Accessible sialic acid content of oral epithelial cells from healthy and gingivitis subjects.

The purpose of the present study was to determine if there were differences in the quantity of accessible sialic acid on superficial epithelial cells collected from different areas of the mouth, and from healthy subjects with good oral hygiene, as compared to subjects with gingivitis. Superficial epithelial cells were collected by gently scraping the tongue dorsum, hard palate, free gingiva and buccal epithelium. The cells were washed and treated with clostridial neuraminidase to release accessible sialic acid; this was quantitated using a fluorometric assay. Buccal cells released an average of 62.6 ng sialic acid per 10,000 cells, which was nearly 3-fold more than cells from the hard palate (24.1 ng), free gingiva (21.9 ng), or tongue (15.4 ng). Buccal and free gingival cells collected from 5 healthy subjects had significantly higher levels of accessible sialic acid on their surface than cells collected from 5 subjects with gingivitis. These differences were significant at the p less than 0.001 and p less than 0.01 levels, respectively. The data obtained suggest that the oral hygiene status of an individual can influence the quantity of accessible sialic acid residues on oral epithelium; this would be expected to influence the attachment and colonization of bacteria which bind to sialic acid-containing receptors.

Hemoglobin concentration and oxygen saturation of clinically healthy and inflamed gingiva in human subjects.

The hemoglobin concentration (Hb index) and oxygen saturation (apparent SO2) in human gingiva were estimated by tissue reflectance spectrophotometry (TRS). The gingiva had significantly lower Hb index and higher apparent SO2 than those in alveolar mucosa, but there was no difference in either parameter among different gingival areas. The reproducibility in repeated measurements was high for both Hb index and apparent SO2 in gingiva. In inflamed gingiva, Hb index was significantly higher than that in clinically healthy gingiva. A lower apparent SO2 was observed in inflamed gingiva. This suggests that the increase in blood supply is insufficient to meet the oxygen demand in inflamed gingiva. There were significant correlations between either the Hb index or the apparent SO2 and the clinical parameters of gingival inflammation such as gingival index, plaque index, Periotron score and probing depth. Thus, TRS may be clinically available to estimate the blood volume and oxygen saturation in inflamed gingiva.

Histochemical localization of hyaluronate in human oral epithelium using a specific hyaluronate-binding probe.

Biochemical data suggest that gingival epithelium contains hyaluronate, but there is little histochemical information about its localization. Hyaluronate was here visualized in gingival and buccal mucosa using a specific probe derived from the hyaluronate binding region of cartilage proteoglycan. Hyaluronate was found both in the gingival and buccal epithelium, but its localization was correlated with the type of keratinization. In the keratinized epithelium of gingiva, whether ortho- or parakeratotic, the intercellular spaces from basal to upper spinous layers displayed strong staining, most intense in the middle spinous cell layer. The uppermost vital cell layers as well as the cornified cell layer remained unstained. In the non-keratinized epithelium of buccal mucosa and the local non-keratinized areas of gingiva, only the basal cells and the lowermost spinous cell layers stained for hyaluronate, whereas the majority of the upper epithelium was negative. Electron microscopic examination of the basal and spinous cell layers displayed hyaluronate, both associated with the cell surface and free in the intercellular space. The subepithelial connective tissue showed positive but diffuse staining in all specimens.

Behaviour of adenylic and pyridinic compounds in gingival tissue after a short-term exposure to air.

Biochemical variations of adenine and pyridine compounds in human gingival grafts during the period between excision and implantation have been studied. These groups of compounds are considered as "indicators" of the metabolic and energetic status of the living cells. Adenylic compounds such as ATP, ADP and AMP are involved in numerous metabolic processes as "modulators" of allosteric enzymes. NAD+ and NADP+ are involved in the carbohydrate metabolism as co-factors of many reactions of oxydoreduction. The exposure to air of the gingival tissue induces modifications in the energy state of the cells as well as in the ox-reox system. No variation is detectable in the intermediates of the pyridine compounds cycle.

