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.     

Silver wire implant of 40 years' duration: influence on local tissues

The retention of an implanted silver wire in human tissue 40 years after surgical fixation of the frontal bone was studied post mortem by histology, transmission electron microscopy, and energy-dispersive x-ray analysis. Corrosion products of the wire were associated with a chronic inflammatory response and were bound to certain connective tissue elements; they were deposited as discrete particles, comprising silver in association with sulphur, on collagen fibrils and vascular basement membranes. Bone structure appeared normal except close to the wire, where it was replaced by a loose connective tissue in which collagen bundles were disorganized.     

Long-term culture of human periodontal ligament cells with autologous root discs

Recent attempts at gaining an understanding of the factors necessary for periodontal reattachment have employed an in vitro system in which connective tissue cells are cultured with dental root discs. The present study was undertaken to optimize culture conditions. Periodontal ligament (PDL) cells and root discs were derived from 2 wisdom teeth extracted from each of 3 patients. Pairs of planed root discs (0.5 mm thick) were gently placed in dishes containing autologous PDL-cells and separated by a 0.5 mm wide gap. After 10 to 13 weeks in culture the specimens were processed for light- and electron microscopy and the collagen fibril diameter was determined. In the interdental space, all cultures contained an aggregate of cells and collagen fibrils. Adherent to root discs with exposed dentin was a loose capsule of cells and collagen fibrils oriented parallel to the disc surface. Adjacent to cellular cementum a discontinuous, dense, short fiber fringe was found along the cultureJroot interface. There was some intermingling between cemental and culture collagen fibrils. The fibril diameter of cemental collagen (59.4 ± 5.0 nm) and culture collagen (31.8 ± 3.5 nm) was very different. Thus, autologous PDL-cell/disc-cultures can be maintained for prolonged periods of time, resulting in the formation of discontinuous patches of condensed collagen fibrils radiating from some portions of the decalcified cemental surface.     

Immunohistochemical localization of the chondroitin sulfate proteoglycan in demineralized rat periodontal tissue

We investigated the immunohistochemical localization of the chondroitin sulfates (chondroitin-4 sulfate, -6 sulfate and dermatan sulfate) in demineralized rat periodontal tissue using monoclonal antibodies (2-B-6, 3-B-3). Also, fixative and demineralized methods were established using these monoclonal antibodies. The result showed that the most effective combination of fixative and demineralized methods was 2% glutaraldehyde 1% para-formaldehyde and 5% EDTA. Chondroitin-4 sulfate and dermatan sulfate were widespread in gingival connective tissue and periodontal membrane, with an especially strong response of dermatan sulfate shown along collagen fiber bundles. Chondroitin-6 sulfate was located in peripheral blood vessels. In alveolar bone, chondroitin-4 sulfate and dermatan sulfate were found inside Hareversion canals, Volkman's canals and lacunae. Chondroitin-6 sulfate was localized at peripheral blood in alveolar bone. In cementum, chondroitin-4 sulfate and dermatan sulfate were found at lacunae of cellular cementum and a part of embedding Sharpey's fiber.     

Immunocytochemical localization of extracellular matrix components in beagle periodontium: I. Collagen types I and III in healthy gingival connective tissue

The localization and identification of types I and III collagen in healthy gingival connective tissue of the beagle dog were investigated using indirect immunofluore-scent and immunoferritin electron microscopic techniques with affinity purified antibody or IgG. Type I collagen was evenly distributed throughout the lamina propria and the underlying connective tissue in the form of thick collagen fibers. In the lamina propria, these fibers appeared to run from the underlying connective tissue in a perpendicular orientation to the overlying gingival epithelium. Localization of type III collagen was restricted to the lamina propria and the connective tissue around blood vessels. Immunoferritin electron microscopy clearly revealed that type I collagen fibrils have a diameter ranging from 40 to 100 nm and a clear 640 Å cross-banding pattern. Thin and short collagen fibrils, 18 to 32 nm in diameter, were labelled with antitype III collagen IgG. A 640 Å cross-banding pattern was rarely observed in these fibrils. Type III collagen fibrils coexisted with type I collagen fibrils in a mixed pattern within the lamina propria and the perivascular space.     

Connective tissue organization of healthy human gingiva

Healthy human gingival connective tissue appears to be arranged in two patterns of organization at the ultrastructural level: Pattern I (PI) and Pattern II (PII). PI is a dense pattern of organization mainly constituted of large, dense bundles of thick collagen fibers, whereas PII is a loose pattern of organization, mainly constituted of short, thin collagen fibers mixed with a fine reticular network, especially located under or around basement membranes. Ultrastructural immunoperoxidase labelling of types I, III, and IV collagen demonstrates that gingival connective tissue is made of an intricate pattern of type I and III collagen where type I collagen fibers are preferentially organized in large dense bundles in PI, whereas a fibrous and fibrillar type III collagen network is predominant in PII. Type IV collagen, which does not exist in fibrous or fibrillar form, appears to be the main collagenous component of the basement membranes.     

