The effect of the outer membrane fraction of Bacteroides gingivalis W50 on glycosaminoglycan metabolism by human gingival fibroblasts in culture

The synthesis of extracellular [35S]-SO4- and [3H]-glucosamine-labelled glycosaminoglycan (GAG) was studied in confluent human gingival fibroblast cultures in vitro. The differential synthesis of the total chondroitin sulphate/dermatan sulphate (CS/DS) and heparan-sulphate (HS) fraction was measured following chondroitinase-ABC digestion, nitrous-acid treatment and column chromatography on Sephadex G50. Control cultures synthesized a CS/DS fraction that represented 78 per cent of the total [35S]-SO4-GAG; the residual 22 per cent was heparan sulphate. Similar cultures were labelled with [3H]-glucosamine and the proportions of a high molecular-weight hyaluronic acid (HA) and proteoglycan fractions measured by gel-filtration HPLC after papain and hyaluronidase digestions. The HA fraction represented 66 per cent of the total isotope incorporated in control cultures. GAG chains released on treatment with papain (24 per cent of the total label incorporated) were of apparent molecular weight 17-20 kDa. All cultures exposed to Bacteroides gingivalis W50 outer membrane at concentrations between 2 and 50 micrograms ml-1 displayed a decrease in the CS/DS fraction and a reciprocal increase in the HS. However, the proportion of HA synthesized was slightly enhanced with a reciprocal decrease in the proteoglycan (papain-digestible) fraction. There was no alteration in the molecular weight of the papain-digestion products or the size distribution of the hyaluronic-acid fraction.     

Qualitative and quantitative analyses of bovine gingival glycosaminoglaycans.

Bovine gingival glycosaminoglycans have been analysed qualitatively and quantitatively by two-dimensional electrophoresis on a cellulose acetate strip. The four spots observed were identified as chondroitin 4-sulphate, dermatan sulphate, hyaluronic acid and heparan sulphate. Neither chondroitin 6-sulphate nor heparin and keratan sulphate were observed.The major components of bovine gingival glycosaminoglycans were chondroitin 4-sulphate, 32–40 per cent; dermatan sulphate, 33–37 per cent; hyaluronic acid, 17–27 per cent. Heparan sulphate was present only in a limited amount. The total uronic acid content of bovine gingiva, however, decreased with age, especially during the first three years of life, possibly due to the marked decrease of both chondroitin 4-sulphate and dermatan sulphate. After 3 years of age, the decrease of these glycosaminoglycans slowed down considerably. Hyaluronic acid decreased rather slowly from the time of birth to 10 years of age, and heparan sulphate decreased initially but increased later.     

Isolation and characterization of the basic proline-rich proteins from rat parotid saliva

Five fractions of basic proline-rich proteins were isolated from rat parotid saliva, obtained by surgical cannulation of the ducts. The purification procedures employed DEAE-Sephadex to isolate a heterogeneous break-through fraction containing the basic proline-rich proteins, followed by gel filtration on Sephadex G-200 to separate the high molecular weight glycoprotein, fraction A, from the other basic proline-rich proteins which were resolved into four additional fractions, SP-1 to SP-4, by ion exchange chromatography on SP-Sephadex. The proteins differed in their amino acid composition and content of neutral and amino sugars. All the proteins were characterized by a high proportion of proline (approx. 40 mol per cent) and glycine (11–23 mol per cent). Four of the fractions were also enriched in glutamic $\text{acid}\text{glutamine}$ (19–26 mol per cent). The exception was fraction SP-4, which contained lower levels of glutamic $\text{acid}\text{glutamine}$ and has no counterpart in human basic proline-rich proteins. Fraction A, the basic glycoprotein, was heavily glycosylated (59 mol per cent), whereas SP-2 and SP-4 were less glycosylated. Fractions SP-1 and SP-3 contained low levels of neutral and amino sugars. Basic proline-rich proteins constitute a smaller percentage of the total protein in rat parotid saliva than they do in human parotid saliva (10.5 versus 40 per cent). Rat basic glycoprotein fraction constitutes less than 1 per cent whereas the human glycoprotein fraction constitutes 17 per cent. Rat basic proline-rich proteins appear to be larger and less basic than most of the human basic proteins, and they resolve into fewer protein fractions (4 versus 9) with SP-Sephadex chromatography.     

