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D M Brunette

Publications and source records attributed to D M Brunette.

At least 55 records · Page 3Linked to original sources
PubMed1987

Effect of cell shape on proteinase secretion by epithelial cells.

Cell proliferation has been found to correlate with increased secretion of proteinases, such as plasminogen activator, in several different cell populations. In addition, the shape of the cell may also play a role in regulating proteinase secretion. However, the relationship between cell proliferation, cell shape and proteinase secretion has not been studied in diploid epithelial (E) cells cultured from porcine periodontal ligament (PL). We have modified PLE cell shape by physical means, such as growth on less-adhesive substrata and mechanical stretching, and by exposure to cholera toxin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Neutral proteinase and plasminogen activator secretion were found to correlate with cell shape, the more round the cells, the greater the amount of proteinase secreted. PLE cells, stimulated to proliferate by cholera toxin or dibutyryl cyclic AMP, were more spread than control cells, but secreted less neutral proteinase and plasminogen activator. TPA stimulated cell proliferation slightly but, in contrast to cholera toxin, increased cell rounding and the secretion of neutral proteinase and plasminogen activator. Thus proteinase secretion was related more to cell shape than to cell proliferation.

Animals↗
PubMed1986

Spreading and orientation of epithelial cells on grooved substrata.

The spreading and orientation of epithelial (E) cells was studied on titanium-coated grooved substrata by light, transmission (TEM) and scanning electron microscopy (SEM). Vertical-walled grooves and V-shaped grooves, 3-60 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of micro-electronic components, and the grooved substrata were replicated in Epon. Photolithography was used to prepare photoresist-based and silicon dioxide-silicon substrata with grooves of approximately 2 and approximately 0.5 micron deep, respectively. Cell clusters were markedly oriented by all the grooved substrata examined, with the orientation index being highest for substrata with grooves of the smallest repeat spacing. Time-lapse cinemicrography showed that the grooves directed the migration of E cells, but the control was not absolute, as some cells crossed over the ridges and descended into the grooves. The 0.5 micron grooves appeared less effective than the deeper grooves in directing cell locomotion. SEM and TEM of E cells spreading on the grooved substrata demonstrated that cell processes, including lamellae and filopodia, were capable of bending around and closely adapting to groove edges. E cells did not flatten as extensively on a substratum with 22 microns deep V-shaped grooves as on a smooth surface, although some cells were markedly elongated. One mechanism proposed to explain contact guidance of fibroblasts is that linear elements of the locomotory system, such as microfilament bundles, are unable to operate when bent. The observed flexibility of epithelial cell processes and the ability of substrata with shallow grooves to orient E cells indicate that contact guidance of E cells on micromachined substrata cannot be explained by the mechanical stiffness of long linear cytoskeletal elements.

Animals↗
PubMed1986

Fibroblasts on micromachined substrata orient hierarchically to grooves of different dimensions.

Contact guidance was studied by light, scanning (SEM) and transmission electron microscopy (TEM) in cultures of human gingival fibroblasts cultured on grooved surfaces. The grooves were originally produced in silicon wafers by micromachining, a process which is based on the methods used to fabricate microelectronic components, and the grooved surfaces were then replicated in Epon. Micromachining enables precise control of groove depth, groove spacing, and groove shape to be obtained. In silicon wafers with appropriate crystal orientation, a second smaller set of grooves, called the minor grooves, is found on the floor of the major grooves. The minor grooves are oriented at a 54 degree angle to the major grooves, so that cells cultured on such surfaces are concurrently exposed to grooves of different dimensions which direct cell migration in different directions. Marked fibroblast alignment with the major grooves was observed both within the grooves and in the intervening flat ridges between the grooves. In addition, shallow and closely spaced grooves in epon or titanium-coated polymer or silicon were also capable of orienting fibroblasts. Although the minor grooves were able to orient fibroblasts in the absence of any other orienting influence, when fibroblasts were concurrently exposed to major and minor grooves the cells aligned themselves with the major grooves. TEM showed that the cellular filamentous cytoskeletal elements reflected the orientation of the cell as a whole. Fibroblasts on grooved substrata appeared to have more filopodia and to round up more frequently than fibroblasts cultured on flat substrata. It is suggested that both the mechanical properties of the cytoskeleton as well as the durability of the cellular attachment to groove edges may play a role in the contact guidance effected by grooved surfaces produced by micromachining.

Cell Adhesion↗
PubMed1985

Mechanical stretching increases the number of cultured bone cells synthesizing DNA and alters their pattern of protein synthesis.

A simple method was devised for applying mechanical stretching to bone cell cultures. Bone cells cultured on the flexible plastic membrane of a Petriperm dish are placed over a template with a convex surface. A lead weight is then placed on top of the dish which causes the membrane and the tightly attached cells to be stretched. Mechanical stretching, applied either intermittently or continuously for a 2-hour period resulted in a 64% increase in the number of cells synthesizing DNA. Stretching the cells also significantly increased incorporation of tritiated proline and tritiated leucine. To assay the ratio of collagenous to noncollagenous protein, medium and cell layers of cultures labeled with tritiated leucine were incubated with collagenase and the digests chromatographed on PD 10 columns. The amount of collagen synthesized by stretched and unstretched cultures did not differ; but an increased synthesis of noncollagenous proteins was observed in the stretched cultures.

