A histochemical study of glycosidases in hydantoin induced hyperplastic, healthy and inflamed human gingiva

Fresh samples of hydantoin induced hyperplastic, healthy and inflamed human gingiva were studied histochemically using various azo dyes for β-glucuronidase (EC 3.2.1.31), for β- and β-glucosidase (EC 3.2.1.20 and 3.2.1.21) as well as β-galactosidase (EC 3.2.1.23) and for N-acetylglucosaminidase (EC 3.2.1.30), in order to add support to the hypothesis that hydantoin induced hyperplasia is always connected with inflammation.
Moderate β-glucuronidase activity was observed in healthy, inflamed and hydantoinhyperplastic gingiva. The distribution of the enzyme activity was similar in all types of the tissue except the stratum corneum. The healthy gingiva did not reveal this activity whereas the inflamed and hydantoinhyperplastic gingiva did. The stratum basale and spinosum of the epithelium, the fibroblasts and the inflammatory cells, especially the macrophages, revealed enzyme activity in all types of tissues. In the healthy tissue only a few inflammatory cells were seen and thus the β-glucuronidase activity was low when compared to inflamed or hyperplastic gingiva.
Weak β-galactosidase, N-asetylglucosaminidase and β-glucosidase activity was seen in all types of gingival samples. Enzyme activity was observed in the same structures as β-glucuronidase with the exception of the stratum corneum, which revealed no activity.
The relatively strong β-glucuronidase activity in the keratinized cell layer of the epithelium of inflamed and hydantoinhyperplastic tissue may be due to the microbial enzyme diffusion into the keratinized cell layer of the injured tissue.     

Subcellular distribution of oxidative enzymes in human, inflamed and dilantin hyperplastic gingiva

The subcellular distribution of cytochrome oxidase (mitochondrial), NADH cytochrome c reductase (nuclei, mitochondrial, microsomal, soluble) and NADPH cytochrome c reductase (microsomal, soluble) marker activities were analysed biochemically in human inflamed and dilantin hyperplastic gingiva. Protein and enzyme distribution patterns were found to be similar in the inflamed and dilantin hyperplastic gingival subcellular fractions. Cytochrome oxidase was found primarily within the mitochondrial fraction, and NADPH cytochrome c reductase was found chiefly within the soluble fraction. NADH cytochrome c reductase distributed in each subcellular fraction, and was found within the nuclear, mitochondrial, microsomal and soluble fractions.Significantly greater activity (cytochrome oxidase, NADH cytochrome c reductase and NADPH cytochrome c reductase) was noted in mildly inflamed dilantin hyperplastic tissue than was observed in mildly to moderately inflamed gingiva. NADH cytochrome c reductase (mitochondrial) was significantly greater than cytochrome oxidase in both tissue fractions, and was rotenone, amytal and antimycin a, insensitive which suggests that the activity may be primarily associated with the outer mitochondrial membrane. The increased specific activities of the enzyme markers in dilantin gingiva may reflect metabolic activity associated with cell mitosis, proliferation, protein (collagen) synthesis and drug detoxification.     

Biogenic amines in human gingiva in healthy and inflamed states

As inflammatory disturbance are of significance in every aspect of periodontal disease, it was deemed pertinent to conceive on experimental study exploring the existence and relationship of biogenic amines, at least in the inflammatory gingiva. Gingival samples from 50 human individuals representing varying grades of inflammatory involvement have been utilised in the present work. From the results of this study, it could be elucidated that biogenic amines (noradrenaline, dopamine and 5-hydroxytryptamine) should show elevated concentration in inflammatory states of the gingiva. Further, these amines turnover was confirmed by studying monoamine oxidase which is a catalyzing enzyme of noradrenaline and 5-hydroxytryptamine; and 5-hydroxy indole aceticacid is a metabolic end product of 5-hydroxytryptamine. This was of a transitory nature indicating increased levels at the early stages of inflammation followed by a decrease at the peak of the gingival inflammation. It is assumed that biogenic amines helps in regeneration of connective tissue of the oral mucosa during the initial development of inflammation rather than final stages of the process, thereby emphasizing its transitory role in the inflammatory process.     