Isolation, chemical and biological characterization of sulphated glycoproteins synthesized by rat buccal and palatal minor salivary glands in vivo and in vitro

35S-labelled sulphated glycoproteins (SGP) were isolated from these glands after the incorporation of radiosulphate in vivo and in vitro by fractionation of tissue and medium extracts on Sepharose 4B and partial purification by DEAE-Sephacel anion exchange chromatography. Fractions were assessed for purity by SDS-PAGE and by cellulose-acetate electrophoresis. Molecular weights ranged from 34,000 to 5 X 10(6). It was notable that the molecular size of SGP from the in vitro media was generally lower than from the corresponding tissue fractions, particularly for the palatal samples. The fractions were heterogeneous and contained no sulphated glycosaminoglycans; they had high levels of aspartic acid, glutamic acid, threonine and serine, but there was no major difference in amino-acid composition between them. Carbohydrate analysis indicated typical components associated with sulphated glycoproteins, including fucose, galactose, glucose, mannose, N-acetylgalactosamine, N-acetylglucosamine and N-acetylneuraminic acid. Protein:carbohydrate ratios ranged from 0.1:1.0-3.5:1.0 and ester sulphate from 0.8 to 16.2 per cent. All fractions exhibited blood-group A reactivity and aggregated Streptococcus sanguis NCTC 7864; several fractions interacted similarly with Streptococcus mutans OMZ61.     

Acid diffusion analysis of different forms of fluoride in human dental plaque

Fluoride concentration was determined by: (a) extraction with diphenylsilanediol after combustion of the plaque in an oxygen bomb; (b) acid diffusion from 0.5 M HClO4 for 16 h at room temperature; (c) acid diffusion from 4.6 M HClO4 for 16 h at room temperature; and (d) acid diffusion from 18 M H2SO4 for 16 h at 60 degrees C. The total fluoride was determined by all the diffusion procedures, and there was no evidence of a large proportion of the plaque fluoride being released only after treatment with strong acid (18 M H2SO4). When approx. 10 mg of plaque was extracted three times with 0.1 ml 0.5 M HClO4, 81 per cent of the fluoride was released by one 5 min extraction. After three extractions no further fluoride was detected when the residue was diffused from 4.6 M HClO4 for 16 h at room temperature. When larger plaque samples (21-66 mg) were extracted four times with 1 ml 0.5 M HClO4, 85 per cent of the fluoride was in the first extract, and none was detected in the fourth. Treatment of the residue with 18 M H2SO4 for 16 h at 60 degrees C released a further, small amount which may constitute up to 3 per cent of the total plaque fluoride. Thus the amount of tightly-bound plaque fluoride, released only by treatment with strong acid, is much smaller than previously believed.     

Stresses in the periodontal ligament

Measurements have been made of the root areas of forty five extracted teeth. These have been used to calculate the theoretical pressures that may occur in the region of the alveolar crest and their increase with progressive loss of alveolar bone when a lateral force is applied to the crown of the tooth. Pressures generated in the periodontal ligament as a result of axial loadings have also been calculated and compared with those due to lateral loadings. The results suggest that a very rapid increase in pressures in the periodontal ligament would occur with bone losses in excess of 55 per cent in the case of lateral loadings and over 80 per cent in the case of axial loading. The implications for clinical practice are discussed.     