[Concentration of thromboxane B2 in gingival tissues in relation to main clinical parameters of periodontal health]. / La concentrazione del trombossano B2 nei tessuti gengivali in relazione ai principali parametri clinici di salute parodontale.

Considering the role played by some metabolites of Arachidonic Acid in the pathogenesis of periodontal disease, the Authors have investigated on the existence of a relation between the Thromboxane B2 concentration in gingival tissues and the principal clinical Indices of periodontal health: Gingival Index, Plaque Index, pocket depth, and Bleeding Time Index. The results showed that just the bleeding Index resulted to be well related to the Thromboxane B2 tissue concentration and then to the level of inflammation of deep periodontal tissues. On the ground of these data the Authors underline the importance held by the bleeding index in the showing to the Clinician the therapy necessity of a periodontal site in order to prevent a possible future attachment loss.

Immunohistochemical localization of chondroitin sulfate and dermatan sulfate proteoglycan in human gingival connective tissue.

This study investigated the immunohistochemical localization of chondroitin sulfate (chondroitin, 4-sulfate and 6-sulfate) and dermatan sulfate proteoglycan (PG) in human gingival connective tissue, using monoclonal antibodies. Dermatan sulfate was found to be widespread in connective tissue, with an especially strong response shown in collagen fiber bundles under the epithelial basement membrane. Chondroitin 4-sulfate occurred widely in connective tissue but showed only a weak response. Chondroitin 6-sulfate was located in peripheral blood vessels. Chondroitin was not detected in gingival connective tissue.

Prostaglandina E2 (PgE2) en encía normal e inflamada: ensayo clínico; revisión bibliográfica / Prostaglandin E2 (PgE2) in normal and inflamed gingiva: clinical assay; bibliographic review

Se analiza el papel de las prostagladinas (Pg) en la patogenia de la enfermedad parodontal, para lo cual se tomaron 92 muestras de encías a igual nùmero de pacientes (48 femeninos y 44 masculinos) cuyas edades oscilaron entre 17 y 74 años de edad. Se clasificaron clìnicamente segùn el índice gingival de Löe. La presencia de prostaglandina se realizó según el método de cromatografía en capa delgada. Se relacionó la presencia o ausencia de PgE2 con el sexo, edad, índice gingival de Löe y el grado de infiltrado inflamatorio. Encontramos que aunque la correlación entre el índice gingival de Löe con el grado de infiltrado inflamatorio no se corresponden exactamente, el primero brinda una útil orientación clínica inicial. En los pacientes con mayor grado de inflamación se correspondieron mayores concentraciones de PgE. Se plantea la posibilidad del uso de drogas inhibidoras de prostaglandina en el tratamiento de la enfermedad parodontal

Immunohistochemical study of neurochemical markers in gingiva obtained from periodontitis-affected sites.

Immunohistochemical methods have been used to study the occurrence of neuronal markers in human gingiva from periodontitis-affected sites. In periodontitis-affected buccal gingiva densely distributed neurofilament (NF)-immunoreactive (IR) fiber bundles were observed in the deeper parts of the propria, while NF-IR single fibers occurred in the superficial propria and occasionally in the buccal epithelium. Periodontitis-affected gingiva obtained from interproximal sites showed only sparsely distributed NF-IR fibers. Single nerve fibers immuno-reactive to the peptides substance P and calcitonin gene-related peptide occurred close to or within the epithelium in both buccal and interproximal gingiva. Around blood vessels neuropeptide Y-, peptide histidine-isoleucine amide- and vasoactive intestinal polypeptide-IR fibers were occasionally observed, while clusters of gamma-melanocyte-stimulating hormone-IR cells were found in the propria, in addition to gamma-melanocyte-stimulating hormone IR nerve fibers. Somatostatin-IR dendritic cells were seen in epithelium and propria of buccal and interproximal gingiva, although a high variability in the number of SOM-IR cells was observed. All neuronal markers studied showed a similar distribution in material obtained from young patients with clinically healthy gingivae, although the number of NF-IR fibers in the propria in these subjects was lower. The results demonstrate that in gingiva obtained from periodontitis-affected sites several different biologically active peptides occur in both nerve fibers and cells. At least some of these substances could possible play a role in the inflammatory process. However, since clinically normal gingiva was shown to contain nerve fibers and cells expressing immunoreactivity to the substances studied, no unique periodontitis-induced expression of the neuronal markers studied was found. Thus, any alteration of these substances during the periodontitis process remains to be elucidated.

