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.     

Immunohistochemical study of chondroitin sulfate and dermatan sulfate proteoglycan in dental pulp and dentin

We investigated the localization of chondroitin sulfate and dermatan sulfate proteoglycans in the dental pulp and dentin of rats, using a combination of an immunohistochemical technique coupled with specific enzymatic digestion. Chondroitin 4-sulfate and dermatan sulfate were found to be widespread in pulpal connective tissue, predentin and dentinal tubules. The response to predentin was found to be particularly strong. Chondroitin 6-sulfate was stretched in pulpal connective tissue and predentin, but showed only a weak response.     

富含亮氨酸的间质蛋白多糖在小鼠牙周组织发育中的组织学定位

目的研究富含亮氨酸的蛋白多糖类物质：纤维调节素（FMN）、核心蛋白聚糖（DCN）及双糖链蛋白聚糖（BGN）在小鼠牙周组织不同发育时期的分布特点，探讨其在牙周组织发育中的作用。方法取出生后第5、10、15、25天的BALB／c小鼠36只，引颈处死，解剖并分离含下颌第一磨牙区的下颌骨，新鲜配制4％多聚甲醛固定24h，甲酸一甲酸钠复合脱钙液脱钙1～3周，常规石蜡包埋，近远中向5μm连续切片。免疫组织化学Power Vision^TM二步法，观察各发育时期第一磨牙牙周组织中3种间质蛋白多糖的组织学分布特点。结果FMN在牙龈结缔组织和牙周膜中呈强阳性表达，且在牙周膜与牙骨质及牙槽骨界面有较强表达；DCN强阳性表达于牙龈结缔组织、靠近牙槽骨面的牙周膜及牙槽骨表面的成骨细胞中；BGN则在各期牙龈结缔组织及牙周膜中表达，在牙槽骨表面及成骨细胞中为阴性。结论FMN可能与DCN及BGN相互作用，调节牙龈结缔组织及牙周膜胶原纤维的网络形成，并可能参与牙槽骨和牙骨质的矿化过程。     

Immunohistochemical localization of collagenous components in healthy periodontal tissues of the rat and marmoset (Callithrix jacchus).

The distribution of collagen types I and III was demonstrated in healthy periodontal tissues of the rat and marmoset using immunofluorescent localization after decalcification of the maxillae and mandiblae in 0.2 N HCl. An intense fluorescence in the alveolar bone and cementum matrix, as well as in the soft periodontal tissue, was demonstrated with anti-collagen type I antibodies. In the gingival connective tissue and in the periodontal ligament thick fibers of collagen type I could be observed. The fluorescent reaction in the rat periodontal ligament was not strong in comparison to the marmoset periodontal ligament. Sharpey's fibers, inserting into the cementum and alveolar bone, were also stained. On the other hand, collagen type III could not be demonstrated in the hard periodontal tissues, but could be in the bone marrow stroma and the incremental lines as well as around the Sharpey's fibers of the cementum, in accordance to previous studies. In the gingival connective tissue a strong staining was evident, especially near the basement membrane. The periodontal ligament showed an intense fluorescence that was, in some areas, continuous with Sharpey's fibers inserting into the cementum. The distribution of collagen types I and III was demonstrated with immunohistochemical techniques in the rat and marmoset periodontium. These results provide necessary information on healthy tissues that will be required for future studies on the effects of pathological, reparative and regenerative processes.     

Immunohistochemical localization of elastin, fibrillins and microfibril-associated glycoprotein-1 in the developing periodontal ligament of the rat molar

