Effects of mechanical stimulation on cell cycle duration in rat gingival fibroblast progenitor cells.

The aim of this investigation was to estimate cell cycle duration in rat gingival fibroblast progenitor cells in steady-state control and during sustained mechanical stimulation. Elastics (0.15 mm thick) were inserted between maxillary M1 and M2 of 8 wk-old male rats which were labelled with H3-TdR and killed in groups of 6-7 animals together with equal-sized groups of labelled control animals at intervals between 1-168 h. Autoradiographs of consecutive mesio-distal sections were used to determine grain counts for H3-TdR-labelled cells in the connective tissue of the gingival papilla between M2 and M3. Median cell cycle times (MCC) were estimated from plots of mean and median grain counts against time. Under steady-state conditions, MCC for heavily-labelled and lightly-labelled paravascular cell populations and for labelled extravascular cells were 144, 76 and 50 h, respectively. Mechanical stimulation caused a significantly faster rate of reduction of total grain counts relative to controls in all three cell populations and a decline of estimated MCC to 115, 50 and 21 h in heavily labelled and lightly labelled paravascular cells and labelled extravascular cells, respectively. These findings indicate that mechanical stimulation induces faster progression of gingival fibroblast progenitor cells through the cell cycle.

Proliferative response of cells of the dentogingival junction to mechanical stimulation.

The aim of this research was to study the proliferative response of junctional epithelium (JE) and gingival connective tissue (GCT) to mechanical stimulation in vivo with regard to the potential occurrence of apical migration of JE and loss of GCT attachment during orthodontic tooth movement. Elastic bands were inserted between the maxillary first and second molars of male rats aged 8 weeks, which were pulse-labelled with 3H-thymidine and subsequently killed in groups, together with labelled control animals (a total of 98 rats) after periods of 1-168 hours. Autoradiographs were prepared from plastic mesiodistal sections, and parameters of cell proliferation for JE and GCT of the papilla between the second and third molars were determined. Although the distance between the apical limit of JE and the most coronal periodontal ligament (PDL) fibres decreased on the pressure and increased on the tension sides of mechanically stimulated animals, the total cross-sectional area of JE remained unchanged compared with controls. In the basal and suprabasal layers of JE, cell proliferation was reduced on the pressure side and showed no change on the tension side. In the apical JE compartments on both sides, mechanical stressing resulted in lower proliferative activity. Cell proliferation in GCT adjacent to JE in stimulated animals did not differ from the corresponding controls. JE rapidly adapted to mechanical stimulation by means of differential local adjustments of cell proliferation without an occurrence of apical migration or hyperplasia. GCT cells in the vicinity of JE maintained their steady-state proliferative activity. These results do not support the concept that orthodontic tooth movement might per se have detrimental effects on the stability of the dentogingival junction.

Marsupialization of percutaneous implants in presence of deep connective tissue.

The primary substratum for epithelial migration during marsupialization of percutaneous implants is adjacent connective tissue. The purpose of this investigation was to test the hypothesis that if the latter is composed of deep connective tissue or other deep tissue, it will inhibit this process. One-hundred-forty-two smooth-surfaced polyethylene implants of a simple geometric design were implanted in dorsal skin of 6-8-week-old male C57/BL/6 mice such that the stems passed through and deep to the panniculus carnosus (PC), which is a deep tissue. Animals were sacrificed in groups between 1 and 11 weeks, and the implants and surrounding tissue were harvested, embedded in historesin, and 3-microns thick step serial sections prepared. No implants were lost by extrusion and 133 were suitable for examination. Epithelial investment of the devices proceeded deep to PC around only five of the implants over the time course. In the remainder, the mean distance of the advancing epithelial edge from the upper border of the PC was 0.25 mm by 1 week and this did not change significantly thereafter (p = 0.647). Significant extrusion of all devices occurred (p = 0.0001). It was concluded that these results are consistent with the hypothesis that deep connective tissue, such as the fascia surrounding the PC or muscle tissue such as the PC itself, can act as a functional barrier to marsupialization, and that exfoliation of such devices is not an epithelial function.

Absence of binding of human salivary glycoprotein to human gingival fibroblast-like cells in vitro.

