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There is growing evidence that apoptosis involves the nuclear transcription factor NF-kappaB in conjunction with related genes. However, in the context of mechanical orthodontic forces, force-sensing target genes assigned to pathways of NF-kappaB and apoptosis have not been fully characterised. To contribute to the identification of putative target genes, we used cDNA arrays specific for NF-kappaB and apoptotic pathways and analysed elevated gene expression in primary human periodontal ligament fibroblasts (PDL-F) after a 6 h application of mechanical force. Among several identified genes (including several caspases), interleukin-1 beta (IL-1 beta) and NF-kappaB displayed significantly higher expression on the NF-kappaB array, whereas higher expression was obtained for BCL2-antagonist of cell death (BAD), member 6 of the TNF-receptor superfamily (FAS) and CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD) on the apoptosis array. Based on a defined cut-off level of a more than 1.5-fold higher expression, this significance in elevated gene expression was corroborated by reverse transcription/polymerase chain reaction (RT-PCR). Here, semi-quantitative (sq) PCR revealed a more pronounced elevation of mRNA gene expression in PDL-F after 6 h of stretch, when compared with 12 h. Moreover, the elevation after 6 h as observed by sq-PCR was convergent with quantitative PCR (q-PCR). q-PCR yielded levels of 5.8-fold higher relative gene expression for IL-1 beta and 1.7-fold for NF-kappaB, whereas that computed for BAD indicated a 5.2-fold, for CRADD a 2.1-fold and for FAS a 2.0-fold higher expression. The data obtained from the expression analysis thus indicate a stretch-induced transcriptional elevation of genes assigned to the NF-kappaB and apoptotic pathways. This elevation may render them target candidates for being addressed by mechanical orthodontic forces.

Although the function and effects of many growth factors and extracellular matrix (ECM) molecules have been described for several periodontal tissues in vivo and in vitro, the molecular interactions involved in the communication between cells of the periodontal ligament and the alveolar bone are poorly understood. To contribute to the identification of such interactions, we have generated co-cultures (CCs) of periodontal ligament fibroblasts (PDLs) and alveolar bone cells (ABCs) and compared mRNA expression for various growth factors, ECM molecules, and matrix metalloproteinase13 (MMP13) after 1 and 2 weeks with matched mono-cultures (MCs) by reverse transcription/polymerase chain reaction. Compared with CCs of 1 week, PDLs and ABCs after 2 weeks revealed relatively high levels of all analyzed mRNAs, viz., for EGF, HGF, VEGF, TGFbeta1, collagen-I (COL1), osteonectin (ON), fibronectin (FN1), and MMP13. At week 2, when compared with MCs, CCs showed an elevation of all tested mRNAs in PDLs and ABCs, except for TGFbeta1 and FN1, which only increased in PDLs. After 1 week, when CCs were compared with MCs, mRNAs for HGF and TGFbeta1 were less abundant in PDLs and ABCs, whereas the other genes exhibited lower expression levels in only one of the cell types. Analysis of our data indicated that the expression of mRNAs for growth factors and for COL1, ON, FN1, and MMP13 was modulated in the distinct cell types with respect to culture time and culture type. The differences in the mRNA expression patterns between CCs and MCs suggest that the respective genes are involved in the molecular interactions of PDLs and ABCs.

Mechanical stress has been shown in vitro to modulate integrin-beta1-mediated activation of p125FAK/FAK. To test the hypothesis whether this also applies to periodontal ligament fibroblasts (PDLs), we subjected human PDLs to mechanical stretch and analyzed stress-induced changes of p125FAK activation by quantitative immunoprecipitation of p125FAK and changes in the topography of molecules localizing in focal adhesions by indirect immunofluorescence. Generally, all components of focal contacts under study-including detection of phosphotyrosine, i.e., integrin-beta1, p125FAK, and paxillin-revealed a relative co-localization during stretch application. Under stretch, we observed a re-distribution of all components from the cell periphery to the cytoplasm following the main axes. Tyrosine phosphorylation of p125FAK was monitored up to 72 hours under stretch. While the amount of p125FAK remained essentially constant, the activation of p125FAK was clearly modulated. Tyrosine phosphorylation of p125FAK increased from 15 minutes up to 1 hour and declined after stretching periods of 24, 48, and 72 hours. The analysis of our data indicated a stretch-induced redistribution of focal adhesion components and a modulation of p125FAK activation, suggesting alterations in focal adhesions and their associated signal cascade.