Chemical and immunochemical characteristics of proteoglycans in bovine gingiva and dental pulp

Proteoglycans were extracted with 4 M guanidinium chloride at 6 degrees C and purified by ion-exchange chromatography and precipitation with cetyl-pyridinium chloride. Chromatography on Sepharose CL-4B under dissociating conditions separated larger (PG1) and smaller (PG2) proteoglycans. Gingival PG2, by virtue of its amino-acid composition and the exclusive presence of L-iduronate-rich dermatan sulphate, was a proteodermatan sulphate (PDS) with a similar molecular weight to periodontal-ligament PDS. Reaction with four monoclonal antibodies to bovine skin PDS confirmed the relationship between these small proteoglycans and that of skin. Their glycoprotein cores, liberated by digestion with chondroitinase ABC, were similar in size (mol. wt = 55,000 by SDS-gel electrophoresis). Pulp PG2 had a small amount of PDS but the main component contained D-glucuronate-rich sulphated galactosaminoglycans. Similar galactosaminoglycans, which included chondroitin sulphate, characterized the larger proteoglycans of gingiva and pulp; significant amounts of L-iduronic acid-rich dermatan sulphate or heparan sulphate were not present.     

A histometric study of the effect of indomethacin and calcitonin on bone remodelling in hamster periodontitis

The action on bone remodelling of indomethacin, a potent inhibitor of prostaglandin (PG) synthesis, was determined in hamster periodontitis and compared to that of calcitonin. The two treatments reduced the extent of bone resorption considerably but not significantly (NS). The reversal phase, the intermediate step between resorption and formation, was decreased by 33 per cent (NS) by indomethacin and 75 per cent by calcitonin (p < 0.02). Bone formation was increased by 270 per cent with indomethacin (p < 0.05) and by 400 per cent with calcitonin (p < 0.03), compared with untreated animals. This exceeded the extent of bone formation activity in control animals. These data strongly suggest that PG are involved in the mechanism of bone destruction in hamster periodontitis and that PG are potent in vivo uncouplers of bone remodelling as they participate both in an increase in bone resorption and a decrease in bone formation. A partial decrease in reversal lacunae indicates that other factors, also acting as uncouplers, probably take part in the mechanism of bone destruction.     

36Cl- and 86Rb+ uptake in rat parotid acinar cells

36Cl- uptake was markedly (85 per cent) inhibited by the loop diuretics, furosemide and bumetanide. Partial replacement of Na+ in the incubation medium (from 137 to 5 mM) reduced 36Cl- uptake 30 per cent; total replacement reduced uptake to 40-45 per cent of control values. Partial replacement of K+ (from 5.8 to 1 mM) decreased 36Cl- uptake approx. 45 per cent; complete replacement resulted in minimal levels of 36Cl- uptake (less than 10 per cent of controls). Ouabain reduced 36Cl- uptake approx. 55 per cent in the absence of bumetanide, but was without effect in its presence. 86Rb+ uptake was reduced approx. 85 per cent with bumetanide; complete replacement of Cl- with I- or gluconate-, decreased 86Rb+ uptake by 55 or 40 per cent respectively. The results support the notion that the bulk of Cl- influx in rat parotid acinar cells is via a loop diuretic-sensitive Na+/K+/Cl- co-transport mechanism as may occur in other secretory epithelia.     

Human oral retention of zinc from mouthwashes containing zinc salts and its relevance to dental plaque control

Subjects using 30 mM zinc phenolsulphonate as a mouthwash retained 12 per cent of the zinc. Salivary zinc concentration was increased by using mouthwashes containing 17-35 mM zinc as the sulphate, phenolsulphonate or citrate. For 17 mM zinc sulphate or phenolsulphonate, the effect lasted 3-4 h. Zinc retained in the mouth gave visible fluorescence after rinsing with 8-hydroxyquinoline and was particularly evident on the tongue, cheek mucosa and dental plaque. The concentration of zinc in plaque was increased 13-19-fold 1 h after using 31 or 18 mM zinc phenolsulphonate. A 3-fold increase was still present 6 h later for the 31 mM mouthwash. Zinc salts inhibited acid production from [14C]-glucose in vitro by plaque at concentrations which were found in plaque in vivo after using the mouthwashes. The effect of zinc on the metabolic activity of plaque may reduce the growth rate of plaque-bacteria and so decrease plaque growth.     

