Connective Tissue Infiltration Research Articles

Stereology of Inflammatory Connective Tissue Infiltrates in Oral Mucosa

In the present summary, different approaches to analyse inflammatory connective tissue infiltrates are reviewed. The most appropriate model for this purpose was found to be the stereological analysis at the electron microscopic level. This method furnishes data comparable with results obtained by other quantifying methods, such as biochemical analysis. The presently used methodology for the analysis of diseased portions of oral mucous membranes, as well as selected results thereof are described. The usefulness and reliability of the method are discussed and future developments are suggested.

Juvenile periodontitis

The microflora of periodontal pockets and some histopathological characteristics of adjacent tissues from individuals with clinical signs of juvenile periodontitis were studied. Adult individuals with ordinary, rapidly advancing periodontitis served as controls. All of the patients had been referred for specialist treatment. Using criteria defined by Baer (19z1), the patients were assigned to one juvenile periodontitis (eight patients), one post-juvenile periodontitis (seven patients) and one adult periodontitis group (seven patients). Only lesions around the first molars and central incisors were studied. Bacterial samples were obtained from each site and were examined by darkfield microscopy and different morphological forms identified. The soft tissue of the diseased sites was excised. The biopsies were placed in a fixative, cut in 1-mm-thick blocks and embedded in Epon. In semithin sections the infiltrated connective tissue (ICT) was identified and the ICT portion further processed for electron microscopy. The numeric and volumetric densities of different cells and structures of the ICT were determined using a morphometric point-counting procedure. The results showed that juvenile periodontitis lesions are associated with a subgingival microflora, the composition of which is different from that of adult periodontitis. Thus, in patients belonging to the adult periodontitis and post-juvenile periodontitis groups, motile microorganisms dominated the subgingival plaque samples, whereas deep pockets in juvenile periodontitis lesions contained a flora dominated by coccoid cells and straight non-motile rods. The most pronounced difference in the composition of ICT between the juvenile on one hand and the post-juvenile and adult periodontitis lesions on the other, was related to the amount of extracellular structures. Thus, in post-juvenile and adult periodontitis lesions, collagen and residual tissue made up around 50% of the infiltrate. In the juvenile periodontitis lesions, however, extracellular structures occupied only around 20% of the volume. In the ICT of the juvenile periodontitis sites, around 70% of the volume was occupied by plasma cells and blast cells. The corresponding figures for the post-juvenile and adult periodontitis sites were 50 and 30%.

Experimental periodontitis induced by silk ligature and topical application of sonicates from oral microorganisms in monkey (author's transl)

The purpose of the present investigation was to study the clinical and histopathological aspects in the progression of a periodontal lesion starting from a healthy periodontium.One monkey weighing 3 kilos was used. During a pre-experimental period of 3 weeks the teeth of the monkey were cleaned once daily with toothbrush and rubber tip. At the end of the preparatory period the gingivae were in excellent clinical health and they were at quadrant divided into experimental and control groups. The silk ligatures were placed around the premolars and the molars on each side of the maxilla and a topical application of the sonicates from Actinomyces viscosus and Fusobacterium nucleatum was used in the mandibular left and the maxillary right sides for 4 hours once a week. The mandibular right side served as a control which was subjected to meticulous toothbrushing.The plaque accumulation, the gingival inflammation, the pocket depth and the distance from the reference point to the gingival crest were assessed at regular intervals during an 3 month experimental period.The animal was sacrificed after 3 months and a serial section of the premolars and the molars with surrounding tissues was prepared for the light microscopic examination. In the sections were measured (1) the size of the infiltrated connective tissue (ICT), (2) the distance from the cemento-enamel junction (CEJ) to the apical cells of the junctional epithelium (JE), and (3) the distance from CEJ to the marginal bone crest (BC).The following findings were obtained:1) At the start of the study no plaque and gingival inflammation could be detected. The gingivae around the teeth showed the presence of abundant amounts of plaque and the signs of pronounced inflammation after 1 month in the ligature and the ligature plus topical application groups. The plaque formation and the gingival inflammation developed with time. The control group did not show a significant increase in the Plaque and the Gingival Indices.2) The pocket depth in both ligature and ligature plus topical application groups rapidly more increased than that in the other groups.3) The distance from the reference point to the gingival crest in the three groups was significantly larger than that in the control group.4) The size of ICT occupied 3.3% in the control group, 12.3% in the topical application group, 27.7% in the ligature group and 26.7% in the ligature plus topical application group.5) The distance from CEJ to JE was similar in the topical application and the control groups. The “histological gingival recession” in the ligature and the ligature plus topical application groups was significantly larger than that in the control group.6) In the biopsies from the ligature and the ligature plus topical application groups it was noted that BC was located slightly below the level in the control group.It is apparent from the present study that the silk ligature and the topical application of the sonicates from oral microorganisms play an important role in the initiation of periodontitis.

