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Biomedical subjects

D M Brunette

Publications and source records attributed to D M Brunette.

At least 73 records · Page 4Linked to original sources
PubMed1980

Orientation of calvaria and periodontal ligament cells in vitro by pairs of demineralized dentine particles.

Rat calvaria cells (RC cells) and monkey periodontal ligament fibroblasts (MPL cells) were cultured for 4-6 days with demineralized dentine, acid-washed glass particles or untreated glass particles to determine whether the presence of these particles can affect the orientation of the cells. Encapsulation of dentine particles began as early as the first day in culture and occasional cells were attached to, and oriented between adjacent particles. Multilayers of cells formed along the periphery of the dentine particles and the more superficial of these cells projected outwards in a radial fashion. Oriented cell sheets were evident between dentine particles after 3 days in culture. Finally, the cell sheets tore away from the surface of the culture fish to give rise to thick multilayered cellular bridges between dentine particles. The nuclear orientation index (ratio of L/W) for each RC cell lying between 15 randomly selected pairs of each particle type in 4-day cultures was measured along 2 axes; the length represented by the shortest distance between the 2 particles and the width perpendicular to this axis. The nuclear orientation index was 1.52 for cells between dentine, 1.13 between acid-washed glass and 1.06 between untreated glass. Control measurements of cells associated with single particles were 1.06, 1.09 and 1.06 respectively. The frequency of cellular orientation occurring between dentine particles was significantly greater than that occurring between acid-washed glass particles (chi 2 > 0.001) which in turn was significantly greater than that for untreated glass particles (chi 2 > 0.001). The observations and results suggest that the development and maintenance of the observed cell orientation depends upon development and maintenance in the cell sheet of tensional forces oriented parallel to the axis between the dentine particles, and that this is related directly to the capacity of the cells to attach to the particles.

Animals↗
PubMed1980

Epithelial rests of Malassez in vitro. Phagocytosis of collagen and the possible role of their lysosomal enzymes in collagen degradation.

Cells from porcine epithelial rests of Malassez were cultured in vitro with collagen prepared from rat tail tendons. Serial sections of the cultures were prepared for examination in the electron microscope. Some of the material was processed to demonstrate activity of acid phosphatase. Electron microscopy showed that the epithelial cells had phagocytosed collagen in vitro, and study of the serial sections indicated that much of the phagocytosed collagen was contained wholly within the cells. Reaction product indicating sites of acid phosphatase activity was found to be associated with intracellular collagen, and some of the intracellular fibrils exhibited nonsymmetrical loss of material and also localized loss of banding, suggesting intracellular digestion of collagen. A lysosomal fraction was prepared from epithelial cells cultured from the rests of Malassez, and this was shown using biochemical assays to contain activities of thiol-dependent cathepain, cathepsin D, beta-D-glucuronidase, aryl sulfatase, and acid phosphatase. The lysosomal fraction had the capacity in vitro to depolymerize and digest collagen obtained from rat tail tendon. It was concluded from these observations and results that epithelial cells cultured from the rests of Malassez can digest collagen in vitro. The findings suggests a possible mechanism whereby the epithelial cells can destroy the extracellular substance of connective tissue during their well known participation in cyst formation in vivo.

Amino Acids↗
PubMed1979

In vitro phagocytosis of exogenous collagen by fibroblasts from the periodontal ligament: an electron microscopic study.

There have been numerous electron microscopic reports of apparent phagocytosis of collagen by fibroblasts and other cells in vivo. We have developed an in vitro system which, to the best of our knowledge, will permit for the first time the study of regulatory mechanisms governing phagocytosis and digestion of collagen fibres. Cells were cultured from explants of monkey periodontal ligament, subcultured, and grown to confluence in alpha-MEM plus 15% fetal calf serum plus antibiotics. The confluent cells were then cultured together with minced rat tail tendon collagen in alpha-MEM lacking proline, lysine, glycine and fetal calf serum for up to 7 days, after which they were processed for electron microscopy. Intracellular collagen profiles could be seen in cultured cells that were associated with exogenous collagen fibrils as early as 24 hours after addition of the collagen. Through electron microscopic examination of serial sections of the culture, we have demonstrated: (1) that fibroblasts can phagocytose collagen; (2) that the observed intracellular collagen is not the result of aggregation of endogenous synthesized collagen; (3) that it is not possible to base a decision as to whether a collagen fibril has been phagocytosed in whole or in part by the type of vesicle with which it is associated; (4) that cleavage of collagen into small pieces may not be a necessary prelude to its phagocytosis.

Animals↗
PubMed1978

Nature of collagens synthesized by monkey periodontal-ligament fibroblasts in vitro.

1. First subcultures of fibroblast-like cells from adult monkey periodontal ligament were incubated in the presence of 14C-labelled amino acids and produced significant amounts of type-I and type-III collagens. 2. The proportion of type-III collagen produced was calculated on the basis of the recovery of procollagens from DEAE-cellulose chromatography to be approx. 20%, and at least 10% when analysed as collagens on CM-cellulose chromatography. 3. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the procollagens, the collagens and their CNBr peptides was used to confirm the identity of the collagen types. 4. In serum-free media extensive conversion of type-I procollagen, but not of type-III procollagen, into collagen was observed, suggesting that a specific type-I procollagen peptidase was produced. 5. The pattern of collagen synthesis was not significantly different from that obtained with fibroblasts derived from skin corium of the same animals.

