The Study Of Collagens And TIMP-1in Gingival Tissue Of Patients With Aggressive Periodontitis

Periodontitis is an inflammatory disease of the supporting tissues of teeth. Aggressive periodontitis (AgP) is a relatively rare inflammatory condition, and is mostly seen in young individuals. It is characterized by early onset, rapid progression, strong damage degree, and familial inheritance. More and more evidence has shown that the degradation of connective tissue and destruction of alveolar bone are induced by host cell enzymes in periodontitis.Matrix metalloproteinases (MMPs) is a family of zinc and calcium-dependent proteolytic enzymes. MMPs and matrix metalloproteinase inhibitor-1(tissue inhibitors of metalloproteinases-1, TIMP-1) have attracted the concern of numerous of scholars from periodontal area. This group of proteolytic enzymes is involved in the process of human’s physiological and pathological processes by regulating the degradation of macromolecules in extracellular matrix. MMPs and TIMPs take part not only in the degradation of matrix proteins during periodontitis but also during normal turn-over in health and wound healing. The imbalance of MMPs and TIMPs is considered to be the cause of the degradation of extracellular matrix, which is closely related to the onset and progress of periodontal diseases.Collagenases (including MMP-1, MMP-8and MMP-13) is the first category and one of the main types of MMPs. MMP-1(FIB-CL) is also known as fibroblast collagenase, which primarily derived from fibroblasts, phagocytic cells and epithelial cells. It preferentially degrades collagen type Ⅲ. MMP-8is also called neutrophil collagenase (PMN-CL), which is mainly produced and secreted by polymorphonuclear neutrophils and is involved in the destruction of collagen type Ⅰ fibers. However, type Ⅰ collagen fibers are also the main composition of periodontal ligament and alveolar bone. MMP-13is related to the rapid transformation of collagen fibers in tissue and is more influential with collagen type Ⅱ than other collagens. TIMP-1is considered to be the one of the most important tissue inhibitors of metalloproteinases and acts on the most of metalloproteinases.It was discovered that the level of MMPs is increased in patients with chronic periodontitis when compared with periodontally healthy adults, However, there is little research on the effect of the level of various MMPs on the occurrence and development of aggressive periodontitis.Thus, this study was to evaluate the expression level of MMP-1, MMP-8, MMP-13and TIMP-1in gingival tissue of patients with aggressive periodontitis, and to analysis the the correlation of the severity of aggressive periodontitis with the above indicator. In addition, MMPs and TIMP-1ratio was analysed as a new indicator in this research, in order to verify the role of the balance between MMPs and TIMP-1in the development process of AgP.Materials and methods:1. Study Population A total of30patients with AgP were selected from patients who visited in Department of Periodontology, Stomatological Hospital of Guangdong Province, following the criteria defined in1999by the International Workshop for a Classification of Periodontal Diseases and Conditions (Average age=29.37). In the control group (n=20), subjects were selected from patients who visited in Surgical department, Stomatological Hospital of Guangdong Province for impacted teeth extraction (Average age=26.35). All of them presented no systematic disease and hadn’t history of antibiotic usage and any periodontal treatment in6months. In addition, informed written consent was granted by each subject after explanations were provided.2. Gingival tissue specimensAll subjects were performed periondontal initial examination by using Florida probe. The indicators concluded probing depth (PD), plaque index (PLI), gingival bleeding on probing (BOP), assessment of clinical attachment loss (AL), tooth mobility, and furcation index (FI). All measurements were performed at six sites per tooth and were carried out by the same investigator to minimize bias. In the experimental group, biopsies were harvested during extraction of teeth with a poor periodontal prognosis. The control group biopsies were collected during extractions impacted teeth. Then washed the specimens with distilled water, and were immediately fixed in4%paraformaldehyde solution