Conditions and pathological features of rapidly destructive, experimental periodontitis in dogs.

In order to analyze the conditions for rapidly destructive periodontitis and to describe the early histopathological changes and bone degradation, periodontitis lesions were produced in five male Beagle dogs, 8 to 12 months of age. Cotton floss ligatures were placed around the crowns of upper and lower third and fourth premolars for periods of 4, 7, 14, 21 days. The buccal gingival tissues, being slightly inflamed initially, were clinically scored for various symptoms of inflammation. Block biopsies of premorlars and the adjacent buccal periodontal tissues were taken and processed for light and electron microscopy. Depending on the ligature position in relation to the gingival margin (above, at, deep below) at the day of biopsy harvest, three groups of selected biopsies were formed, and randomly selected sections of these were used for various histometric measurements, for stereological estimation of the size and composition of the connective tissue infiltrate and the osteoclast density, and for general histopathological evaluation. The resulting data and observations implied that (1) onset and maintenance of this periodontitis lesion depend on subgingival ulceration, (2) rapid bone desstruction is the result of osteoclast activity stimulated by acute inflammation, and (3) bone degradation occurs independently of the loss of collagen fiber attachment and the apical migration of the junctional epithelium.

Histologic characteristics of experimental gingivitis in the juvenile and adult beagle dog

An earlier study (Matsson & Attström 1979) revealed an unexplained difference between juvenile and adult dogs in the propensity to develop clinical signs of gingivitis. The aims of the present investigation were to depict the structural composition of clinically normal gingiva and to analyze the histologic changes in the gingiva during plaque development in juvenile and adult dogs. Six beagle dogs were used. Two periods of discontinued oral hygiene were studied, the first at 3 and the second at 12 months of age. Biopsies were sampled on days 0, 4, 7, 14, 21 and 28 of each period. Sections from the biopsies were analyzed at two levels of magnification. Compared to adult dog gingiva, juvenile gingiva seemed to display: 1) a thicker keratinized layer of the oral epithelium, 2) a junctional epithelium that structurally resembles the oral epithelium, 3) a cuticular structure at the surface of the junctional epithelium, 4) a limited mononuclear inflammatory cell response during experimental gingivitis, and 5) a delayed establishment of an infiltrated connective tissue portion during experimental gingivitis. In addition, during experimental gingivitis, subgingival plaque formed along the tooth surfaces to a lesser extent in the juvenile stage compared to adult dogs.

The effect of systemic antimicrobial therapy on plaque and gingivitis in dogs

Six dogs with varying degrees of gingivitis wer divided into 3 groups of 2 dogs each. One pair of dogs received tetracycline HCI 250 mg, 2 times/day, for 4 weeks. The controls reveived no medication during the experimental period of 4 weeks. The results showed a decrease in the proportions of coccoid cells and straight rods in the periodontal flora with increasing gingival inflammation, while the proportions of spirochetes and motile bacteria were markedly reduced. Metronidazole appeared to be somewhat more effective in eliminating spirochetes than tetracycline. Treatment with the antimicropbial drugs tended to derease the size of th infiltrated connective tissue as well as the proportion of junctional epithelium occupied by inflammatory cells, but did not succeed in eliminating the inflammatory reaction entirely. At the initial examination around 60% of the leukocyte volume in junctional epthelium consisted of neutrophilic granulocytes. At the final examination less than 30 % of the leukocyte volume consisted of neutrophis. It was not clear from the results if the improvement in the clinical and histological parameters was due to the specific suppression of ertain organisms or to an overall. non‐specific reduction in volume of the periodontal flora.