The aim of this study was to determine whether human high molecular weight salivary glycoprotein binds in vitro to human gingival fibroblast-like cells. Primary monolayer cultures of 2 human gingival fibroblast-like cell lines were incubated with a high molecular weight fraction of salivary glycoprotein which expressed blood group A activity and glycoprotein-cell binding probed using an FITC-conjugated mouse monoclonal antibody to human blood group A antigen. Surface fluorescence of protein-treated cells was found to be no greater than that of untreated or serum-treated control cultures. As significant binding of salivary glycoprotein to gingival fibroblast-like cells does not occur in vitro, saliva-mediated inhibition of fibroblast attachment to hydroxyapatite is not dependent on specific ligand-lectin interactions.

An in vitro investigation of the role of high molecular weight human salivary sulphated glycoprotein in periodontal wound healing.

High molecular weight human salivary sulphated glycoprotein (SGP) inhibits attachment of fibroblasts to cementum in vitro and thus may enhance periodontal wound healing by repair with long junctional epithelium. However, competitive inhibition by serum constituents might prevent adequate binding of SGP for this effect to occur in vivo. The primary aim of this study was to investigate the co-adsorption in vitro, of SGP, fibronectin (FN), and albumin (ALB) to synthetic hydroxyapatite (HA) from solutions of SGP/FN (62.5/2 micrograms/mL respectively), or SGP/FN/ALB (62.5/2/4 micrograms/mL), or from individual solutions of the agents as controls: Desorption of SGP and FN was studied by preabsorbing HA with SGP or FN and subsequently exposing it to the other agent, or to buffer only as control. Adsorbates were assayed after incubation periods of up to 26 hours using specific ELISAs. SGP displaced previously adsorbed FN after 3 hours and significantly inhibited adsorption of FN and ALB compared with controls. Neither FN or ALB had a significant effect on SGP adsorption. These results are consistent with the possibility that salivary adsorption to cementum during surgery might interfere with repopulation of the root by connective tissue cells and thus contribute to wound healing by repair rather than regeneration. Separate studies taken to validate the ELISA used for SGP determination showed that HA-bound SGP contained all constituents of native SGP as revealed by SDS-PAGE and that ion exchange chromatography of SGP gave 6 fractions (I through VI), of which only the most ionic (VI) was able to inhibit cell attachment in vitro.

Migration and proliferation of progenitor cells in the connective tissue of rat gingival papilla.

The aim of this investigation was to determine whether gingival fibroblast progenitor cells undergo division and whether there is evidence of migration of their progeny from the paravascular sites within the interdental gingival septum (IGS). 30 male hooded Lister rats aged 6 weeks, were killed in groups of ten, 3, 75 and 171 h after a single injection of tritiated thymidine. Autoradiographs were examined of transverse Historesin sections of the papilla between second and third mandibular molars in 29 specimens, taken from equidistant intervals between the col and alveolar bone crest. Median grain counts in the whole papilla were greatest within 10 microns of blood vessels at 3 h (p < 0.02) and decreased throughout the papilla with time (p < 0.0001). The median grain counts at level 5, nearest the periodontal ligament, were greatest at all times (p < 0.01). The distance of labelled nuclei from blood vessel walls increased with time, most notably at level 5 (p < 0.001). The mean Clustering Index increased (p < 0.01) from 4.6% at 3 h to 12.3% and 11.6% at 75 h and 171 h respectively. At 75 h, the median grain count of clustered nuclei was smaller (p < 0.01) than that of non-clustered nuclei. These data are consistent with the occurrence of clonal division and migration within the IGS and the presence of a slowly cycling cell population near to the periodontal ligament.

Cell generation within the interdental gingival septum of the rat.

The aims of this investigation were to determine whether connective tissue progenitor cells in the interdental gingival septum (IGS) have a paravascular origin, and how the distribution of 3HTdR-labelled cells within the IGS changes with time after a single injection. 30 male hooded Lister rats aged 6 weeks, were killed in groups of ten, 3, 75 and 171 h after a single injection of tritiated thymidine. Autoradiographs were examined of 3 transverse Historesin sections of the papilla between second and third mandibular molars in 29 specimens, taken at equidistant intervals between the col and alveolar bone crest. At all times and levels, 73.9-93.0% of labelled cells and 72.0-79.0% of unlabelled cells lay within 50 microns of blood vessels (BVs). The highest percentages of labelled cells (PLCs) occurred within 5 microns of BVs (P < 0.001) although mean nuclear density here was lowest (P < 0.017), and there was a significant diminution of PLC (P < 0.05) with increasing distance from BVs, occurring most precipitously 5-10 microns from vessel walls. Sites of significantly increased PLC at 3 h also approximated to sites in which mean BV densities were greatest. At 3 h, a number of discrete sites with significantly increased PLCs (P < 0.05) were found within two zones, each equivalent to 40% of IGS volume and juxtaposed with each tooth. At later times, additional sites of raised PLC appeared throughout the IGS. Overall PLCs in the upper two levels and within 5 microns of BVs also increased over the time-course (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The validity of periodontal probing as a method of measuring loss of attachment.

