Strain response in fibroblasts indicates a possible role of the Ca(2+)-dependent nuclear transcription factor NM1 in RNA synthesis.

On the mechanistic level, response of periodontal fibroblasts permanently exposed to mechanical strain forces in vivo still lacks in clarity. Therefore, we first investigated putative strain modulation of proteins by combined 1D gel electrophoresis-based protein profiling and electrospray tandem mass spectrometry (ESI-MS). Thereafter, the exponential-modified protein abundance index (emPAI) identified strain modulation of cytoskeleton-associated molecules, including decrease in talin and microtubule-associated protein 4 (MAP4), and significant increase in myosin IC (Myo IC), the latter ones regulated by Ca(2+). These findings were corroborated by western blotting (WB) and indirect immunofluorescence (IIF). Regarding the dual function of Myo IC as actin-based cytoplasmic motor protein and nuclear transcription factor NM1, WB and IIF revealed inverse correlation for Myo IC and NM1. During strain application, cytoplasmic increase of Myo IC was counteracted by nuclear NM1 deprivation, the latter coinciding with a decline in RNA quantity. Independent on strain, cytoplasmic Myo IC and nuclear NM1 abundance could be abrogated by the Ca(2+) channel blocker nifedipine, suggesting Ca(2+) dependency of cytoplasmic and/or nuclear Myo IC/NM1 expression. Mechanistically, we conclude that, application of strain appears as causative for the decline in RNA by impacting NM1, thereby indicating the possible role of NM1 in RNA synthesis.

Mechano-transduction in periodontal ligament cells identifies activated states of MAP-kinases p42/44 and p38-stress kinase as a mechanism for MMP-13 expression.

BACKGROUND: Mechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated. RESULTS: In the present study we show by western blot (WB) analysis and/or indirect immunofluorescence (IIF) that mechanical strain modulates the amount of the matrix metalloproteinase MMP-13, and induces non-coherent modulation in the amount and activity of signal transducing molecules, such as FAK, MAP-kinases p42/44, and p38 stress kinase, suggesting their mechanistic role in mechano-transduction. Increase in the amount of FAK occurs concomitant with increased levels of the focal contact integrin subunits beta3 and beta1, as indicated by WB or optionally by IIF. By employing specific inhibitors, we further identified p42/44 and p38 in their activated, i.e. phosphorylated state responsible for the expression of MMP-13. This finding may point to the obedience in the expression of this MMP as extracellular matrix (ECM) remodelling executioner from the activation state of mechano-transducing molecules. mRNA analysis by pathway-specific RT-profiler arrays revealed up- and/or down-regulation of genes assigning to MAP-kinase signalling and cell cycle, ECM and integrins and growth factors. Up-regulated genes include for example focal contact integrin subunit alpha3, MMP-12, MAP-kinases and associated kinases, and the transcription factor c-fos, the latter as constituent of the AP1-complex addressing the MMP-13 promotor. Among others, genes down-regulated are those of COL-1 and COL-14, suggesting that strain-dependent mechano-transduction may transiently perturbate ECM homeostasis. CONCLUSIONS: Strain-dependent mechano-/signal-transduction in PDL cells involves abundance and activity of FAK, MAP-kinases p42/44, and p38 stress kinase in conjunction with the amount of MMP-13, and integrin subunits beta1 and beta3. Identifying the activated state of p42/44 and p38 as critical for MMP-13 expression may indicate the mechanistic contribution of mechano-transducing molecules on executioners of ECM homeostasis.

Soft micropillar interfaces of distinct biomechanics govern behaviour of periodontal cells.