Effect of tetracycline and/or scaling on human periodontal disease

Abstract Twelve adult patients with severe chronic periodontitis were examined prior to treatment, and after 8 and 25 weeks following tbe start of treatment. Six subjects received tetracycline during weeks 1 and 2 and weeks 7 and 8, while the other six did not. All subjects were instructed in oral hygiene and received a series of scaling and root planing treatments on one side only of their dentition. The contralateral side received no scalings at any time. The experiment was designed to provide clinical and microbiological data at the 0‐, 8‐ and 25‐week intervals for at least six sites in each of four groups, namely untreated sites (T0S0), sites which were scaled only (T0S1), sites which received tetracycline only (T1S0) and sites which were scaled and were exposed to tetracycline (T1S1). In addition, biopsies of initially diseased sites which had been treated or left untreated were evaluated by light and electron microscopy at all time intervals.The results indicated that T0S0 sites remained more or less unchanged with respect to all parameters during the 25‐week period, with the exception of a decrease in PII scores due to improved oral hygiene. T0S1 sites showed a marked clinical improvement between time 0 and 8 weeks, i.e. reduced PII and GI scores and reduced probing depth; the microbial flora showed an increase in the proportion of coccoid cells with a concomitant decrease in motile rods and spirochetes; the plasma cell‐dominated infiltrated connective tissue (ICT) showed a significant decrease in the proportion of plasma cells with a corresponding increase in lymphocytes; evidence of collagen deposition was also observed histologically. This 8‐week status persisted after 25 weeks, but in addition the tissues showed an increase in the proportion of fibrobiasts and a decrease in the proportion of lymphoblasts. T1S0 sites showed a similar improvement in the clinical and microbiological parameters at 8 weeks, but tbe ICT showed only a moderate reduction in the proportion of dead and unidentified cells. After 25 weeks, tbe clinical parameters remained unchanged from the 8‐week interval, but the microbial composition and the tissue characteristics showed a significant rebound toward the values observed at baseline. T1S1 sites showed essentially similar changes in the clinical, microbiological and tissue characteristics as the T0S1 sites for all time intervals. However, in the presence of the antibiotic the 8‐week proportions of coccoid cells were higher and those for motile rods and spirocbetes lower. Evaluation of all biopsied sites revealed a positive correlation between the proportion of plasma cells in the ICT and the proportion of spirochetes in the associated microflora.The results suggest that the microflora of healthy and periodontally diseased sites differ and that some of these differences are associated with detectable differences in the composition of the ICT. Mechanical debridement and/or treatment with tetracycline cause changes in the clinical, microbiological and histological parameters. Discontinuation of treatment with tetracycline leads to a return of the microbial and histological parameters toward values observed prior to treatment. These changes appear prior to detectable clinical changes.

Intravas device studies in rabbits: II. Effects on sperm output, fertility and histology of the reproductive tract

Solid (rod) and open (tube) prototype gold-platinum intravas device housings of three different surface designs were surgically inserted into the vasa deferentia of rabbits to test the feasibility of controlling male fertility. The devices were inserted through a short longitudinal slit in each vas deferens, secured with 4-0 silk suture and remained in situ for various periods ranging from 56–426 days. The solid devices completely inhibited the fertility of 6 rabbits in 20 of 21 matings. The fertile mating resulted after the loss of a device in one animal. In contrast, 7 of 9 rabbits receiving identical, but open (i.d. 0.8 mm), devices were fertile in numerous matings. Reduced sperm numbers or sperm-free ejaculates were usually obtained for 2–3 weeks after surgery, but returned to normal following this period. However, sperm blockage due to connective tissue infiltration and eventual encapsulation of the open devices was subsequently observed in about 50% of the animals. Histological changes of the vas deferens at the sites of some of the devices included occasional sperm granulomas, mild focal submucosal edema, mucosal erosion and mild mucosal hyperplasia due to fibroblastic proliferation.