Cholinergic components in human gingiva in healthy and inflamed states

Acetylcholine (ACh) and acetylcholinesterase (AChE) are main components in cholinergic nervous system. ACh is a natural constituent of many parts of the nervous system and its chief role is neurotransmission. It is not entirely unique in function to the cholinergic tissues of the human body. Gingiva is the part of the oral mucosa which contains numerous mast cells. They contain a variety of biologically active substances including neurotransmitters such as 5-hydroxytryptamine, histamine etc. In the dental literature accessible to authors no data were found on ACh and AChE in the different oral structures in health and inflamed conditions. Therefore gingiva samples from 50 human individuals representing varying grades of inflammatory involvement have been utilised in the present study. ACh and AChE were estimated in the gingiva tissues by flurometric and spectrophotometric methods. This study established hithero unknown "norms" for the ACh and AChE contents of the clinically normal gingiva, which are found to be 0.85 +/- 0.06(SE) ug/g and 210 +/- 18(SE) micromoles ACh hydrolysed/hr/gm/wet tissues. Results also revealed that the range of variations of ACh is high and AChE is low in all the inflamed states of gingival tissues.     

Collagenase activities in healthy and inflamed gingiva of dogs

The effects of inflammation on collagenase and gelatinase were studied in dog gingiva. Inflammation was induced by the placement of cervical ligatures. Collagenase activity was measured using soluble [¹⁴C]-acetylated collagen with high specific activity as substrate and separating the reaction products by 50% dioxane (Terato et al. 1976). Two forms of collagenase, a soluble enzyme free from the substrate and an insoluble enzyme bound to the substrate, were extracted by neutral salt solution and by sequential sonication in low and high salt buffers, respectively. Treatment with a high concentration of trypsin was necessary for detection of collagenase activities in both extracts. The collagenase activities in the neutral salt extract and the first sonic extract were significantly higher in inflamed gingiva after 1 week of plaque formation than in healthy gingiva, but that in the second sonic extract showed no significant increase. After 3 weeks of plaque formation, the activities in neutral salt extract and the first sonic extract of inflamed gingiva similarly showed significant increases relative to controls. On the other hand, gelatinase activity did not vary significantly with the induction of inflammation.     

Some histological and histochemical observations on the connective tissue of chronically inflamed human gingiva

This investigation was undertaken during the tenure of a Research Fellowship in the Institute of Dental Surgery, University of London, and a Leverhulme Research Fellowship in the Department of Dental Science, Royal College of Surgeons of England. Some of the findings were included in a Thesis submitted to the University of London for the Degree of Doctor of Philosophy.
One hundred and fifty biopsies of human gingiva, removed randomly from patients undergoing gingivectomy, were examined in the light microscope. Cold microtome and paraffin sections were prepared. Eosin, Mallory's PTAH, acid solochrome cyanine and chlorantine fast red, the Gomori aldehyde fuchsin, resorcin fuchsin and orcein methods for elastic fibres, and silver impregnation were used to colour collagen fibres; fibrin was stained by the DMAB method, and mucosubstances by the paS, DMPD, alcian blue and the aldehyde fuchsin methods. All the interdental gingival septa exhibited an ulcerated col. The intercellular substance of the connective tissue was not separated in cold microtome sections, as in paraffin sections, and probably more faithfully reflected the state of the tissue in vivo . However, cell detail in paraffin sections was clearer than in cold microtome sections. The collagen fibres in inflamed connective tissue were found to disintegrate and to lose their affinity for stains with which they usually react, but were not coloured by stains for elastic fibres during the process, nor by the DMAB method. The distribution of neutral and acid mucosubstances was found to overlap in healthy connective tissue. Neutral mucosubstances became less reactive in inflamed areas, but the reactivity of acidic mucopolysaccharides, which could also be demonstrated by two of the three stains used for elastic fibres, increased at the periphery of these areas, and was thought to be associated with fibrogenesis.     