Sugawara Y, Sawada T, Inoue S, Shibayama K, Yanagisawa T. Immunohistochemical localization of elastin, fibrillins and microfibril‐associated glycoprotein‐1 in the developing periodontal ligament of the rat molar. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2008.01196.x. © 2009 John Wiley & Sons A/S Background and Objective: Elastic system fibers are a major component of the periodontal ligament, but little information is available about their detailed composition or the mechanism of elastogenesis in the developing periodontal ligament. The purpose of this study was to investigate immunolocalization of elastin, fibrillins and microfibril‐associated glycoprotein‐1 (MAGP‐1) in the developing periodontal ligament of the rat molar. Material and Methods: Frozen sections of demineralized as well as non‐demineralized periodontal ligament of Wistar rats of various ages from 19 days to 7 weeks were incubated with anti‐elastin, anti‐fibrillin‐1 and ‐2 and anti‐MAGP‐1 antibodies followed by peroxidase‐conjugated secondary antibodies. After incubation with diaminobenzidine solution, immunoreaction products were observed with a light microscope. Results: In the developing periodontal ligament of 19‐day‐old rats, fibers immunopositive to elastin were not present, but fibers positively stained for fibrillin‐2 and MAGP‐1 were widely distributed throughout the ligament. The latter fibers were arranged in the apico‐occlusal direction along with blood vessels. In 3‐week‐old rats, fibers stained for elastin were observed for the first time in the apical region of the ligament. The number and distribution pattern of these elastin‐positive fibers was basically the same as those in rats aged 5 and 7 weeks. In contrast, fibrillin‐2‐ and MAGP‐1‐positive fibers were more extensively distributed in the ligament, and their pattern of distribution was comparable to that of reported oxytalan fibers. Fibrillin‐1 was, however, not detected either in demineralized sections or in non‐demineralized sections, indicating its absence in periodontal ligament. Conclusion: Elastin expressed in the periodontal ligament assembled into elaunin fibers in the vicinity of blood vessels. Both fibrillin‐2 and MAGP‐1 are structural components not only of the elastin‐associated microfibrils but also of elastin‐free microfibrils, with possible roles in elastogenesis and in periodontal ligament homeostasis.     

Immunohistochemical localization of epithelial rests of Malassez in human periodontal membrane

The aim of the present study was to describe the localization and extension of epithelial rests of Malassez (ERM) in the periodontal membrane (PDM) in normal human third molars. The material consisted of 24 normally developed human third molars surgically removed from patients with an age range from 15 to 27 years (six females and six males). The root lengths were developed from close to half-length to complete apex closure. The extracted teeth were fixed in 10 per cent neutral-buffered formalin, decalcified in ethylene diamine tetra acetic acid EDTA, paraffin embedded and cut sagittaly in 5 microm serial sections. Immunohistochemistry was performed using polyclonal rabbit anti-bovine cytokeratin (wide-spectrum screening, WSS) and the EnVision+ dual link system. The results were based on the visual comparison of WSS in the tissue sections using a light microscope. It was demonstrated that the ERM cells were distributed in the PDM in a network-shaped manner along the root surface and in the furcation region. The distribution of ERM was more prominent in teeth with incomplete root formation. The ectodermal tissue layer might influence not only the morphology of the tooth but also tooth eruption. The reaction of this epithelial layer in connection with ankylosis and orthodontic tooth movement may be of future interest.     

Occlusal hypofunction causes changes of proteoglycan content in the rat periodontal ligament

The biological functions of proteoglycans and glycosaminoglycans are closely associated with mechanical stress on the tissue. In order to reveal the relationship between proteoglycans in the periodontal ligament and mechanical stress such as occlusal stimuli, occlusal hypofunction of rat unilateral mandibular molars was induced by extraction of the opposing first, second and third maxillary molars. Immunohistochemical analyses were performed using antibodies for chondroitin sulfate, decorin, biglycan, heparan sulfate and keratan sulfate, and hyaluronic acid-binding protein. Chondroitin sulfate, observed more strongly in the cervical side than in the apical side of the periodontal ligament of the unextracted sides of mandible, and uniformly present in the extracellular matrix of the periodontal ligament, decreased significantly from 1 wk post-extraction of the antagonists, with a decrease in thickness and disarrangement in fibrous components. Decorin core protein, uniformly present in the periodontal ligament of the unextracted sides, decreased as early on as 2 d post-extraction. Heparan sulfate, mainly localized on the cell surface of vascular endothelial cells and osteoclastic cells as well as in the extracellular matrix of the unextracted sides, decreased significantly in association with the decreased number of blood vessels and osteoclastic cells as early on as 2 d post-extraction. Biglycan, keratan sulfate and hyaluronic acid, uniformly distributed in the periodontal ligament of the unextracted sides, showed little change after the extraction. These results demonstrate that occlusal hypofunction causes tissue remodeling of the periodontal ligament, with a significant decrease of chondroitin sulfate, decorin and heparan sulfate.     