[Prostaglandin E2 (PgE2) in normal and inflamed gingiva. Clinical assay. Literature review]. / Prostaglandina E2 (PgE2) en encia normal e inflamada. Ensayo clinico. Revisión bibliografica.

The role of prostaglandins (Pg) in the pathogeny of periodontal disease is analyzed. For that purpose, 92 samples of gingivae were taken to the same number of patients (48 women and 44 men) aged 17-74 years, and were clinically classified according to Löe gingival index. Presence of prostaglandins was determined by thin layer chromatography. Presence or absence of PgE2 was related to sex, age, Löe gingival index and degree of inflammatory infiltrated. Although correlation between Löe gingival index and degree of inflammatory infiltrate has not an exactly correspondence, the first one offers a useful initial clinical orientation. To patients with higher degree of inflammation corresponded higher PgE2 concentrations. Possibility of using prostaglandin inhibiting drugs in the treatment of periodontal disease is stated.

Método técnico para la obtención y recuento de leucocitos en el surco gingival / Technical method for the attainment and counting of leukocytes in the gingival sulcus

Se presenta una nueva técnica de recuento celular que permite determinar la concentración de los leucocitos en el interior del surco gingival. Para ello se utiliza un método de extracción del líquido del surco en el cual se utilizan micropipetas. La concentración de leucocitos se determina mediante la aplicación de la técnica de la cámara de New Bauer. Este método ya ha sido aplicado con éxito en 2 trabajos de investigación

Histochemical localization at the electron microscopic level of sulfated glycosaminoglycans in the rat gingiva.

Using the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique, we investigated ultrastructural localization of sulfated glycosaminoglycans (GAGs) in the rat gingiva shortly after eruption, especially those associated with internal and external basal laminae. In the apical portion of the internal basal lamina, HID-TCH-SP stain deposits were distributed mainly in the region of the lamina lucida located between the lamina densa and the distal surface membrane of the junctional epithelium and inside the depression of the distal surface membrane adjacent to the basal lamina. Stain deposits were also detected on the surface membrane of the cytoplasmic protrusion. Interestingly, the density of HID-TCH-SP stain deposits in the internal basal lamina was highest in the apical portion of the junctional epithelium and decreased in the coronal direction, finally tending to disappear completely. On the other hand, in the external basal lamina the deposits were localized in the whole region of the basal lamina or at both sites of the lamina densa. HID-TCH-SP stain deposits were also detected external to the lamina densa in the basement membrane associated with capillaries and in the connective tissue where they were distributed in close relation to collagen fibrils. Testicular hyaluronidase digested most HID-TCH-SP stain deposits in the connective tissue, whereas those in the region of basement membranes resisted this enzymatic digestion.

A method for the refinement of data arising from electrophoresis of biological fluids.

Gingival crevicular fluid (GCF) is being analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) in our laboratory. We wish to characterize the major protein species in GCF and to investigate their association with the progression of disease. Laser densitometry of SDS/PAGE provides estimates of log molecular weight (LMW) and relative abundance for each band in each sample. Our aim was to develop a method for the refinement of these data into clusters which could be treated as distinct protein species, whose relation to disease progression could be assessed. A reproducibility study showed that the estimated LMW would fall within 3% of the true LMW 95% of the time. The method clusters the estimated LMWs of each band in each sample so that clusters form at LMWs which occurred frequently. The data from each band in each sample are thus refined into cluster number and relative abundance. Application of the technique to data from the reproducibility study showed that clusters formed around the individual components in the mixture, with little misclassification. The technique was then applied to two data sets: from SDS/PAGE of 104 GCF samples from 74 adolescents without progressive periodontal disease, and from 2 patients suffering from advanced progressive disease. The clusters appeared accurately to reflect the appearance of the gels, and clear differences were observed between the two data sets. The method will enable changes in the composition of biological fluids to be associated with external factors such as disease status and should be widely applicable.