Linear probe measurements are used to assess the severity and extent of attachment loss in chronic periodontitis and to identify, retrospectively, sites of disease activity. The use of the probe for these purposes is based on the implicit assumption that there is a direct and predictable relationship between linear probe measurements of attachment loss and the area of cemental surface which has been denuded of periodontal ligament. The aim of this study was to test this assumption by determining the correlation between loss of attachment as expressed by probe readings, and that expressed as the area of denuded root surface. The areas of denuded root surface of 236 teeth of different morphotype in 41 human dried skulls were determined by a rubber base impression technique and compared with their corresponding probe measurements, made at 10 sites per tooth. Although the majority of correlations between linear and area measurements were statistically significant for some individual morphotypes and categories of bone loss, there was overall, no consistent pattern of correlation between the two parameters. Furthermore, many correlations which were statistically significant had low values of the correlation coefficient: Kendall's T. It was concluded that probe readings are not a very precise measure of attachment loss, particularly with increasing severity destruction. These results cast doubt on the ability of individual linear measurements to represent the true severity of attachment loss, and thus on the precision of loss of attachment charts for retrospectively identifying sites of periodontal disease activity.

Inhibition of attachment of human gingival fibroblast-like cells in vitro by saliva and salivary-sulfated glycoprotein in the presence of serum.

We have previously shown that human whole saliva and a high molecular weight sulfated glycoprotein (SGP) salivary component inhibits attachment of human gingival fibroblast-like cells to plastic substrata in serum-free conditions. The purpose of this study was to investigate the influence of saliva on attachment of these cells to tissue culture plastic in the presence of serum. Individual wells of multiwell dishes were coated with either sterile whole saliva or SGP, sequentially with fetal bovine serum followed by saliva or SGP, sequentially with the latter agents applied in the reverse order, with mixtures of saliva and serum or SGP and serum. Washed wells were seeded with 1.0 x 10(5) fibroblasts in alpha-MEM and numbers of adhering cells determined after 30 minutes. Saliva or SGP inhibited cell adherence as previously reported. Cell adherence in wells treated sequentially with saliva or SGP followed by serum, or with the latter followed by the salivary agents, was reduced significantly compared with that in untreated control wells. Wells treated with mixtures of serum and saliva or SGP exhibited progressive reduction in numbers of adhering cells as the concentration of the salivary agents increased. Significant suppression of attachment compared with controls also occurred when cells in alpha-MEM containing 15% serum were plated onto saliva- or SGP-treated wells. These results are consistent with the hypothesis that adsorbed salivary glycoprotein may bring about periodontal wound healing by repair rather than by regeneration by inhibiting fibroblast attachment to root surfaces in vivo.

Creation of a chimaeric periodontium in the rat by isotopic tooth germ transplantation.

The purpose of this work was to develop and test a chimaeric periodontium in which it would be possible to distinguish between connective tissue cells of odontogenic and oral mucosal origin. The recombinant periodontium was created by transplanting first maxillary molar tooth germs with their follicles from 1-3-day-old hooded Lister rats into the corresponding evacuated crypts of 6-9-day-old histocompatible recipients of the same strain. Of 71 transplants, 22 had formed erupted teeth 3 weeks later, with dentogingival junctions and periodontal ligaments histologically similar to those of control teeth. The recombinant nature of the graft periodontium was confirmed by incubating tooth germs in vitro with tritiated thymidine before grafting them, and then demonstrating radiolabelled nuclei in the dentogingival junctions formed by the transplants. Labelled cells were randomly distributed within the periodontal ligament and predominantly near to the basement membrane of junctional epithelium.