Collagen fiber reunion in wound repair: transmission electron microscopic study of rat gingiva

Abstract – The purpose of this study was to characterize connective tissue at the site of reunion following surgical injury of the transseptal fiber system in rat molars. Eight conventional albino rats aged 50 days were incised interdentally and the lesions allowed to heal for 14 days followed by specimen preparation for transmission electron microscopy. Four specimens with identifiable areas of fiber attachment at the light microscopic level of magnification were selected for a detailed ultrastructural examination. Transitional connective tissue consisted of thin (250-375 Å) and thick (450 Å) collagen fibrils at a ratio of 41/59 mainly arranged as aggregates of uniformly sized fibrils. Fibril thickness in the vicinity of fibroblasts ( n = 40) varied at a ratio of 65 (thin)/35 (thick). Distribution and spatial arrangement of fibrillar components indicate that the site of reunion is characterized by separate aggregates, of fibrils rather than alignment of individual collagen fibrils of varying thickness suggestive of covalent linkage.     

Immunohistochemical study of types I, III and IV collagen in fibrosis of diseased gingiva during chronic periodontitis: A light and electron microscopic study

The distribution of type I, III and IV collagen and their ultrastructural organization have been studied in fibrotic gingival connective tissue of patients with longstanding cases of chronic periodontitis. The use of an immunofluorescent procedure has shown that the diseased connective tissue was made up of both type I and type III collagen. Type I collagen was strongly fluorescent and appeared to be the main gingival collagenous component, equally distributed in all layers of the tissue, whereas type III collagen was weakly fluorescent and mostly found in gingival papillae, but not around blood vessels. Standard electron microscopy and immunolabelling using the peroxidase procedure have shown that the large and dense bundles of type I collagen of P₁, which is the main pattern of organization of the gingival connective tissue, were infiltrated by small bundles of microfilaments (10–15 nm) identified as the fibrillar form of type III collagen. However, in P_II, the second pattern of organization of the gingival connective tissue, type I collagen fibers were predominant and gave a fibrous feature to this area. Type IV collagen was exclusively located in the thickened, degraded Lamina densa of basement membranes.     

Distribution of collagens type V and VI in the normal human alveolar mucosa: An immunoelectronmicroscopic study using ultrathin frozen sections

The ultrastructural localization of collagens type V and VI in normal human gingival mucosa was investigated by immunoelectron microscopy. Twenty biopsies were fixed in dimethylsuberimidate and shock-frozen in slush nitrogen. Collagen type V was mainly located to meshworks of uniform nonstriated microfibrils of 12 to 20 nm width, which preferentially appeared in larger spaces between cross-striated major collagen fibrils. Occasionally single microfibrils of collagen type V fanned out from the ends of major collagen fibrils, which may indicate a role as a core fibril. Collagen type V was not found in the subepithelial basement membrane and the immediately adjacent stroma. Collagen type VI was detected in a loose reticular network of unhanded microfilaments that were morphologically distinguishable by knoblike protrusions every 100–110 nm. These microfilaments were found in the vicinity, but not as an intrinsic component, of the subepithelial basement membrane. Single filaments of collagen type VI filaments appeared to form bridges between neighboring cross-striated major collagen fibrils, suggesting an interconnecting role for this collagen type. The method presented appears to be excellently suited to study the normal and pathological supramolecular organization of the oral extracellular matrix.     

The structure of the interstitial surfaces of the epithelial basement membranes of mouse oral mucosa, gingiva and tongue.

It is known that the gaps between epithelium and the underlying connective tissue usually occur between the epithelial cells and the basement membrane, resulting in exposure of the cellular surface of the lamina densa. After dithiothreitol separation, the epithelia of oral mucosa, gingiva, and tongue were mechanically peeled off from the underlying connective tissues. This treatment severed the connections between the basement membrane and the underlying connective tissue and the anchoring fibrils were pulled off from the collagen layer. In contrast, connections between the epithelial cells and basement membrane were preserved, resulting in exposure of the interstitial surfaces of the laminae densae. Scanning electron-microscopic observations of those interstitial surfaces were possible using the specimens prepared as above. The basement membranes of these three oral epithelia were morphologically the same not only by transmission but also by scanning electron microscopy. Scanning electron-microscopic observations revealed that their laminae densae were composed of fine fibrils and demonstrated three-dimensionally the projection of the anchoring fibrils from the laminae densae to the interstitial side. These findings coincide with those for epidermal basement membrane, which had already been observed with the same method.     

In vivo and in vitro glycosaminoglycans from human dental pulp.