Reaction of vaginal tissue of rabbit and of cheek pouch of hamster to inserted collagen sponges treated with either zinc or copper

Resilient collagen sponges either untreated (CS) or treated with zinc (Zn-CS) or copper (Cu-CS) were inserted intravaginally into 41 adult New Zealand white rabbits. Ultimately, a distal vaginal ligature proved the most effective method of keeping the CS in place. A group of sham-operated control animals had ligated vaginas without sponges. After 10 days, the animals with ligated vaginas were killed. Vaginal secretions and vaginal and uterine tissues were analyzed for zinc and copper content. The morphology of vaginal tissue and of the various CS was examined grossly and microscopically. The CS caused minimal but definite irritation to the vagina, seen mostly in localized lysis of epithelial cells, the presence of a few leukocytes in the submucosal layer, and cellular infiltration of the CS. The morphology of vaginas with Zn-CS inserted did not differ from those of sham-operated animals. No signs of inflammation in vaginal tissue and no infiltration of leukocytes appeared. Conversely, Cu-CS produced striking inflammatory changes with diffuse necrosis of epithelial lining, leukocytic infiltration of the lamina propria, and extensive cellular infiltration of the sponge. The semiquantitative rating of the morphology of the Cu-CS was 14, hence unacceptable, while that of CS was six and that of Zn-CS was only two. A similar set of sponges was inserted for three days in the cheek pouches of golden hamsters. Mucosal cell linings were not affected by CS or Zn-CS. However, Cu-CS produced marked inflammation, ulceration of epithelial cells, and leukocytic infiltration of subepithelial connective tissue and of inserted sponges. We conclude that Zn-CS did not produce a topical toxic effect in either rabbit vaginas or hamster cheek pouches. Cu-CS proved unacceptable because of the toxic effects that these caused in both models.

Eosinophilic Fasciitis: A Scleroderma-like Illness

To the Editor.— Shulman 1 recently described two patients with scleroderma-like illnesses characterized by subcutaneous induration of the extremities, eosinophilia, hypergammaglobulinemia, and absence of visceral disease. Biopsy specimens showed fascial thickening and mononuclear cell infiltration between tela subcutanea and muscle; the skin was normal. The appearance of 15 additional cases, 2-5 occasionally with prominent eosinophilic connective tissue infiltration, has justified consideration of a new entity: eosinophilic fasciitis (Shulman syndrome). A patient with the typical clinical and histopathologic features of eosinophilic fasciitis is reported. Report of a Case.— AA 36-year-old male physician sought medical attention in April 1976, for possible scleroderma. Previously healthy and enjoying daily vigorous exercises and jogging, he first noted the onset of hand paresthesiae in January 1976. Digital stiffness and progressive subcutaneous induration of the hands and distal forearms ensued shortly thereafter. Over the next month, induration involving the upper arms and anterior neck was noted. Preceding

An ultrastructural study of Langerhans cells crossing the basal complex in oral lichen planus

Langerhans cells, identified by the presence of the characteristic granule, were found in oral lichen planus lesions, both within the epithelium and in the inflammatory cell infiltrate of the connective tissue. They varied in numbers in different specimens and also in different areas of the same specimen. They were most numerous in the prickle cell layer. However, where Langerhans cells were increased, some were in the basal cell region, some extended through the basal complex and some lay wholly within the connective tissue infiltrate. The present findings suggest that Langerhans cells are capable of moving freely from the epithelium to the connective tissue and vice versa.

Morphological and Functional Changes of the Liver Following Small Intestinal Bypass for Obesity

Liver biopsies of 6 patients were obtained at time of jejuno-ileal bypass and compared with liver biopsies obtained at 6, 12, and 18 months after the operation. The extent of fatty changes, cellular infiltration, connective tissue infiltration, and parenchymal cell damage was recorded. In biopsies taken 6 and 12 months postoperatively a considerable increase was observed in steatosis and cell damage, biopsies obtained at 18 months after the operation showing reduction or clearing of fat. Transient increase in periportal cell infiltration was observed, and in one patient a manifest cirrhosis developed, necessitating partial reversal of the shunt. An attempt was made to correlate the histological changes with alterations in standard liver function tests. The tests were of limited value in predicting the histological changes unless the morphological changes were very serious and the liver in a state of insufficiency. The importance of liver biopsies in the follow-up of bypass patients is stressed.

