymphocytes and released extracellularly to the pulpal collagen matrix. Healthy tissues displayed mild intracellular IL-2 labelling and no extracellular labelling. Both healthy and inflamed pulpal tissues exhibited moderate i mmunogold IL2R labelling on the surface membrane of inflammatory cells as well as in small aggregates in the extracellular connective tissue matrix. TheobservedresultssuggestthatlL-2isreleasedfrom pulpal T-cell associated with irreversible pulpitis. No difference in intensity or localization of immunogold l L-2R could be determined when irreversibly inflamed tissue sections were compared to normal samples. Therefore, IL-2 may be suitable as a marker for inflammatory pulpal disease and IL-2R should not substantially interfere with released IL2 associated with irreversible pulpitis.
Abstract #67 - The effects of three retrofilling materials on selected oral bacteria. C.U. Hong*, M. Torabinejad, J.D. Kettering Loma Linda University, Loma Linda, California An antibacterial effect of filling materials has been considered as an ideal property. The purpose of this study was to compare the effects of amalgam (AM) Super EBA, and a mineraltrioxide (MT) aggregate on five facultative oral bacteria. Lactobacillus sp (Ls), Streptococcus fecaelis (SF), Streptococcus mitis (Smi), Streptococcus mutans (Smu), and Streptococcus safivanus (Ss) were grown overnight in a broth culture. The bacteria were standardized (O.D. = 0.02 at 590 mm). 0.1 ml aliquots of each bacteria were inoculated onto separate blood agar plates (BAP) and spread evenly over the surface. The three materials were mixed and placed in a mold to form a plug 1 cm in diameter x 5 mm high. Materials were tested using freshly mixed samples. The materials were placed in the center of the BAP and incubated at 37 ° for 24 hours. Antibacterial effect was demonstrated by a zone of inhibition (no bacterial growth around the materials). The diameter of the zones were measured using a ruler and analyzed statistically (ANOVA). Fresh EBA had no antibacterial effect on Ls, Sf and Smu, but slight effect on Smi (1 mm) and Ss (1 mm). AM had no antibacterial effect on any organism tested. MT aggregate, however, had an anti-bacterial effect on all microorganisms used (Ls = 4.58 + .20 mm, Sf = 1.38 + .14 mm, Smi = 3.17 + .20 mm, Smu = 3.5 + .32 mm and Ss = 4.84 + .19 mm). In conclusion, AM and EBA demonstrated no antimicrobial properties against five facultative oral microorganisms, whereas MT aggregate had more effect with Ls, SmL Smu and Ss being more sensitive than Sf (p < O.05).