The effect of centrifugal force on mRNA levels of collagenase, collagen type-I, tissue inhibitors of metalloproteinases and beta-actin in cultured human periodontal ligament fibroblasts.

BACKGROUND: The aim of orthodontic treatment is to relocate teeth abnormally positioned in the jaws. This is achieved by application of continuous force on the tooth, which is immediately being sensed by the periodontal ligament (PDL), bone and the gingiva. Since the bony response is mediated by the PDL, tooth movement is primarily a PDL phenomenon. OBJECTIVES: Thus, the purpose of the present study was to evaluate the direct effect of force (excluding the in vivo tissue response) on the molecular level of matrix metalloproteinase-1 (MMP-1) and collagen type-I (Col-I) in human PDL fibroblasts. METHODS: PDL cell culture flasks were centrifuged for 10, 20, 30, 60, 90 and 120 min by horizontal microplate rotor. The effect of force on mRNA levels of beta-actin, MMP-1, Col-I, tissue inhibitors-1 and -2 (TIMPs) genes was analyzed by RT-PCR. RESULTS: The results showed that force had no effect on the mRNA levels of beta-actin during the first 90 min of application of force, indicating for the first time the use of beta-actin gene as an internal invariant control. It increased the mRNA levels of MMP-1 while almost no effect on Col-I and TIMPs was observed. CONCLUSIONS: The results indicate that PDL remodeling following application of orthodontic force could be partly attributed to the direct effect of the force on MMP-1 gene expression in fibroblasts.

Basic fibroblast growth factor suppresses tropoelastin gene expression in cultured human periodontal fibroblasts.

Growth factors are known to play a major role in the regeneration of the periodontium. Basic fibroblast growth factor (bFGF) is a polypeptide growth factor considered to have a role in chemotaxis and mitogenesis of periodontal ligament (PDL) cells. The aim of this study was to assess the effect of bFGF on the transcription level of tropoelastin. As known controls, we assessed the transcription levels of collagen type I, collagen type II and the housekeeping gene, actin. Initially, PDL cells were cultured without bFGF for 3, 7 and 14 days. At each time point. total RNA was extracted and the levels of transcription were assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. The results showed that tropoelastin mRNA is transcribed in PDL cells and its levels increased from minimal amounts by day 3 to maximal amounts by day 14 of culture. We further examined the effect of the addition of 10 ng/ml bFGF to the culture media by day 14. The results showed that the addition of bFGF suppressed the transcription level of tropoelastin. At that time, as expected, a decrease in collagen type I transcription level was shown, while the transcription level of collagen type III was not affected. The findings that elastin is transcribed in vitro by PDL cells, but only negligibly in vivo, imply mechanisms that downregulate or even shut down the expression of the elastin gene in the functioning PDL. Basic FGF might be one of the cytokines involved in control of elastin expression in vivo.

Cementum attachment protein enriches putative cementoblastic populations on root surfaces in vitro.

We tested the capacity of cementum attachment protein (CAP) to recruit putative cementoblastic populations to root surfaces in vitro by determining the phenotypic expression of periodontal ligament cloned cell populations. The clones were derived from cells that attached to either CAP-coated (experimental) or uncoated (control) root slices. Root slices were co-cultured with primary human periodontal ligament cells. Cloned and parent populations were analyzed for their capacity to express alkaline phosphatase (AP), osteopontin, bone sialoprotein (BSP), and CAP and to form mineralized tissue in vitro. The percentage of CAP- and BSP-positive clones was significantly higher in the experimental clones than in the controls. The percentage of cells positive for AP, BSP, and CAP was higher in the experimental clones than in their control counterparts. Mineralized tissue formation was observed only in the cell populations derived from the CAP-coated root slices. These results indicate that CAP is capable of recruiting putative cementoblastic populations on root slices in vitro and therefore might play an important role in cementogenesis during periodontal homeostasis and wound healing.

Inverse dose- and time-dependent effect of basic fibroblast growth factor on the gene expression of collagen type I and matrix metalloproteinase-1 by periodontal ligament cells in culture.

BACKGROUND: Growth factors are known to play a major role in the regeneration of the periodontium. Basic fibroblast growth factor (bFGF) is a polypeptide growth factor considered to have a role in chemotaxis and mitogenesis of periodontal ligament cells (PLC). The aim of this study was to assess the dose-dependent effect of bFGF administration on the levels of gene expression of collagen type I (a1) (col I), collagen type III (col III), and collagenase-1 (MMP-1) in PLC. METHODS: PLC were cultured in different concentrations of bFGF (0.1 to 10 ng of bFGF) for 14 and 21 days. At each time point, the gene expression of the examined molecules was assessed semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULTS: The results indicated that bFGF exhibits an inverse time- and dose-dependent effect on the gene expression of col I and MMP-1: it simultaneously downregulates the gene expression of col I and upregulates the gene expression of MMP-1. On the other hand, bFGF had no dose-dependent effect on col III gene expression. The effect of bFGF on the expression of the three genes was modulated by the time of incubation with bFGF. CONCLUSIONS: These results suggest that bFGF is one of the important regulators involved in the active remodeling of col I in the periodontal ligament and possibly in other connective tissues.