Immunohistochemical distribution of keratin proteins in clinically healthy human gingival epithelia.

In clinically healthy/subclinically inflamed biopsies of marginal gingiva, the immunohistochemical distribution of keratin proteins was studied in junctional (JE), sulcular (SE), oral gingival (OGE) and in a few samples of alveolar mucosal epithelium (AE) by means of various mouse monoclonal anti-keratin antibodies in an indirect fluorescence technique. All regions stained in a nearly similar way with AE3 (keratins 1-8, all cells) and BE14 (keratin 5, basal and supra/parabasal cells). AE8-staining (keratin 13, supra/parabasal and spinous cells) was primarily confined to the stratified, nonkeratinized epithelia SE and AE, but also a variable part of JE and less frequently OGE were positive. The parakeratinized OGE was distinct in showing a homogeneous staining with AE2 (keratins 1/2, 10) and AE5 (keratin 3) throughout spinous cell layers. These antibodies did not stain JE and AE whereas SE stained in a scattered way with AE5 and sometimes also with AE2. The latter finding might indicate initial keratinization at molecular level. The JE was distinct in retaining basal characteristics throughout the epithelium with PKK2 (keratin 7, 16, 17, 19) and BE14 (keratin 5) although some initial suprabasal maturation, as observed with AE8, cannot be excluded. Differences in keratin staining of gingival epithelia and the AE was found with respect to AE1-reactivity (keratins 10, 14-16, 19) which was suprabasal in JE, SE and OGE but basal in AE.

The distribution of Langerhans cells and CD1a antigen in healthy and diseased human gingiva.

The distribution of CD1a antigen in gingival epithelium of clinically healthy gingiva was examined and compared with the distribution in gingival epithelium of adult periodontitis lesions. Cryostat sections were examined with monoclonal antibodies to CD1a antigen using the ABC immunoperoxidase technique. In healthy gingiva, CD1a was limited to Langerhans cells (LC) which were observed throughout the length of the external epithelium and orosulcular epithelium. The numbers of LC expressed either per unit length of orosulcular epithelium or per mm2 were similar to the numbers in external gingiva. Junctional epithelium contained few if any dendritic LC. The numbers of LC in pocket epithelium of adult periodontitis lesions were significantly lower compared with orosulcular epithelium of healthy tissue and compared with external gingiva of diseased tissue (p less than 0.005). In many sections, no LC were identified in pocket epithelium. In 5 of 8 adult periodontitis sites, CD1a was also observed in association with the membranes of suprabasal keratinocytes in external and pocket epithelium in areas where no LC were identified. These findings provide further evidence that changes in gingival epithelial cells occur in periodontal disease which are analogous to those documented in dermatological diseases and suggest that epithelium may play a role in gingival homeostasis and in the pathogenesis of periodontal diseases.

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application.

We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome. The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.

A biochemical study of glycosaminoglycans in the palatal rugae of the monkey (Macaca fascicularis).

Glycosaminoglycans (GAG) were extracted from the connective tissue of the palatal rugae, separated by electrophoresis and compared with the results obtained for the remaining palatal mucosal and gingival connective tissues. The GAG content of the rugae (3.01 mg/g defatted dry weight) was higher than in the remaining palatal mucosa (2.33 mg/g defatted dry weight) or gingiva (1.68 mg/g defatted dry weight). Dermatan sulphate was the predominant GAG in both the palatal rugae (48% of total GAG) and the remaining palatal mucosa (50%) followed by hyaluronic acid (33 and 31% respectively). The results do not support previous histochemical observations in which the rugae appeared to be rich in hyaluronic acid.