Application of Stereologic Methodology to Inflammatory Infiltrates Residing in Oral Mucous Membrane Lesions Structural and Morphometric Comparison of Benign Mucous Membrane Pemphigoid and Reticular Lichen Planus

In the present study, stereologic procedures are employed to estimate quantitatively the composition of chronic connective tissue infiltrates characterizing various oral mucous membrane diseases. One case each of benign mucous membrane pemphigoid (BMMP) and reticular lichen planus (RLP) served as examples to demonstrate the applicability of this methodology. In both lesions which are described clinically, histologically and on the ultrastructural level and which display a markedly different infiltrate composition, fibroblasts were affected and either proliferated (BMMP) or became cytopathologically altered (RLP). The quantitative data presented, besides providing new information, argue in favour of the stereologic approach.

Histology of air sac lesions induced in chickens by contact exposure to Mycoplasma synoviae.

Chickens were housed when 1 day old with chickens experimentally infected with Mycoplasma synoviae. Air sacs from 16 of the 2-week-old and eight of the 3-week-old contact-exposed chickens were given gross lesion scores and embedded in glycol methacrylate for slide preparation. Histologic lesions in air sacs with gross scores of 0-2 were mild edema resulting in a two to eightfold increase in air sac thickness, capillary proliferation, and exudate consisting largely of heterophils and necrotic debris. Histologic lesions in air sacs with gross scores of 3 and 4 were marked hyperplasia of epithelial cells and diffuse infiltration of the air sac connective tissue by mononuclear cells. Nine of 10 air sacs with gross scores of 0 had no histologic evidence of inflammation. The most severe histologic lesions were in those air sacs with gross scores of 4. The glycol methacrylate procedure resulted in 2-mum sections with excellent cellular detail.

A study of the morphological changes in the small intestine of the spontaneously diabetic Chinese hamster.

Several morphological changes were observed microscopically in the small intestine of some diabetic Chinese hamsters. Although some alterations lacked statistical significance due to variation, most diabetics displayed a greater incidence and severity compared with nondiabetic controls. The following structural deviations were seen in the small intestines of some diabetics: increased surface area, elevated number of goblet cells per villus, decreased muscle thickness with connective tissue infiltration, reduced number of Auerbach's plexuses, lymphocyte aggregations accompanied by blunted villi, blood vascular lesions and deformed villi due to excessive loss of epithelial cells.

Experimental gingivitis in young dogs.

The aim of the present study was to analyze and express in quantitative terms some of the structural alterations which develop in an initially normal gingiva during a phase of continuous plaque accumulation. Four beagle dogs were used. The animals had from birth been twice daily subjected to meticulous mechanical tooth cleaning. When the dogs were 10 months of age their gingiva were in excellent health as evaluated by Gingival Index and Gingival Exudate measurements. Gingival tissues were harvested from the premolar and molar regions in the right jaws. The tooth cleanings were then terminated and plaque allowed to accumulate. Clinical examinations were performed and gingival biopsies sampled after 4, 7, 14, 21 and 28 d. The composition of the gingival biopsies was analyzed in a sampling microscope. After 4 d of plaque accumulation significant amounts of gingival exudate could be sampled. The exudation then gradually increased during the following weeks. Biopsies representing day zero did not contain any inflammatory cell infiltrates. However, after 4 d of the experiment leukocytes were found in the collagen-poor connective tissue immediately beneath the junctional epithelium. The size of the infiltrated connective tissue (ICT) gradually increased during the experiment. The volumetric density of collagen in the noninfiltrated connective tissue (NCT) was always much higher than in ICT. In ICT, however, this density parameter remained rather constant throughout the study. On days 4 and 7 neutrophilic granulocytes constituted 60-70% of the leukocyte population. On day 28, however, the infiltrate comprised mainly mononuclear leukocytes, especially plasma cells, neutrophils at that time occupying only a small fraction of the infiltrate.