for12～24h.3. ImmunohistochemistryDehydration, transparent, embedded by paraffin of the fixed specimens. Serial paraffin sections of biopsies were cut3um thick, and immunohistochemistry was performed using a three-step immunoperoxidase procedure. First, the tissue section was deparaffinized, rehydrated and incubated with normal horse serum diluted to1:50in phosphate-buffered saline (PBS) containing0.1%bovine serum albumin (Zhongshan, Beijing, China) for20min at room temperature to block non-specific binding. Subsequently, the immunohistochemistry of TIMP-1, MMP-1, MMP-8and MMP-13were performed.4. Locate and semi-quantitative analysisRandomly selected10positive cells, which cytoplasm were stained yellow, vision of each slice under microscope at x400times. Analysed by IPP (Image Pro plus) computer system. Calculated the average IOD of MMP-1, MMP-8, MMP-13and TIMP-1, and the ratio of MMP-1/TIMP-1, MMP-8/TIMP-1, MMP-13/TIMP-1.5. StatisticsStatistical analysis was performed using SPSS Software (Version13.0). One-way AN OVA or two independent samples non-parameters rank sum test was applied in all of the clinical indicators and TIMP-1, MMP-1, MMP-8, MMP-13expression and the MMP-1/TIMP-1, MMP-8/TIMP-1, MMP-13/TIMP-1ratio. In addition, Spearman’s correlations were run between MMP levels and clinical parameters. Inspection standards are in alpha of0.05.Results:1. The expression and distribution of TIMP-1, MMP-1, MMP-8and MMP-13in the gum tissue. All of the four peptides were detected in all gingival specimens. Two types of expressing cells were observed:gingival epithelial cells and fibroblast cells.①The expression of TIMP-1peptides in periodontal healthy tissues were observed in the cytoplasm of the epithelium and lamina propria. The positive cells with MMP-1was weakly expressed seen scattered in the epithelial cells and lamina propria cells, and there was a decreasing trend of staining intensity of the expression in the cortex from the shallow of the prickle cell to the deep prickle cell. MMP-8and MMP-13were weakly expressed mainly in the connective tissue area.②AgP group gum tissue. TIMP-1was weakly expressed and the major expression sites was in inflammatory connective tissue. There was no statistically significant of the difference between two groups (P>0.05). MMP-1and MMP-8expression were strongly positive, presents a large number of positive cells with cinnamon brown cytoplasm in the epithelium and lamina propria, they were mainly distributed with in the densely populated areas of inflammatory cells in the lamina propria. Compared with gums healthy group,there was statistically significant of the difference (P<0.05). MMP-13expression was strongly positive, the distribution of a large number of positive cells in the epithelium and lamina propria, the main expression site are in the inflammatory cell infiltration area. There was no statistically significant of the difference between two groups (P>0.05).2. The correlation between clinical indicators and experimental indicators. The relationship between the PD, BOP, PLI and AL and the production of MMP-1, MMP-8and MMP-13were analyzed by means of Pearson’s correlation coefficient. The increased PD, BOP, PLI and AL was significantly correlated with MMP-1MMP-8and MMP-13in periodontal disease. There was a significant correlation between MMP-1, MMP-8, MMP-1/TIMP-1and MMP-8/TIMP-1and probing pocket depth and attachment loss (P<0.05), there was a significant relationship between MMP-1, MMP-8, MMP-13, MMP-1/TIMP-1, and MMP-8/TIMP-1and BOP (P<0.05). There were some correlations among various MMPs, such as MMP-1and MMP-8, MMP-1and MMP-13, and MMP-8and MMP-13(P<0.05).3. According to the ROC curve analysis, the AUC (MMP-8/TIMP-1)=0.947> AUC(MMP-8)=0.752, AUC(MMP-1/TIMP-1)=0.870> AUC(MMP-1)=0.835, illustrating that using of the ratio of MMPs and TIMP-1could enlarge AUC; and According to the ROC curve, MMP-8/TIMP-1, MMP-1/TIMP-1, MMP-1and MMP-8may be as the objective indicators of periodontal tissue destruction and inflammation. Conclusion:1. MMP-1, MMP-8, MMP-13and TIMP-1may be involved in normal and pathological alterations of the periodontal tissues in AgP.2. When biomarkers of host and/or microbe origin are combined, the detection of periodontitis may be significantly improved. MMPs and TIMP-1ratio as a new factor would provide a new perspective for the pathological mechanism of AgP.