A. H. Melcher

Fibroblasts contracting three-dimensional collagen gels exhibit ultrastructure consistent with either contraction or protein secretion.

Periodontal ligament fibroblasts contracting collagen gels changed their phenotype in accordance with the stages of gel compaction. In the early and active phase of gel compaction, the fibroblasts exhibited numerous cell processes containing microfilamentous material and few organelles, little RER and Golgi membranes, but many free ribosomes, numerous structures resembling gap junctions, and a rich cell coat. Fibroblasts changed to a more rounded, relaxed state after 24 hr when the gel was compacted. At this stage, extensive RER and Golgi membranes, small peripheral microfilament bundles, little cell coat, and few, if any, gap junctions were observed. On the other hand fibroblasts plated on plastic exhibited prominent RER and Golgi membranes, peripheral microfilament bundles, and junctions of the adhaerens type. The results suggest that so-called “myofibroblasts”, which have been described as being prominent in the granulation tissue of contracting wounds, represent a reversible phenotypic modulation of fibroblast-type cells to a contractile state.

An in-vitro model for tooth eruption utilizing periodontal ligament fibroblasts and collagen lattices

An in-vitro model that simulates tooth eruption was devised to determine the possible role of periodontal ligament fibroblasts (PLF) in providing a major force for eruption. The model consisted of agarose-coated wells in multiwell tissue-culture plates to which wire mesh was attached at the rim and a slice of demineralized root was attached to the floor. When the wells were filled with a collagen lattice containing PLF, the lattice contracted and elevated the root slice. The root slice in models that were unattached at the rim was not elevated. Cultures without cells did not contract and root slices were not elevated. Elevation was inhibited by cytochalasin D and Colcemid. Histologically, cultures that had elevated showed orientated cells and collagen fibres. The results suggest that PLF may be capable of generating forces that are sufficient to produce tooth eruption.

Dome formation by oral epithelia in vitro.

SummaryMulticellular, 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 10−6M) and abolished by ouabain (10−10M), 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.

Effect of ascorbic acid on protein synthesis and collagen hydroxylation in continuous flow organ cultures of adult mouse periodontal tissues.

SummaryA continuous flow organ culture system (CFCS) was used to determine the effect of ascorbic acid on the synthesis of collagen and noncollagenous protein by bone of the alveolar process and periodontal ligament in organ cultures of adult mouse periodontium. For the last 24 h of 2 day cultures, 5 µCi/ml3H-proline was added to the medium. Highly purified collagenase was used to separate the collagenous and noncollagenous proteins and the incorporation of isotope into each fraction measured. Collagen synthesized in the presence of less than 10 µg/ml ascorbic acid was found to be highly under-hydroxylated (pro:hypro sp. acts. 2.3–3.1) in both tissues. When the ascorbic acid levels were between 25 and 100 µg/ml, the synthesis of collagenous proteins was selectively stimulated and hydroxylation significantly improved (pro:hypro sp. acts 1.72–1.89). The effect of ascorbic acid was not related to tissue viability since tissues cultured initially in the absence of ascorbic acid were able to recover completely when compared to controls given ascorbic acid continuously. The proportion of radioactivity in collagen and noncollagenous protein, collagen hydroxylation, and percentage of collagen synthesized as type III (av. 23%) in bone of the alveolar process was similar to that found in vivo. However, in the periodontal ligament in vitro the proportion of noncollagenous protein synthesized was increased from 70% to 87% and the percentage of type III collagen increased from 14% to 26% compared to in vivo results.

A CONTINUOUS-FLOW CULTURE SYSTEM FOR ORGAN CULTURE OF LARGE EXPLANTS OF ADULT TISSUE: EFFECT OF OXYGEN TENSION ON MOUSE MOLAR PERIODONTIUM

SummaryA modified continuous-flow culture system (CFCS) was developed to maintain large explants of periodontium from adult mouse in organ culture. The culture medium was stored in a reservoir outside of the incubator, pumped via polyvinyl tubing into small glass culture chambers that were placed in the oxygenator and then collected in a waste flask. Medium was analyzed for pO2, pCO2 and pH during the culture period. Three-molar and singlemolar explants of periodontium were maintained for 48 hr in the CFCS at two different pO2 ranges: 100 to 120 mm Hg and 400 to 420 mm Hg. [3H]Proline was added 24 hr prior to sacrifice. Light-microscope morphological and radioautographic observations suggested that cell viability and incorporation of [3H]proline, probably into newly synthesized protein, increased with an increase in pO2 and was related to a pO2 gradient extending from the periphery to the center of the explants.

Method for maintaining large explants of mature mixed tissue in organ culture: Molar tooth and supporting structures from the mouse

SummaryA method has been developed for successfully maintaining for 4 days in organ culture the first molar and supporting tissues of adult mouse. The explant, which comprises the tooth, periodontal ligament, bone and, on occasions, gingiva, was cultured in a modified New circulator in Waymouths medium MB 752/1 supplemented with ferrous sulfate, ascorbic acid, hydrocortisone, HEPES, 10% calf serum, and antibiotics. Various tensions of O2 were used, the tissues being best maintained in 95% O2+5% N2.