The metabolism of proteoglycans and glycosaminoglycans in inflamed human gingiva

Glycosaminoglycan (GAG)and proteoglycans (PG)were extracted from both relatively uninflamed and severely inflamed human gingiva. The constituent GAG, Hyaluronic acid, dermatan sulphate and chondroitin 4' sulphate, were present in the same total amount and porportions in both tissues. In contrast the PG underwent substantial breakdown in the serverly inflamed tissue as judged by anion exchange chromatography and cellulose acetate electophoresis. The findings indicate theat whereas the GAG remain apparently unchanged during inflammation, the protein moiety of the PG is catabolised leading to a loss of structural integrity.     

Glycosaminoglycan patterns in gingival proteoglycans of rat with age.

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-, 6-, 8-, 12- and 18-week-old rats were incubated for 4 h in medium containing [3H]-glucosamine and [35S]-Na2SO4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.     

Sulphated glycoproteins in rat saliva

Intraperitoneal injection of sodium [³⁵S] sulphate into rats was followed by the rapid appearance of radioactivity in the saliva. Dialysed, concentrated saliva collected by oral rinsing during a 6 hr period after injection was fractionated on Biogel P-150 followed by ion-exchange chromatography on DEAE-cellulose. Four electrophoretically and chemically distinct radioactive sulphated glycoproteins were obtained. One glycoprotein had a sulphate content of 24.7 per cent while the remainder possessed sulphate contents in the range 3–8 per cent. The four glycoproteins differed with respect to molecular size and charge.     

Ultrastructural morphology of vascular endothelial junctions in periodontal ligament

The TEM was used to categorize vessels and their junctions in normal and tensioned rat maxillary molar periodontal ligament. In tensioned periodontal ligament mean luminal diameters of capillaries were significantly smaller (p< 0.001). Goniometer tilting of sections with apparent tight regions revealed that only 16 per cent were actual tight junctions. The other regions proved to be close junctions (85 per cent) and open junctions (4 per cent). No gap junctions were found. These findings establish that morphologically the periodontal ligament contains a microvascular bed of 'leaky' endothelium with a potentially high permeability factor.     

Glycosaminoglycan synthesis by adult rat submandibular salivary-gland secretory units

The synthesis of glycosaminoglycans (GAG) by a preparation of purified, functional submandibular-gland secretory units (acini and intercalated ducts) was examined. Such units were isolated from Sprague-Dawley rats by digestion of minced gland with hyaluronidase and collagenase followed by gentle sieving of the digest through a graded series of Teflon screens. They incorporated amino acids into exocrine proteins which could be released by stimulation with isoproterenol as in vivo, indicating their functional integrity. Secretory units, incubated for 2 h in medium containing [35S]-sodium sulphate alone or in combination with [3H]-glucosamine, were then washed, homogenized and digested in pronase. The resulting material was then sequentially digested by specific enzymic and chemical procedures and analysed by chromatography on Sephadex G-50 columns to identify the various GAG synthesized. Secretory units synthesized a GAG mixture which was 20-25 per cent hyaluronic acid, 70-75 per cent heparan sulphate, and only 3-5 per cent chondroitin or dermatan sulphates, similar to that synthesized in vivo. No GAG was present in the secretory material, suggesting that all the GAG synthesized was destined for the basement membrane or cell surface.     

The effect on intrusive tooth mobility of surgically removing the cervical periodontal ligament in monkeys (Macaca fascicularis)

The intrusive mobility of 10 teeth was recorded at half-hourly intervals under 4N loads following reflection of the gingiva in 2 animals. After 3 h, the periodontal ligament surrounding the coronal area of the root was removed with a steel bur to a depth of 4 to 5 mm; loadings were then continued. For 5 teeth, little or no change in mobility occurred. Displacement increased in the other teeth which could be accounted for by tilting, the teeth not being vertical. The residual healthy ligament seemed to withstand the small vertical loads as though the periodontium was intact, whereas cutting the ligament, mesially and distally in a previous experiment, but not removing it, substantially weakened the supporting mechanisms.     

