The purpose of this study was to monitor the secretion of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) by human periodontal ligament (PDL) fibroblasts stimulated with Prevotella nigrescens lipopolysaccharide (LPS), and to examine the effect of calcium hydroxide treatment on P. nigrescens LPS.

LPS was extracted and purified from anaerobically cultured P. nigrescens. PDL fibroblasts were stimulated by the LPS (0, 0.1, 1, 10 ug/ml) or LPS (10 ug/ml) pretreated with 12.5 mg/ml of Ca(OH)₂ for 3 days, for various periods of time (12, 24, 48 h). Immunoprecipitation were performed for protein level analysis of MMP-1, MMP-2 and TIMP-1. Total RNA was isolated and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 mRNA.

According to this study, the results were as follows:

1. The production of MMP-1 by stimulation with P. nigrescens LPS increased in time-dependent manner, and showed maximum value at 48 h in both protein and mRNA level. But there was no dose-dependent increase.

2. MMP-2 production time-dependently increased when stimulated with 1 and 10 ug/ml LPS, but there was no dose-dependent increase.

3. TIMP-1 production increased to 24 h, but decreased at 48 h. It increased when stimulated with 0.1 and 1 ug/ml LPS, but suppressed at 10 ug/ml.

4. P. nigrescens LPS pretreated with Ca(OH)₂ markedly downregulated MMP-1 gene expression.

The purpose of this study is to monitor the secretion of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) produced by human osteosarcoma cell line (MG63) stimulated with Prevotella nigrescens lipopolysaccharides (LPS), and to compare the level of secretion before and after the treatment of calcium hydroxide on P. nigrescens LPS.

LPS was extracted and purified from anaerobically cultured P. nigrescens. MG63 cells were stimulated by the LPS (0, 1, 10 µg/ml) or LPS (10 µg/ml) pretreated with 12.5 mg/ml of Ca(OH)₂ for 3 days.

Total RNA was isolated from the cell, and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 and TIMP-1.

The results were as follows.

1. MMP-1 mRNA expression at 48 hr was highly increased by stimulation with P. nigrescens LPS. The increase was dose-dependent.

2. When stimulated with 1 µg/ml of LPS, TIMP-1 mRNA expression was highly increased at 24 hr and 48 hr. However, TIMP-1 expression was suppressed at higher concentration (10 µg/ml).

3. When P. nigrescens LPS was pretreated with Ca(OH)₂, MMP-1 and TIMP-1 gene expression was downregulated.

The results of this study suggest that transcriptional regulation of MMP-1 and TIMP-1 by P. nigrescens LPS could be one of the important mechanisms in bone resorption of periapical inflammation. The result of calcium hydroxide on MMP-1 and TIMP-1 gene expression suppression shows that calcium hydroxide detoxified bacterial LPS and thus should be used the medication of choice for intracanal dressings in root canal infected with black-pigmented bacteria.