A soft micropillar extracellular environment of distinct biomechanics is established by fabricating polydimethylsiloxane (PDMS) interfaces with pillar distances of 5, 7, 9 and 11 microm and elasticity moduli of 0.6, 1.0 and 3.5 Mega Pascal. To allow for cell adhesion, the biomimetic concept of pillar head fibronectin (FN) biofunctionalisation is applied. This environmental set-up aims at the analysis of favourable conditions for cell behaviour of three periodontal cell-types, here reflected by the establishment of regular cell morphology and optimal collagen gene expression. Biomechanics of these predefined functionalized model surfaces reveal progressive deterioration of regular cell morphology with increasing pillar distance, independent from pillar elasticity and cell type. Analysis of collagen gene expression demonstrates interdependency to the elasticity and the micropattern of the extracellular environment in all cell types under study. The results suggest that biomechanics of the extracellular environment govern tissue-specific cell behaviour in different periodontal cell types. Moreover, they form the basis for the creation of new biomaterials which address distinct cell functions by specific biomechanical properties.

Gingival fibroblasts established on microstructured model surfaces: their influence on epithelial morphogenesis and other tissue-specific cell functions in a co-cultured epithelium: an in-vitro model.

OBJECTIVE: The objective of this study was to investigate how gingival fibroblasts cultured on microstructured model surfaces affect epithelial morphogenesis and other cell functions in a cocultured epithelium while conducting a molecular analysis of interactions between biomaterials employing periodontal tissue cells. MATERIALS AND METHODS: We are the first to have successfully co-cultured gingival fibroblasts together with gingival keratinocytes on biofunctionalized, microstructured model surfaces and, in the resulting co-cultured epithelium, examined the molecules of tissue homeostasis, the differentiation marker keratin (K) 1/10, and involucrin after 1- and 2-week periods of cultivation. Desmoplakin was perceived as evidence of cell-to-cell contact and thus as proof of epithelial integrity. We also analyzed the basement membrane component laminin-5. The aforementioned co-culture model without gingival fibroblasts was used as a control set-up. RESULTS: On the protein level, indirect immunofluorescence demonstrated the presence of K1/10, involucrin and the basement membrane component laminin-5 in the co-cultured epithelium in both culture periods. Furthermore, we observed that these epithelial markers had become re-oriented toward the suprabasal cell layers which, in turn, reflects the native in-vivo gingival epithelium. We identified cell-to-cell adhesion as a function of desmoplakin after just 1 week. In the mRNA analysis using quantitative RT-PCR after 2 weeks of cultivation, we noted a considerable rise in relative gene expression that was time-dependent for the early keratinocyte differentiation marker K1 and late marker involucrin. CONCLUSIONS: Our findings demonstrate that gingival fibroblasts on microstructured model surfaces are vitally important for tissue- specific cell functions such as epithelial morphogenesis and other biological cell functions such as differentiation and epithelial integrity. These study findings can thus contribute to the optimization and/or new development of biomaterials currently used in dental medicine.

Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents.

Ethanol treatment of immortalised human gingival keratinocytes (IHGK) yields in an epithelium-like (EPI) and fibroblast-like (FIB) phenotype. With respect to the stratified gingival epithelium, putative structural and molecular differences assigning cells to these phenotypes have not, to date, been analysed in a three-dimensional tissue/epithelial context. Therefore, we generated epithelial equivalents (EEs) in organotypic co-cultures of IHGK, EPI and FIB cells for 1 and 2 weeks and conducted protein and gene expression studies on the EEs for epithelial biomarkers including keratin K14, integrin subunits alpha6 and beta1, E-cadherin, and mesenchymal vimentin. As in the EEs of IHGK and EPI, indirect immunofluorescence revealed continuous expression of beta1 integrin in EEs of FIB cells. However, FIB cells exhibited a significant down-regulation in K14 and integrin alpha6 protein and a loss of E-cadherin at week 2, whereas vimentin was increased. FIB EEs were devoid of transcripts for E-cadherin at both time points, although transcription of the other genes remained constant in all phenotypes. Thus, the FIB phenotype exhibited a poor epithelial structure coinciding with disturbances in the expression of epithelial biomarkers and the persistence of mesenchymal vimentin. Transcription analysis revealed post-transcriptional regulation of vimentin in IHGK and EPI and of K14 and alpha6 in FIB cells. Our findings indicate that differences in the epithelial integrity and expression of molecules in EEs allow for the discrimination of EPI and FIB cells. This suggests that FIB cells share features of epithelial-mesenchymal transition and reflect a more progressive stage in epithelial cell transformation.