Immunohistochemical localization of keratin in the taste buds of rat vallate papillae

Keratin detected with anti-human whole keratin serum, raised in rabbits by injection of the isolated human whole keratin, was used as a histologic marker to study the origin of the cells in taste buds. Rat vallate papillae, including surrounding tissue, were processed for indirect immunofluorescent staining of ketatin. In the taste buds, most basal cells and some of the elongated cells were immunoreactive with anti-keratin serum, showing that most taste-bud cells, if not all, originate from cells of epithelial origin.     

Immunohistochemical localization of C-type natriuretic peptide in the rat submaxillary salivary gland

To define the localization and characteristics of C-type natriuretic peptide (CNP) in the rat submaxillary gland, immunohistochemistry and gel permeation-high-performance liquid chromatography were used. Immunoreactive (IR)-CNP was localized in cells of the granular convoluted tubule, striated duct and endothelial cells of the capillary, where atrial natriuretic peptide (ANP) was colocalized in consecutive sections, but not in acini. Gland extracts co-eluted with synthetic CNP and its content was 60.3+/-4.9 pg/mg protein (n=4). Molecular profiles of immunoreactive material showed two peaks corresponding to synthetic CNP((1-53)) and CNP((1-22)). These results indicate that CNP is colocalized with ANP in the duct and endothelial cells of the rat submaxillary gland. Therefore, CNP may have a physiological role in the submaxillary gland by interacting with ANP and/or other biologically active substances in the ducts and granular convoluted tubule cells.     

Immunohistochemical localization of the matrix glycoprotein tenascin in the skull of the growing rat

Tenascin is an extracellular matrix glycoprotein which interacts with other matrix molecules and with cells, and which appears to play important roles in growth and differentiation. The immunohistochemistry of sections of newborn, 5-day and 20-day-old rats showed that accumulation of tenascin was largely restricted to bones, cartilages and teeth. It was also present in the periosteal and endosteal surfaces of membrane bones, in perichondrium, and ion the dental pulp, but was absent from mature bone, cartilage and dentine. In nasal cartilage, tenascin was present only in the perichondrium, whereas in the condylar cartilage staining was observed in the proliferating and maturing cell layers but not in the hypertrophied cartilage. These differences may reflect differences in the growth mechanisms of primary and secondary cartilages. Accumulation of tenascin was particularly striking in areas where the periosteum or perichondrium was thickened such as sites of some muscle attachments, sutures and condylar cartilage. The restricted distribution of tenascin is unlike the patterns observed for other extracellular matrix molecules. Tenascin may have a unique role in bone growth and remodelling in the craniofacial region.     

Maturation of periodontal connective tissue in new-born rat incisor.

The organization of collagen and ground substance in the periodontium of the rat incisor was studied during the first 12 days of postnatal life. Tissues were rapidly frozen in isopentane at ?150 °C, and cut in a cryostat without decalcification. Maturation of fibres and ground substance was followed using correlated biophysical (polarization microscopy), histochemical (paS, toluidine blue and collagen immunofluorescence), and empirical (Van Gieson and silver impregnation) methods.Periodontal ligament cells contained glycoprotein granules, probably precursors of ground substance. Glycogen was prominent in young bone cells. At birth, the periodontal ligament consisted of unorganized isotropic argyrophilic fibres strongly reactive to collagen immunofluorescent staining; with further development many fibres became oriented and showed anisotropy. They were mainly disposed parallel to the tooth axis with lateral ramifications into bone. Concurrently, collagen of cementum, bone borders, and pericellular matrix of osteocytes showed intense immunofluorescence. When taken together with distinct metachromasia seen in these areas, this indicates a loose aggregation of their macromolecular constituents. Ground substance of the periodontal ligament acquired metachromasia a few days after birth.Principal fibres were subjected to heat, a variety of enzymes and one fixative. Collagenase abolished birefringence and immunofluorescence. Principal fibres were not equally stable during heating. Some fibre groups became isotropic at 62 °C (T_s), others were unaffected or partially affected. Hyaluronidase treatment lowered T_s to 57 °C, and eliminated the heat resistance of thermostable fibres. Evidently, ground substance stabilized the principal fibres. It appears that collagen-ground substance interactions affect stable-labile transformations involved in fibre remodelling, and may influence eruption.     