Animals↗
PubMed1978

Isolation and identification of epithelial-like cells in culture by a collagenase-separation technique.

An operational criterion for the identification and isolation of epithelial-like (E) cells, based on their ability to cover and protect a collagen gel from the action of collagenase, has been developed. The E cells isolated by this collagenase-separation technique (CST) exhibited the ultrastructural features, including desmosomes and abundant tonofilaments, that are considered characteristic of this cell type. Unlike confluent cultures of fibroblast-like (F) cells, E cells were not found to have large external transformation-sensitive (LETS) protein on their surface membranes. The CST provides a nondestructive and efficient means of identifying and isolating E cells from mixed populations.

Cell Separation↗
PubMed1977

Location of progenitor cells in periodontal ligament of mouse molar stimulated by wounding.

Wounds were made in the alveolar process overlying the mesial root of the mandibular first molar of 35-g mice to expose periodontal ligament. Dividing cells were arrested in metaphase by injection of vinblastine sulphate three hours before death three, five and seven days after wounding. Serial 1-micron plastic sections of the wound were stained with hematoxylin and eosin. The distance between the mitotic figures and the nearest blood vessel, bone and cementum was measured in every fourth section. No mitotic figure was measured twice. The distances of at least 500 interphase cells from the closest blood vessel were measured in each wound. At all time periods the distribution of mitotic cells was significantly closer to blood vessels than that of interphase cells (p less than 0.01). From three days to seven days after wounding there was a significant shift of mitotic activity away from the bone side of the ligament, but not away from blood vessels (p less than 0.05). Approximately 10% of all mitotic cells lay close to cementum, but not close to blood vessels. The majority of the dividing cells were confirmed to be paravascular, and appeared to belong to two populations, one adjacent to bone, the other in the body of the ligament. A third population, not paravascular, lay adjacent to cementum.

Animals↗
PubMed1977

Determination of L-ascorbic acid levels in culture medium: concentrations in commercial media and maintenance of levels under conditions of organ culture.

The method of Deutsch and Weeks was modified to provide a reliable and reasonably quick method for assaying the L-ascorbic acid content of culture medium. The modified method was used to determine the decay of L-ascorbic acid under various conditions of culture and the concentration of the vitamin in commercially prepared media. The half-life of L-ascorbic acid in a modified New circulator gassed with 95% O2 + 5% CO2 was 1.5 hr.; and when gassed with 20% O2 + 5% CO2 + 75% N2, about 2 hr. In Petri dishes gassed with 20% O2 + 5% CO2 + 75% N2, the half-life of L-ascorbic acid was 0.9 hr. About 4% of the L-ascorbic acid was lost per day when medium was stored at 0 degrees C and about 9% per day when stored at 5 degrees C. When medium with an initial content of 300 microng per ml was stored at room temperature, the half-life was found to be 15.5 hr. The L-ascorbic acid in five commercially available media, which contain the vitamin in their formulations, was assayed immediately after their delivery to the laboratory. The values of L-ascorbic acid measured in these media were in all cases far lower than prescribed. A continuous-flow organ culture system has been designed which allows the provision of a relatively constant level of L-ascorbic acid to explant by taking advantage of the slow oxidation of L-ascorbic acid at 0 degrees C.

Ascorbic Acid↗
PubMed1977

Parathyroid hormone- and prostaglandin E1-response in a selected population of bone cells after repeated subculture and storage at -80C.

Five different cell populations, designated I to V, were isolated from minced newborn rat calveria by 5-sequential 20 min incubations with an enzyme mixture containing collagenase, elastase and DNAse. In primary culture, all five populations responded to parathyroid hormone (PTH) to a different degree, population IV giving the highest increase in cyclic-3'5'-adenosine monophosphate (cAMP) level. None of the five populations gave any response to calcitonin. Upon subsequent subcultures, all populations, except population IV, either lost or considerably decreased their response to PTH. Population IV gave a two to three-fold increase in cAMP concentration in response to PTH up to the third subculture. No morphological differences could be observed among the five populations. The third subculture of population IV cells that had been stored in 10% glycerol at -80C for four months was subsequently thawed and subcultured to the sixth subculture. These cells still responded to PTH with an increase in cAMP level. In a second experiment, 5 different cell populations designated I to V were isolated in a similar way by incubation with collagenase and DNAse. The maximum response to PTH was found in population 3. The preservation of the PTH-responsiveness of this population, after subculturing, freezing, storing in 10% glycerol at -80 C and subsequent subculturing, was likewise demonstrated. The hormone-responsiveness of cells from the sixth subculture of previously frozen and thawed population IV cells was further analyzed. These cells responded to PTH at a concentration of 0.1 U/ml to 5U/ml and to prostaglandin E1 (PGE1) at a concentration of 0.1 microng/ml to 10 microng/ml. The time course of action on population IV of PTH was found to be different from that of PGE1, suggesting a possible difference in the regulation of intracellular cAMP levels by these hormones.

Bone and Bones↗