Stimulation of collagen and glycosaminoglycan production by phenytoin 5,5-diphenylhydantoin in monolayer cultures of mesenchymal cells derived from embryonic chick sternae

Cultures grown with or without phenytoin (PHT) at a concentration of 5 micrograms/ml from the fifth to the eighth day after plating were labelled with [14C]-proline (0.2 microCi/ml) from the sixth to eighth day. Collagenase digestion indicated that collagen content increased approx. 2-fold after PHT exposure. Increases in sulphated glycosaminoglycan product in response to PHT were approx. 1.5-fold; PHT also stimulated protein production. Both actinomycin D and cycloheximide blocked incorporation of [3H]-leucine, [3H]-proline, and H2(35)SO4 approx. 90 per cent with or without PHT. Continuous sucrose density-gradient fractionation indicated that PHT produced quantitative but not qualitative changes in cellular RNA.     

A quantitative chemical study of glycosaminoglycans in the articular disc of the bovine temporomandibular joint

Glycosaminoglycans were prepared from the disc by digestion with papain. The disc contained 5% of its dry weight as glycosaminoglycan. Fractionation by ion-exchange chromatography, followed by precipitation at varying concentrations of ethanol, together with chemical and enzymatic analyses, showed this glycosaminoglycan to consist of approx. 5% hyaluronic acid, 14% dermatan sulphate, 79% chondroitin sulphate and 2% keratan sulphate. Disaccharides obtained from the chondroitin sulphate were 75% 6-sulphated, 21% 4-sulphated and 4% non-sulphated. Chemical analysis showed a low average degree of sulphation of chondroitin sulphate. Immunohistochemical staining of sagittal sections of the disc showed chondroitin sulphate to be distributed throughout, whereas dermatan sulphate (as proteodermatan sulphate) appeared to be concentrated in the periphery.     

Organ culture study of effect of vitamin-A-deficiency on rat third molar development

A culture procedure for rat third molars suitable for nutritional-developmental studies is described. Unerupted third molars from 12-day-old rats were cultured in BGJb media containing 20 per cent rat serum and supplemented with 25 mM HEPES buffer, 25 mg ascorbic acid, 20 mg L-glutamine, 12 mg penicillin G and 10 mg streptomycin sulphate per 100 ml of media. Molars were cultured at the liquid-gas interphase using a 50 per cent O2, 45 per cent N2, 5 per cent CO2 gas mixture at 10 lb-psig (pounds per square inch guage). Molar cultures were maintained successfully for 9-14 days without evidence of necrosis, although they developed at a slower rate than in vivo. Molars cultured in 50 per cent O2 compared to those cultured in 21 per cent O2 for periods of 2, 4, 6 and 8 days had higher values for protein, alkaline phosphatase (AP), Ca, P and Ca/P. Vitamin-A-deficiency gave lower values for AP, Ca, P, Ca/P, 45Ca, 35S and [14C]-proline uptake. Histologically, A - molars had atrophic ameloblasts, some foci of squamous metaplasia and abnormal keratin formation. Thus, deficiency of vitamin A imposed during in-vitro development of rat third molars retarded dentinogenesis and interfered with early mineralization of enamel and dentine.     

Surface area and pore size analysis for human enamel and dentine by water vapour sorption

The water sorption apparatus described allowed water sorption isotherms to be plotted on several samples simultaneously, with the added possibility of recording their nuclear magnetic resonance spectra for any points on the isotherm. Water sorption showed that human dentine had a saturated water content (removable in vacuo at room temperature) of approx. 10 per cent w/w (21 per cent v/v), probably in micropores or associated with the organic phase, and a specific surface of approx. 150 m2/g. A similarly measured water content for outer enamel was approx. 0.9 per cent w/w (2.7 per cent v/v), with a specific surface of 5.5 m2/g. For whole enamel, the slightly larger values of 1.15 per cent w/w (3.4 per cent v/v) and 8.7 m2/g were obtained. Results are shown to depend upon the conditions of measurement. Pore-size distribution analysis of enamel and dentine by water sorption, in this study and in investigations by others, is shown to be subject to many uncertainties and of doubtful value. Some uncertainty is also believed to apply to surface area measurements.