Elevated expression of genes assigned to NF-kappaB and apoptotic pathways in human periodontal ligament fibroblasts following mechanical stretch.

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.

Early keratinocyte differentiation on micropillar interfaces.

We employed topographical patterning to analyze early keratinocyte differentiation on top of microfabricated pillar arrays. Fibronectin immobilized on pillar "heads" yielded a nucleus-associated granular keratin 1 (K1) pattern in immortalized human gingival keratinocytes (IHGK) at pillar interspaces of 14 mum. Decreasing distances of 11and 8 mum revealed cytoplasmic extension of the early differentiation marker K1 on poly(dimethylsiloxane) (PDMS) pillars. The most extensive cytoplasmic K1 protein distribution noted at the smallest pillar scale coincided with higher ratios of K1 mRNA gene transcription. These experiments suggest that early keratinocyte differentiation was governed by the topographical characteristics of the pillar pattern. Moreover, they form the basis to study cell functions such as differentiation in a defined topologically structured environment.

Organotypic co-cultures allow for immortalized human gingival keratinocytes to reconstitute a gingival epithelial phenotype in vitro.

We report here that the organotypic co-culture (OCC) system allows for significant preservation of the tissue-specific phenotype of human gingival keratinocytes (IHGK) immortalized with the E6/E7 gene of the human papillomavirus type 16 (HPV16). The approach adopted is based on the OCC system facilitating spatially separated cell growth and cell-to-cell interactions via diffusible growth factors. Generally, IHGK reveal transcription of the HPV16 E6/E7 gene at rising passages. Fluorescence in situ hybridization performed for chromosomes 1, 8, 10, and 18 demonstrates that disomic fractions differ between the tested chromosomes but otherwise remain fairly constant. Monosomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromosome 10 and 18 are detected in early passages 25 and 27. In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, proliferate, and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for growth factors interleukin 1beta, granulocyte-macrophage colony stimulating factor, fibroblast growth factor 7, and EGF in OCCs is different from that in MCs. When grafted onto nude mice, IHGK develop hyperplastic, differentiated surface epithelia devoid of malignant growth. We are not aware of any other OCC system comprising of IHGK, which allows for site-specific expression of gingival epithelial markers. This substantiates reconstitution of a gingival epithelial phenotype in vitro.

Incisor trauma and the planning of orthodontic treatment.

Because of the frequency of dental injuries during infancy and adolescence, traumatized teeth with variable long-term prognoses present a problem for orthodontic treatment planning. Orthodontic therapy can remain unaffected, or be complicated, by traumatized teeth. In some cases, following dental injury, orthodontics can also be used to enhance (prosthetic and) restorative treatment results. The orthodontic challenges involved in treating patients with a history of dental trauma are complicated by the consequences of trauma on dentition development and the different treatment options that must be considered. In this paper, we provide actual examples of the effects dental trauma can have on orthodontic treatment planning.

Molecules contributing to the maintenance of periodontal tissues. Their possible association with orthodontic tooth movement.