Microvascular volumes in healthy and inflamed gingiva in humans

It has been suggested that the vascular alterations found in inflamed gingiva may be of significance in the enhanced extension of the pathological process into the periodontium. The purpose of this investigation was to measured the changes in blood vascular volume occurring in gingiva with the onset of gingivitis and its resolution. Twenty-six individuals participated in this study. Gingival biopsies were taken following a 21-day experimental gingivitis, following resolution of a 21-d experimental gingivitis and during a 6-month experimental gingivitis and a 6-month period of optimal oral hygiene. A total of 126 biopsies was obtained, from which 378 sections were cut at 2 microns for stereological analysis. At low magnification, reference volumes were estimated using point counting procedures and expressed as mm3 of gingiva per mm length of vestibular gingiva, in a vestibulo-lingual plane. At higher magnification the ratio between the volume of vessels and connective tissue was calculated. The final results were expressed as mm3 of vessels per mm length of vestibular gingiva, in a vestibulo-lingual plane. The mean vessel volume expressed per unit length of vestibular gingiva ranged from 0.010 to 0.024 mm3/mm. No statistically significant differences in vascular volumes were found between inflamed and non-inflamed gingiva. It was concluded that the changes in vascular architecture during early gingivitis described in the literature had either taken place in the subjects prior to the time of experimentation or that any vascular changes (cytologic or functional) which had taken place may be compensatory for the changes in architecture described in the literature.     

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.     

Metabolism of testosterone by human healthy and inflamed gingiva in vitro

Testosterone was incubated with homogenate and mitochondrial, microsomal and soluble-fraction preparations of healthy and inflamed gingiva in human subjects of both sexes. In the inflamed preparations, the metabolic activity was higher than in the samples from healthy gingiva. The gingiva contained the following enzymic activities: Δ⁴-5α-steroid hydrogenase, Δ⁴-5β-steroid hydrogenase, and 3α-, 3β- and 17β-hydroxysteroid dehydrogenase, principally in the soluble fraction. Mitochondrial and microsomal preparations were less active. Both the healthy and especially the inflamed samples were able to convert testosterone to 17/gb-hydroxy -5α-androstan-3-one, suggesting that gingiva may be a target tissue for androgens.     

Cathepsin D, L and B in inflamed human gingiva

Cathepsin D, cathepsin L, and cathepsin B activities were detected in homogenates of human gingival tissue from 20 patients with different degrees of periodontal disease and in one patient with juvenile periodontitis. Clinical plaque index (PI), gingival index (GI), and pocket depth were determined. Patients were divided into three groups with regard to the severity of the disease. Averaged values for the activities of all three lysosomal proteinases increased from the group with less damaged tissue to the group with more advanced periodontal disease. When the correlations between the specific activities of the cathepsins and pocket depth in each of 20 patients were considered by the linear regression analysis, the correlation coefficient for cathepsin D was much higher than for the other two cysteine proteinases. Cathepsin D was also extracted from the homogenates by specific immuno-adsorbtion on anticathepsin - D antibody - Sepharose and quantitatively determined; the average amount of enzyme ranged from 118.5 to 184.5 μg per mg of total protein extracted from the tissues and was positively correlated to pocket depths.     

Topical distribution of FcgRI, FcgRII and FcgRIII in inflamed human gingiva

The topical distribution of Fc gamma receptor types I, II and III (Fc gammaRI-III) was analyzed by means of immunohistochemistry in human gingival tissue obtained from 12 patients with chronic periodontitis. CD68+ macrophages expressing all three classes of Fc gammaR were found throughout the whole gingival connective tissue (CT), whereas dense infiltrates of polymorphonuclear granulocytes (identified by staining for neutrophil elastase) with strong staining for Fc gammaRIII and Fc gammaRII were found subjacent to the apical part of the pocket epithelium (PE) and in the PE itself. CD19+ B lymphocytes with variable staining intensity for Fc gammaRII were observed in clusters subjacent to the PE and extending into the central part of the CT. Only a few scattered CD3+ T lymphocytes stained for Fc gammaRIII. Some spindle-shaped cells (CD68-, therefore non-macrophages) and apparently non-cellular fibrous tissue elements stained for Fc gammaRI and Fc gammaRII. In the epithelium, Fc gammaRII+ dendritic cells were frequently observed in the entire oral gingival epithelium and in the coronal part of the PE. Occasionally, some keratinocytes which stained for Fc gammaRII and Fc gammaRIII were found. The observations indicate that Fc gammaR of the various classes are amply expressed on numerous cell types in inflamed gingival tissue. The specific distribution pattern detected suggests that Fc gammaRs may play a role in the mediation of chronic inflammation in the periodontal lesion.