Immunohistochemical study of types I, III and IV collagen in diseased human gingiva of patients with rapidly progressive periodontitis: a light and electron microscopic study.

The distribution of type I, III and IV collagens and their ultrastructural organization have been studied in diseased gingival connective tissue of patients with rapidly progressive periodontitis. This disease is characterized by acute destruction of the gingival collagenous components. The use of an immunofluorescent procedure has shown that the diseased connective tissue was made up of both type I and III collagens but that type III collagen was less resistant to acute inflammation. Ultrastructural immunolabelling, using the peroxidase procedure has shown that the large, dense bundles of type I collagen of PI, the main pattern of organization of the gingival connective tissue offered a better resistance to acute destruction than PII, a loose pattern of organization mainly composed of type III collagen. Type IV collagen was exclusively located in degraded lamina densa of basement membrane.

Miokamycin penetration into oral cavity tissues and crevicular fluid.

Diffusion of miokamycin into gum, maxillary-mandibular bone and crevicular fluid was studied in human beings. The antibiotic concentrations were determined in specimens at different times after oral administration of 600 mg in a single dose of miokamycin. Peak serum levels (2.32 +/- 0.67 mcg/ml) were found at the first hour after dosage. In healthy gum tissue the highest antibiotic levels (1.44 +/- 0.34 mcg/gr) were observed at the second hour, while in the inflamed gum miokamycin penetrates more rapidly, being, as in serum at the highest levels detectable during the first hour. In the bone of the maxilla or mandible the highest levels of miokamycin (0.88 +/- 0.13 mcg/gr) were detected at the second hour after treatment. In the crevicular fluid miokamycin showed a similar profile as that in serum, since the peak levels were reached at the first hour (2.4 +/- 0.88 mcg/ml, but the decrease of the antibiotic occurred more slowly than in serum. Miokamycin rapidly penetrates into tissues and fluids of oral cavity. A single oral dose of 600 mg guarantees antibacterial levels against susceptible bacteria over six hours.

A reevaluation of the distribution of 3H-proline in the transseptal ligament of the mouse in radioautography.

It is generally accepted that there is uniform collagen metabolism within the periodontal and transseptal ligaments. The present study suggested regional variations in the incorporation and removal of 3H-proline within the transseptal ligament in radioautography, suggesting variable rates of collagenous protein remodeling coincident with physiological tooth movements. Highest numbers of silver grains were over the middle third of the ligament during both incorporation and removal phases (p less than 0.001). Rates of grain removal were greater in the middle than in mesial or distal thirds (p less than 0.001). The half-life of labeled proteins was significantly less in the middle than in mesial or distal thirds (p less than 0.005). Because there were no significant regional differences in cell numbers, regional variability in grain incorporation and removal within the transseptal ligament likely indicates regional differences in cellular synthetic or degradative activity coincident with remodeling of the transseptal ligament during physiological drift and suggests that the center of this ligament may experience more stress and, thus, remodels more rapidly.

The detection of platelet-activating factor in inflamed human gingival tissue.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a biologically active lipid, like the prostaglandins, which mediates allergic and inflammatory reactions. Aggregation of washed rabbit platelets was induced by a lipid prepared from inflamed gingiva. The mobility of the active lipid was coincident with that of authentic PAF on thin-layer chromatography. The aggregation was dose-dependent and inhibited by pretreatment with a specific PAF antagonist, ONO 6240, but not by indomethacin or creatine phosphate/creatine phosphokinase, which inhibit the platelet aggregation due to arachidonic acid or ADP, respectively. Thus the active lipid was identified as PAF; the amount of PAF detected was 118.1 +/- 79.7 pg/50 mg tissue (n = 6, mean +/- SD), the amount in normal tissue being 13.0 +/- 11.3 pg/50 mg tissue (n = 6). There was therefore a significant difference between the tissues. Lyso PAF, the metabolite of PAF with acetylhydrolase, was not detectable in either gingival tissue. Thus PAF was produced more in inflamed gingival tissue than in normal tissue; PAF may be involved in the occurrence and maintenance of periodontal disease.