This review is aimed at providing a depiction of molecules and their topography which characterize native gingiva and PDL fibroblasts, to describe their function in tissue maintenance, and to discuss their possible modulation due to orthodontic tooth movement. Maintenance of the human periodontium requires the balance of proliferation and differentiation in the respective tissues' cells. Moreover, the cells must synthesize the extracellular matrix molecules and receptors that facilitate adhesion. To describe the molecules that contribute to periodontal tissue maintenance, we illustrate the localization of their expression and topography on frozen sections from native gingival tissue and primary cell cultures derived from periodontal ligament. In native gingival epithelium, proliferation is confined to basal and parabasal cells. Keratin K14, when used as structural marker, is visible in the entire epithelium, while K13, an indicator of early differentiation, is restricted to the suprabasal cell compartment. Vimentin indicates mesenchymal cells in the subgingival connective tissue. Concerning the matrix molecules, collagen type-IV is abundant at the epithelium-lamina propria interface, and fibronectin is apparent throughout the mesenchyme. The matrix receptor integrin beta1 reveals a pericellular localization in basal and parabasal cells, while focal adhesion kinase p125FAK is seen pericellularly in all epithelial layers. Cultures of primary periodontal ligament (PDL) fibroblasts (PDL-F) reveal expression of vimentin, strong proliferation, synthesis and extracellular deposition of collagen type-I and fibronectin. The integrin subunits beta1 and p125(FAK) are largely detectable at the cell periphery.

Indications for digital volume tomography in orthodontics.

Disturbances in tooth eruption and tooth impactions make great demands on radiographic diagnostics. There is often need for radiographic images in various projections to assess the exact position of unerupted and impacted teeth. Digital volume tomography (DVT) is a method for localizing hard tissue structures such as bone and teeth on various planes. Moreover, it makes a three-dimensional image of the teeth, jaws and the viscerocranium possible. From an orthodontic point of view, digital volume tomography is indicated to detect impacted and ectopic teeth and to demonstrate the amount of bone available for orthodontic tooth movement. In patients with cleft lip and palate, DVT can be used to visualize the size of the alveolar cleft and to evaluate the position and development of multiple teeth, as these patients often suffer from disturbances in tooth eruption.

[Psychosocial problems and coping strategies of young patients with cleft lip and palate]. / Probleme und Bewältigungsverhalten von Jugendlichen mit Lippen-Kiefer-Gaumenspalte -- Ein Vergleich der Sichtweisen von Müttern und deren Kindern.

OBJECTIVE: The purpose of this study was to improve the psychological care of young patients with cleft lip and palate by gaining insight into those problematic areas in which social conflicts arise and coping-strategies become necessary for the patients. METHODS: The procedure included semi-standardized interviews with 20 patients aged between 12 - 17 and their mothers. Every interview was recorded on tape, transcribed, analyzed contextually and categorized. RESULTS: Four areas of main problems showed up: the time of surgery, the experience of being rejected or teased by peers, remaining visible defects and how to integrate them into self-percept, the problems of the siblings. The mothers showed more pessimistic perceptions regarding the coping strategies of their children and the helpful role of parental support. Consequences for individual support and family therapy are derived.

Applications for direct composite restorations in orthodontics.

BACKGROUND AND AIM: Besides prosthetic and indirect, laboratory-produced restorations, the focus of dental therapy is increasingly on restorative measures and direct restorations as minimally invasive treatment concepts. Thus, the use of direct composite restorations with modern restorative materials for the shaping and recontouring of teeth in combination with orthodontic treatment offers a diversified, extensive sphere of application. The aim of the study was to demonstrate applications for direct composite restorations with reference to selected cases. MATERIAL AND METHODS: The composites used were hybrid composites, which offer increased abrasion resistance and color stability and are applied incrementally. Special attention was paid to the shape, color and structure of the tooth. CASE REPORTS: The case reports present patients in whom relatively narrow or peg-shaped teeth were built up with composite to correct various tooth size discrepancies or cuspids were recontoured by means of direct composite restorations following orthodontic space closure in cases with missing lateral incisors. Similarly, space closure was achieved using orthodontically repositioned lateral incisors recontoured to resemble central incisors after traumatic loss of upper central incisors. Finally, direct composite restorations were used for retention following completion of orthodontic treatment. CONCLUSIONS: Observations over recent years confirm the stability of composites in both form and color, as well as their ability to maintain gingival health. Our case reports demonstrate that, subject to a corresponding indication, recontouring single teeth using direct composite restorations can optimize orthodontic treatment results.