Fatty haemorrhagic liver syndrome in laying hens on diets supplemented with rapeseed products

Livers of laying hens of Hy-Line No 934E on low erucic acid rapeseed meals and rapeseed oil were studied. Gross lesions in the livers of hens on experimental diets were moderate to severe fatty degeneration, focal necrosis and moderate to severe haemorrhage. Histological examination revealed oedematous foci and lysis of hepatocytes along with large amounts of lipid droplets in the necrotic lesions. Necrotic lesions were not always associated with large haemorrhages. Connective tissue infiltration of older degenerative and haemorrhagic lesions was not extensive. Abdominal haemorrhage from livers occurred when extensive necrosis in the form of hepatocyte lysis and some vascular changes were present, suggesting hepatocytic degeneration caused by toxic products or their metabolites present in rapeseed by-products.

Clinical and stereologic analysis of the course of early gingivitis in dogs

In the present investigation an attempt was made to follow the sequence of events occurring in the gingival tissues of dogs reacting to the reaccumulation of microbial plaque. The experiment was performed on 5 young dogs which twice a day during a preparatory period of 5–6 months had been treated with a 2 % chlorhexidine mouthrinse. When the eruption of the permanent teeth was in a final phase a clinical examination (Plaque Index, Gingival Index, Gingival exudate measurements) was performed, biopsies sampled from predetermined tooth regions and the chlorhexidine regimen terminated. On days 1, 3, 5, 7, 14, and 28 the clinical examinations were repeated and biopsies of buccal gingival tissues taken from incisors, canines, and premolars. The gingival tissues were subjected to a stereologic analysis based on morphometric point counting procedures. On day zero small amounts of plaque were detected on most teeth. Following the termination of the chlorhexidine regimen plaque accumulation as well as gingival exudation increased. The morphometric data revealed that (1) all biopsies contained a small portion of ICT (infiltrated connective tissue) which did not increase in size with time (2) the cellular composition of ICT fluctuated but did not change its general pattern with time (3) immunoblasts were rarely seen but an unusual lymphoid cell (X‐lymphocyte) was regularly present in ICT. The possibility was discussed that chlorhexidine not only interferes with plaque formation but also may suppress some aspects of the inflammatory and immunopathologic response of the gingiva.

Relationship between histological degree of inflammation and epithelial proliferation in macaque gingiva.

Epithelial proliferation of various regions of macaque gingival epithelium, as measured by uptake of locally injected H3‐thymidine, showed regional differences which could be explained in part by the size and nature of the inflammatory infiltrate invariably found in gingival biopsies. The proliferative activity of oral sulcular epithelium was greater than that of oral epithelium and was negatively correlated with the size of the area of infiltrated connective tissue. On the other hand labelling of oral epithelium was positively correlated with size of infiltrate, the effect decreasing with distance of the epithelium from the infiltrate

Experimental gingivitis in the monkey. Relationship of leukocyte counts in junctional epithelium, sulcus depth, and connective tissue inflammation scores.

Rhesus monkeys receiving an oral hygiene program which included brushing, interdental cleansing and topical applications of chlorhexidine gluconate demonstrated a clinically normal gingiva for periods of up to 3 months. Wide fluctuations within individual dental units were noted with respect to histological sulcus depth, degree of connective tissue infiltration with lymphocytes and plasma cells, and total leukocyte or polymorphonuclear leukocyte (PMN) counts in the Junctional epithelium, even when the plaque and gingivitis index scores were 0. Of 18 clinically normal dental units sampled, only 6 appeared free of connective tissue inflammation. However, more than half of the tissue blocks obtained from dental units with a gingivitis and plaque index of 0 also showed scores of 0 with respect to the connective tissue inflammation (CTI) score and the number of PMNs in the Junctional epithelium. Within three days following discontinuation of oral hygiene procedures, rhesus monkeys developed a clinically noticeable gingivitis which began in the interdental papillae. Increases in CTI scores and number of leukocytes in the Junctional epithelium were evident after 2 days without oral hygiene. These values tended to increase further during the experimental period. A slight, but significant increase in sulcus depth was also noted during this time period. Regardless of the clinical state of the gingiva, a positive correlation was established between CTI scores and the number of PMNs and leukocytes in the Junctional epithelium. In early gingivitis, the plasma cells did not appear to outnumber lymphocytes, as has been reported for chronic gingivitis of longer duration.