Experimental study of periodontal tissue regeneration after the application of enamel matrix derivative in rat periodontal defects

The purpose of this study was to evaluate periodontal wound-healing after the application of enamel matrix derivative (EMD) in rats. Periodontal defects were surgically created on the mesial side of the first maxillary molar of 24 male Long-Evans rats. EMD was applied to cover the denuded root surfaces in the experimental group. The contralateral molar was used for the control group, which received the same treatment without EMD. The rats were sacrificed at 2 weeks (8 rats), 4 weeks (8 rats), and 8 weeks (8 rats) after the surgery. Demineralized paraffin sections were stained with Masson's trichrome. Histological analysis and histomorphometric measurements were performed on the periodontal sections. Using an immunohistochemical technique, the localization of osteocalcin (OC) was also examined. The formation of new cementum was statistically significant in the experimental group, especially new cementum with extrinsic fiber. Both acellular and cellular cementum were also rather strongly observed in the experimental group. Epithelial down growth was also strongly inhibited in the experimental group at 8 weeks after the surgery. OC-positive cells and matrix were limited at the bottom of the defects in the control group, while positive reaction was detected not only at the bottom but also spreading to the coronal portion of the defects in the experimental group. These results suggest that EMD has potential to promote cementum regeneration, especially fiber-inserted cementum, and to create a favorable environment that will promote periodontal regeneration.     

Immunohistochemical studies of the periodontal membrane in primary teeth

Objectives. To describe the periodontal membrane of human primary teeth immunohistochemically, while focusing on the epithelial layer of Malassez, fibers, and peripheral nerves, and to compare the findings with those of a previous study of human permanent teeth. Material and methods. Nineteen human primary teeth extracted in late childhood in connection with treatment were fixed, decalcified, dehydrated, and embedded in paraffin. Paraffin sections were stained with wide spectrum screening (WSS), Vimentin, and NeuN in order to mark the epithelial layer of Malassez, fibers, and peripheral nerves. Results. For root surfaces without resorption, the epithelial rests of Malassez appeared as small scattered islands. The fibers varied from tightly packed close to the root surface to a messy and loose organization. Innervation could be seen in close proximity to the root surface. The epithelial cells of Malassez were not usually seen along root surfaces with resorption. The fibers were sparse or not present. Innervation was seen in close proximity to the root. In regions with repair of resorption lacunae, the immunohistochemical reactions for epithelial cells of Malassez, fibers, and innervation pattern could be identical to those in regions with no resorption. Conclusion. In regions without resorption, spatial organization of the periodontal membrane of primary teeth was similar to that of permanent teeth, although the number and distribution of epithelial cells and fibers differed. In regions with repair of root resorption, the epithelial cells of Malassez, fibers, and innervation appeared as root surfaces without resorption.     

Immunohistochemical localization of fibromodulin in the periodontium during cementogenesis and root formation in the rat molar