A qualitative assessment was made of the type of glycosaminoglycans (GAG) present in normal human dental pulp using electrophoresis on cellulose-acetate plates. A comparison was also made between the GAG derived directly from the dental pulp (in vivo) and those derived from cultured pulp fibroblasts from the same individual (in vitro). The results of this study showed four main types of GAG in normal human dental pulp tissue, which were dermatan sulfate, heparan sulfate, hyaluronic acid, and chondroitin sulfate. GAG synthesis from cultured pulp fibroblasts in vitro was different from the GAG present in the dental pulp (in vivo). Extracellular GAG, as well as pericellular GAG consisted of dermatan sulfate, hyaluronic acid, chondroitin sulfate, and heparin. Cellular GAG, however, contained only dermatan sulfate, hyaluronic acid, and chondroitin sulfate. There was no difference in type of GAG from the second and fourth passaged pulp fibroblasts.     

Non-specific cleavage of collagen by proteinases in the presence of sodium dodecyl sulfate

Abstract – Proteolytic degradation of collagen normally requires specific collagenases of either mammalian or microbial origin. We have observed, among many detergents, a unique effect of sodium dodecyl (=lauryl) sulfate after preincubation of insoluble collagen (type I) with detergent. When treated fibrils were isolated and resuspended in detergent-free buffer, they were very sensitive to cleavage by non-specific proteases like trypsin, elastase, and subtilisin. We suggest that dodecyl sulfate causes a structural change in the collagen molecule which abrogates the resistance of collagen to most proteolytic enzymes. These observations may have implications for collagen and connective tissue research in general and especially to periodontal research. Lauryl sulfate (2-5%) is included in the majority of toothpaste formulations as a foaming agent, and our results again raise the question whether toothbrushing with lauryl sulfate may accelerate periodontal destruction by synergism with bacterial and host proteases. It may also bind to the collagen in exposed root surfaces and thus affect the stability of their hard tissues.     

Collagen deposition during wound repair in rat gingiva

Abstract – The aim of this study was to examine day-by-day changes at incisional wound edge in rat molar interdental tissue with special reference to early interactions of new and pre-existing collagen fibrils. Twenty rats aged 50 days were divided into experimental groups each comprising two animals. Following incision and post-injury observation periods of 1–10 days, the rats were given an overdose of sodium pentothal and specimens prepared for light and electron microscopy. In 1-3-day post-injury specimens fibrin was replaced by inflammatory cells. Fibroblasts were found along the cut edge in 4-day post-injury specimens. Fibrillogenesis had started at 5 days post-injury. During the period of 5–10 days post-injury increasing amounts of new collagen fibrils were laid down at the wound edge. No granular material was identified at the interface of new and preexisting collagen. In standardized mesiodistal sections, bundles of newly formed fibrils regularly appeared cross-cut in relation to the tangentially cut transseptal fiber system, suggestive of a buccolingual fibril orientation at the wound edge. The ultrastructural observations indicate that during early stages of wound repair, connective tissue continuity is generally established without direct splicing of severed fibers.     

Localization of fibronectin in gingival connective tissue of the beagle dog. Ultrastructural detection with ferritin and peroxidase-conjugated antibodies

Ferritin and peroxidase-conjugated antibodies were used in an indirect antibody method to localize fibronectin in gingival connective tissues. Fibronectin was found in the basal lamina beneath the epithelium and endothelium. Collagen fibrils associated with the basement membranes were also heavily coated by fibronectin. Amorphous patches of fibronectin were found adjacent to the plasma membrane of epithelial cells as well as free in the interepithelial spaces. Fibronectin was present throughout the connective tissue in close association with individual collagen fibrils, apparently serving as an interfibrillar cementing substance. Patches of fibronectin were located at the cell surface of fibroblasts, plasma cells, lymphocytes, endothelial cells, smooth muscle cells, and neutrophils. These amorphous patches were observed to connect adjacent cells across narrow spaces and to connect cells to collagen fibrils. The heavy labeling for fibronectin visualized by fluorescent microscopy around gingival blood vessels (Cho et al., 1985) can be accounted for by a heavy coating of fibronectin on the collagen fibrils and basal laminas associated with endothelial cells, as well as by the presence of abundant deposits of fibronectin along the cell membranes of endothelial cells and in the intercellular spaces of the vessel wall.     

Root resorption leading to linkage of dentinal collagen and gingival fibers?