Animals↗
PubMed1984

Cholera toxin and dibutyryl cyclic-AMP stimulate the growth of epithelial cells derived from epithelial rests from porcine periodontal ligament.

The epithelial cells (E-cells) grew best at high (greater than 5 per cent) concentrations of fetal bovine serum (FBS). Growth could be obtained at low concentrations of dialysed FBS (DFBS) if the medium (alpha-MEM) was modified so that the levels of Ca2+ and K+ were reduced to 0.1 and 1.0 mM, respectively (beta-MEM). The addition of 0.5 per cent DFBS to the beta-MEM did not initiate good growth but was sufficiently supportive to enable the effects of various growth promoters to be tested. Cholera toxin and dibutyryl cyclic-3'5'-adenosine monophosphate (Bt2cAMP), which are known to increase intracellular cAMP levels, at concentrations of 1 ng/ml and 0.5 mM, respectively increased cell number. Cholera toxin caused the E-cells to be more flattened when viewed by phase-contrast; this appeared to be due to spread of the cells. No difference in cell-size distributions obtained between the trypsinized E-cells grown in the presence or absence of cholera toxin was observed. Epithelial proliferation that occurs in dental cysts could result from a rise in intracellular cAMP levels in epithelial cell rests.

Animals↗
PubMed1984

Ultrastructural study of the attachment of human gingiva to titanium in vivo.

A study was conducted to determine if the behavior of epithelium in vitro is similar to its attachment behavior in vivo by the use of small sections of titanium-coated implants that could be inserted in human gingiva. The size of the implants allowed their insertion in a limited region and enabled the fixation and embedding procedures that are necessary for electron microscopy to be effective. Examination of the thin sections obtained from this material demonstrated that the epithelial cells attached to titanium in a manner similar to that observed in vitro and similar to the way that epithelium attaches to the tooth in vivo (Fig. 2). That is, there was a formation of hemidesmosomes and basal lamina. Because the ability of the oral epithelium to form such an attachment with an implant may be a crucial factor in the determination of clinical success or failure, this study provides further evidence that one reason for the apparent clinical success of titanium dental implants may lie in the firm attachment of epithelial cells to this material.

Adult↗
PubMed1984

Mechanical stretching increases the number of epithelial cells synthesizing DNA in culture.

The influence of mechanical stretching on epithelial (E) cells was examined by culturing E cells derived from the epithelial cell rests of Malassez on a flexible plastic substrate and stretching the substrate by means of an orthodontic screw. A significant increase in the number of E cells synthesizing DNA was observed after just 30 min of stretching. In 17 experiments the ratio of cells labelled with tritiated thymidine in cultures stretched for 2 h to the number of labelled cells in unstretched controls was 1.92 +/- 0.34. An increase in labelling as a result of stretching was found for E cells cultured at either high or low cell-population densities but the effect was most pronounced for E cells cultured at higher concentrations of foetal bovine serum. Morphometric analysis of electron micrographs of stretched and unstretched cultures indicated that the stretched cultures had a higher volume fraction of filamentous structures and more desmosomes per unit length of cell membrane than unstretched cultures. The behaviour of E cells in response to stretching in vitro appears to be similar to the response of the epithelial rests in vivo when the latter are exposed to tension as a result of forces produced by orthodontic techniques.

Animals↗
PubMed1982

Dome formation by oral epithelia in vitro.

Multicellular, cystic structures, termed domes, have been described previously in epithelia cultured from various tissues that have a known transport or secretory function in vivo and in vitro. We report for the first time dome formation in cells cultured from "covering" and "rest" epithelia of oral tissues: porcine gingival and alveolar mucosa epithelium and epithelial rests of Malassez. As demonstrated by light- and electron microscopy, the morphology of the domes varied with the location of their lumen and the number of cells or cell layers involved in their structure. Sequential observations using phase contrast microscopy and time lapse cinematography of living cultures showed that the domes were dynamic structures with expansion-collapse cycles of between 30 min and 17 h duration. Dome formation in oral epithelia was stimulated by dibutyryl cyclic AMP (dbcAMP, 10(-3) to 10(-6) M) and abolished by ouabain (10(-10) M), an inhibitor of sodium transport. The morphological features and the dynamic nature of domes found in oral epithelia, and their dbcAMP and ouabain responsiveness are similar to those demonstrated previously in several other epithelia that have a known transport function in vivo and in vitro. Such fluid transport is not thought to be a property of oral epithelia in vivo. Our data, however, suggest a similar function of these epithelia cultured in vitro, and perhaps in pathological cyst formation in vivo.

Animals↗