BACKGROUND: Cementum is essential for periodontal regeneration, as it provides anchorage between the root surface and the periodontal ligament. A variety of macromolecules present in the extracellular matrix of the periodontium, including proteoglycans, are likely to play a regulatory role in cementogenesis. Recently, the small leucine-rich proteoglycan, fibromodulin, has been isolated from bovine periodontal ligament and localized in bovine cementum, as well as in human periodontal ligament. OBJECTIVE: The aim of this study was to examine the distribution of fibromodulin during cementogenesis and root formation. METHODS: A standard indirect immunoperoxidase technique was employed, using an antifibromodulin polyclonal antibody on sections of molar teeth from rats aged 3, 5 and 8 weeks. RESULTS: Immunoreactivity to fibromodulin was evident in the periodontal ligament in all sections. An intense positive stain was observed in the extracellular matrix where the periodontal ligament fibers insert into the alveolar bone and where the Sharpey's fibers insert into the cementum. There was no staining evident in the mineralized cellular and acellular cementum. The intensity of immunoreactivity to the antifibromodulin antibody increased proportionally with increasing tissue maturation. CONCLUSION: The results from this study suggest that fibromodulin is a significant component of the extracellular matrix in the periodontal ligament during development, and may play a regulatory role in the mineralization process or maintaining homeostasis at the hard-soft tissue interface during cementogenesis.     

Appearance of osteoclasts by injections of lipopolysaccharides in rat periodontal tissue

The effect of lipopolysaccharides (LPS) on periodontal tissue was studied in 11-week-old Wistar rats. Four injections of 500 micrograms LPS induced marked osteoclastic alveolar bone resorption, whereas no alveolar bone resorption occurred after four injections of physiological saline. The osteoclast count increased progressively during the four injections of 500 micrograms LPS. After eight injections, however, the osteoclast count fell from the maximum level after four injections, although osteoclasts continued to increase in size and demonstrate an increased number of nuclei. Comparison of the osteoclast number between a series of four injections of 5, 50 or 500 micrograms LPS each revealed a dose-dependent increase. This strongly suggests LPS induction of osteoclastic bone resorption in vivo. Combined use of indomethacin, a prostaglandin synthesis inhibitor, with LPS injections inhibited both the LPS-induced alveolar bone resorption and osteoclast increase, suggesting a possible participation of prostaglandins in osteoclast-mediated bone resorption.     

Gingival tissue proteoglycan and chondroitin-4-sulphate levels in cyclosporin A-induced gingival overgrowth and the effects of initial periodontal treatment

OBJECTIVES: Cyclosporin A (CsA) is a potent immunosuppressive drug used in organ transplant patients to prevent graft rejection. CsA-induced gingival overgrowth is one of the side effects of this drug and its pathogenesis is still unclear. The present study was planned to comparatively analyse total proteoglycan (PG) and chondroitin-4-sulphate (C4S) levels in CsA-induced overgrown gingival tissue samples obtained before and after initial periodontal treatment and to compare these findings with the situation in healthy gingiva. MATERIAL AND METHODS: Gingival tissue samples were obtained from nine patients with CsA-induced gingival overgrowth before and 4 weeks after initial periodontal treatment including oral hygiene instruction and scaling and also from 10 healthy control subjects. Total PG and C4S levels were determined by biochemical techniques. PG levels were analysed using modified Bitter and Muir method. C4S assay was carried out using chondroitin sulphate lyase AC and chondroitin-6 sulphate sulphohydrolase enzymes. The results were tested statistically using non-parametric tests. RESULTS: All clinical measurements in the CsA-induced gingival overgrowth group demonstrated significant reductions 4 weeks after initial periodontal treatment (p<0.05). There was no significant difference between the levels of baseline total PG in CsA-induced gingival overgrowth and healthy control groups (p>0.05). The gingival tissue levels of PG in CsA-induced gingival overgrowth group decreased significantly 4 weeks after treatment (p=0.043). Gingival tissue C4S levels in the overgrowth group were significantly higher than the healthy control group at baseline (p=0.000). C4S levels of the overgrowth group were significantly reduced after treatment (p=0.033), but these levels were still significantly higher than the healthy control group (p=0.000). CONCLUSION: The observed prominent increase in gingival tissue C4S levels may be interpreted as a sign of an increase in C4S synthesis in CsA-induced gingival overgrowth. Furthermore, remission of clinical inflammation by means of initial periodontal treatment had a positive effect on tissue levels of these extracellular matrix molecules.     

Immunohistochemical localization of cytokeratins in ameloblastomas

Cytokeratins (CKs) are a group of intermediate filaments expressed in epithelial cells. There are 20 CK types that vary in molecular weight.