Fibroblasts isolated from human periodontal ligament (PDL) were cultured under a medium supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone. The cultures were assessed for their ability to elaborate a mineralized matrix. Cell cultures stained positive when analyzed for alkaline phosphatase activity throughout the culture period. After about 3 weeks in culture, the cells produced a calcified matrix. Light microscopy showed formation of clusters of different shapes and sizes. Von Kossa staining revealed mineral deposits as amorphous brown-black precipitates. Transmission electron microscopy showed cells in multilayers and mineralized formations in close association with a dense network of collagen fibers. Scanning electron microscopy revealed smooth formations rising over the cultures with an abundant fiber matrix. We conclude that human PDL fibroblasts can be induced to form a mineralized matrix which shares features with bone mineralized matrix but most likely represents a more immature type of in vitro mineralization. Moreover, the present study further supports the osteoblastic potential of these cells.

Identifying the biological properties of the cells residing within the periodontal ligament (PDL) will help in understanding the role that these cells play in the various functions of the periodontal ligament, and will improve the success of clinical procedures such as orthodontic tooth movement. For this purpose, fibroblasts isolated from human periodontium were cultured and characterized both histochemically and biochemically with respect to their putative osteoblast-like properties. Histochemically, cultured PDL fibroblasts showed an intense staining for alkaline phosphatase (ALP). Biochemically, the basal ALP activity increased in culture over time. ALP levels after stimulation with 1 alpha, 25-dihydroxyvitamin D3 were significantly higher than those of control cultures. Moreover, immunofluorescence against osteocalcin (a highly reliable osteoblastic marker) was strongly positive. Von Kossa staining of the cell cultures revealed the formation of mineral-like nodules. These results indicate that human PDL fibroblasts exhibit in vitro phenotypic characteristics consistent with osteoblast-like cells, thus suggesting that such cells have the potential to differentiate into osteoblasts and/or cementoblasts.

To investigate molecular aspects of the mechanism(s) involved in orthodontic tooth movement, periodontal ligament fibroblasts (PDL) were isolated and grown on culture dishes with a flexible bottom. The cells were stimulated by stretching the bottom of the culture dishes and whole cell extracts were prepared and subjected to sodium dodecyl sulfate polyacrylamide (SDS-PAGE) electrophoresis, electrotransferred to nitrocellulose membrane and immunoprobed with antibodies specific for vimentin and alpha- and beta-tubulin. No apparent alterations in the expression profile of the above mentioned major cytoskeletal elements were evident after mechanical stretching. Moreover, immunofluorescence against the same proteins revealed no changes in their topographical organisation between stretched and unstretched cell cultures.

The loss of periodontal support, especially of the periodontal ligament, presents the orthodontist with a constraining factor precisely in the treatment of patients who have reached adult and senior citizen age. Given this situation it was decided to take small biopsies of periodontal ligament cells, grow them, amplify them in vitro, and then with the help of different types of membranes, some of which consisted of resorbable materials, which served as cell carriers, transplant them onto tooth roots. The work reported here constitutes a starting point for research aimed at actively and directly re-populating tooth roots with periodontal ligament fibroblasts and thus, by improving the overall periodontal condition, widen the spectrum of treatment possibilities open to the orthodontist.

Cells of the periodontal ligament and the alveolar bone lie in close vicinity in the periodontium. The goal of this study was to create an in vitro model to facilitate the study of the morphogenesis and proliferation of these two cell types under more in-vivo-like conditions. This was accomplished by the generation of organotypic co-cultures of primary human periodontal ligament fibroblasts (PDL) and alveolar bone cells (BC) and matched mono-cultures after 1, 2 and 3 weeks. Indirect immunofluorescence (IIF) for vimentin indicated that PDL cells exhibited sustained stratification only in the presence of BC cells, suggesting an important role for BCs in maintaining the stratification of PDL cells. In mono-cultures, only BC cells showed progressing stratification. They also displayed the most pronounced contraction of the cell culture matrix. Moreover, Ki-67 antigen detection by IIF revealed that these features coincided with cell proliferation localized on the matrix surface at the onset of cell stratification. These findings suggest that, in addition to proliferation, a further prerequisite for stratification may be cell migration. Furthermore, the maintained cell stratification, proliferation, and compartmentalization noted for PDL cells in organotypic co-cultures and BCs in mono-cultures can only be observed in a three-dimensional culture system. Thus, our system represents a novel experimental tool to further elucidate the underlying mechanisms of the growth and differentiation of PDL and bone tissue. [ABSTRACT FROM AUTHOR]