A maxillary central incisor in a 55-year-old Caucasian female was extracted in modified block as part of a histologic study of gingival responses to subgingival crown placement. The facial portion of this block showed preclinical pocket depth of 1.0 mm with moderate gingival inflammation. The block was extracted 2 weeks after crown placement and prepared for histologic evaluation. Histologic responses to subgingival crown placement were, among others: gingival recession (1.0 mm), reformation of crevicular depth (0.7 mm) and remodelling of the gingival and periodontal attachment apparatus. Histologic evaluations of the gingival unit using cellular and connective tissue stains revealed an area of root resorption immediately apical to the junctional epithelium at a portion of the facial surface. Root resorption had progressed into dentin. Gingival connective tissue abutted the dentinal surface. Collagen stains showed the presence of tufts of collagen fibrils which appeared to arise from the dentin. These fibrillar elements seemed to splice with collagen fibrils found in the gingival connective tissue. The specimen thus suggests the possibility of a fiber linkage attachment involving exposed dentinal fibers as part of gingival repair following injury.     

Ultracytochemical localization of basal lamina anionic sites in the rat epithelial attachment apparatus

The basal lamina anionic sites of the epithelial attachment apparatus (EAA) were investigated at the electron microscopic level in adult rat periodontium. After 1M NaCl junctional epithelium detachment, an irregular and fluffy basal lamina-like structure appeared to cover the cementum surface. This structure reacted positively with polyethyleneimine (PEI), a strongly cationized ultrastruc-tural tracer, appearing to be composed of highly electron-dense microaggregates. Depending on section plane, double-tracked structures of undefined length were found within PEI precipitates and closely related to cementum collagen fibrils. After nitrous acid de-N-sulphation, 8 nm wide sets of two parallel lines were clearly identified. "Double tracks", i.e., sets of paired lines with peripherical PEI electron-dense material, were found to self-assemble to form dimers, clusters or more complex organizational patterns. From sensitivity towards nitrous acid oxidation and positive control observations, it was concluded that basal lamina anionic sites in the EAA. represented by PEI microaggregates, contain heparan sulfate proteoglycans (HSPGs). Furthermore, high resolution ultrastructural images demonstrated that HSPGs adopt a morphological appearance of "double tracks" in the tissue. On the other hand, the present findings suggest that HSPGs clusters, never found in the mucosal basement membrane used as positive control, may be related to a functional specificity of the tissue at the dento-gingival junction.     

Glycosaminoglycans in the lamina propria and submucosal layer of the monkey palatal mucosa.

Glycosaminoglycans (GAGs) in monkey palatal lamina propria plus both fatty and glandular zones of palatal submucosa were compared. Chemical analysis revealed that GAG contents of the lamina propria and glandular zone were higher than that of the fatty zone. The four GAGs identified by electrophoretic analysis were hyaluronic acid, dermatan sulfate, chondroitin sulfate and heparan sulfate. Each mucosal layer contained all four GAG components. The predominant GAG in both the lamina propria and glandular zone was dermatan sulfate followed by hyaluronic acid. The reverse situation (predominant hyaluronic acid, less prominent dermatan sulfate) was noted in the fatty zone of the submucosa. The three tissue regions showed different molar ratios of unsaturated chondroitin sulfate disaccharides. The ratio of delta Di-4S to delta Di-6S was lower in the lamina propria than in either the fatty or glandular submucosal zones.     

Do collagen fibrils of the periodontal ligament shrink with age?

Do collagen fibrils of the periodontal ligament shrink with age? Certainly not! Based on the quantitative estimation of over 25000 periodontal ligament collagen fibrils at 3 periodontal levels of 28 freshly extracted human teeth, using 10- to 75year-old subjects and high resolution electron micrographs evaluated planimetrically, the present study demonstrates with data obtained from 2 sampling procedures that the diameter of the average collagen fibril, i.e., 54 to 59 nm, is rather constant throughout the human life span. This is particularly true for the vast majority of comparatively large fibrils (i.e., 57 to 64 nm), while a numerically limited population of small fibrils (i.e., 40 to 48 nm) in fact increased in diameter with age. These results are discussed in view of analogous data for other connective tissues and of the functional and metabolic status of such tissues.     

Collagen fiber reunion in wound repair: transmission electron microscopic study of rat gingiva

The purpose of this study was to characterize connective tissue at the site of reunion following surgical injury of the transseptal fiber system in rat molars. Eight conventional albino rats aged 50 days were incised interdentally and the lesions allowed to heal for 14 days followed by specimen preparation for transmission electron microscopy. Four specimens with identifiable areas of fiber attachment at the light microscopic level of magnification were selected for a detailed ultrastructural examination. Transitional connective tissue consisted of thin (250-375 A) and thick (greater than or equal to 450 A) collagen fibrils at a ratio of 41/59 mainly arranged as aggregates of uniformly sized fibrils. Fibril thickness in the vicinity of fibroblasts (n = 40) varied at a ratio of 65 (thin)/35 (thick). Distribution and spatial arrangement of fibrillar components indicate that the site of reunion is characterized by separate aggregates of fibrils rather than alignment of individual collagen fibrils of varying thickness suggestive of covalent linkage.