Human gingival glycosaminoglycans in cyclosporin-induced overgrowth

Glycosaminoglycans in normal and cyclosporin‐induced gingival overgrowth were extracted by papain digestion and purified by Mono Q‐FPLC chromatography. The purified glycosaminoglycans were analyzed by agarose gel electrophoresis and by the pattern of degradation products formed by chondroitin lyases on HPLC chromatography. Our results on the glycosaminoglycan composition showed presence of chondroitin 4‐ and 6‐sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in both normal gingiva and cyclosporin‐induced gingival overgrowth. The total and relative amounts of glycosaminoglycans were similar between normal and overgrown gingiva. This suggests that the glycosaminoglycan composition is not changed in cyclosporin‐induced gingival overgrowth. Our present biochemical results conflict with histochemical and biosynthetic data previously reported by other groups. Those studies suggested that the affected tissues contained higher levels of glycosaminoglycans and that cyclosporin induced comparably high levels of these compounds in in vitro cultures of gingival fibroblasts. Therefore, these discrepant results suggest that a cyclosporin‐induced increase on gingival glycosaminoglycans still remains an open question. The implications of these conflicting results are discussed.     

The effect of chronic inflammation on gingival connective tissue proteoglycans and hyaluronic acid

Proteoglycans have been isolated and analysed from extracts of normal and chronically inflamed human gingiva in order to determine the effects of chronic inflammation on these important soft connective tissue extracellular macromolecules. The uronic acid content of glycosaminoglycans isolated by papain digestion of normal and inflamed gingiva did not differ significantly. Likewise, electrophoretic analysis revealed that the content of hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfute was similar. The sulfated glycosaminoglycans from both sources eluted from a Sepharose C1-6B column with a Kav of 0.45 (approximate M_r 25,000). However, hyaluronic acid from normal gingiva was predominantly of a large size eluting in the void volume of a Sepharose. CL-6B column, while that isolated from inflamed tissue was mostly a small molecular weight species which elutccl in the included volume of a Sepharose CL-6B column. Using dissociative conditions, intact proteoglycans could be more readily extracted from inflamed tissues (90% of the total tissue uronic acid) than from normal tissues where only 80% of the total tissue uronic acid was extractable. Even though DEAE-Sephacel ion-exchange chromatography revealed no differences in charge between normal and inflamed gingival proteoglycans, Sepharose CL-4B chromatography revealed more molecular size polydispersity in samples from inflamed tissue than from normal tissue. Taken together, these results indicate that while hyaluronic acid is depolymerized in inflamed tissue, no evidence of sulfated glycosaminoglycan degradation was found. Therefore, the most likely cause for disruption to the molecular integrity of the proteoglycans is via proteolytic alteration to the proteoglycan core protein.     

Isolation, identification, and quantitation of glycosaminoglycans synthesized by human gingival fibroblasts in vitro

The glycosaminoglycans synthesized by diploid fibroblasts obtained from healthy human gingivae of three donors were isolated, identified, and quantified. Degradation with specific enzymes identified the glycosaminoglycans as hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; hyaluronic acid predominating. The distribution of the sulfated glycosaminoglycans in the cell layer and the medium was not the same. The cells contained mainly heparan sulfate (48.3%) and the medium mainly dermatan sulfate (47%).     

Proteoglycans and glycosaminoglycans in phenytion-induced gingival overgrowth

Proteoglycans and glycosaminoglycans in normal gingival and phenytoin-induced gingival overgrowth were studied by gel electrophoresis and HPLC methods after extration with guanidinium hydrochloride and subsequent cesium chloride gradient centrifugation. The results showed that normal gingival. The relative collagen content was decreased in the phenytoin lesion. These results are in agreement with our revious stereological study, which reported an accumulation of the non-collagenous matrix chondroitin sulfates Containing non-sulfated, 4-sulfated and 6-sulfated disaccharide units, dermatan sulfate, hyaluronic acid and presumably also heparan sulfate in both normal gingival. and phenyton-induced gingival overgrowth. The increased amounts of PGs seen in the PHT lesion were associated with an increase mainly in chondroitinase sensitive glycosaminoglycans of high molecular weight and with a relative increase in iduroinc acid content. This study is consistent with the view that the PHT-lesion represents a tissue with an altered composition of the connective tissue.     

Molecular size distribution of proteoglycans in human inflamed gingival tissue

Proteoglycans were extracted from human gingiva with 2 M CaCl2. The extracts were examined by gel filtration on Sephacryl S-400 in 2 M CaCl2 under dissociative conditions. The 280 nm absorbance profiles of clinically uninflamed, inflamed and severely-inflamed tissues showed that material was present with molecular weights of between 2 X 10(6) or greater, and 16,000. Proteoglycans were examined by cellulose-acetate electrophoresis with subsequent identification of the constituent glycosaminoglycans after protease digestion, and finally by chondroitinase AC digestion of the liberated glycosaminoglycans. The relative proportion of each glycosaminoglycan was calculated by scanning each cellulose-acetate sheet on an integrating densitometer. Heparan sulphate was found only in fraction I (mol. wt 2 X 10(6) or greater), together with hyaluronic acid and chondroitin-4-sulphate, these being present in all of the glycosaminoglycan-containing fractions (I-IV). Dermatan sulphate was absent from fraction I, but present in II-IV, apparently existing on the same protein core as chondroitin-4-sulphate. The relative proportions of these two glycosaminoglycans was related to molecular size, and with the degree of inflammation for a given molecular species.     

Molecular size distribution analysis of human gingival proteoglycans and glycosaminoglycans in specific periodontal diseases

Abstract. In order to determine the molecular-size distribution of gingival proteoglycans (PGs) and glycosaminoglycans (GAGs) both in periodontal health and disease states, gingival tissues were obtained from patients with early onset periodontally (EOP) and adult periodontitis (AP) and also from periodontally healthy subjects. Gel filtration chromatography of gingival PGs revealed different profiles for periodontally diseased and healthy gingiva. Healthy gingiva was mainly composed of high-molecular size proteins and PGs. while diseased gingival tissue presented a decrease in high-molecular size PG forms and a shift towards low-molecular size proteins and PGs. This indicates the degradation of PG macromolecules during periodontal disease activity. Furthermore, this shift towards low-molecular size forms was more intense in EOP patients when compared to AP patients. Gel filtration of gingival GAGs also demonstrated depolymerization of GAGs, with low-molecular size GAGs being more intense in periodontally diseased gingival, while healthy gingival GAGs profile was mainly composed of high-molecular size GAGs. Similar to the profile of gingival PGs. low-molecular size gingival GAGs. were more prominent in gingival tissue from patients with EOP, These findings suggest that both PGs and GAGs. essential components of the extracellular matrix (ECM). are depolmerized during eriodontal disease activity, which is more prominent in EOP, Since the basie feature of periodontal disease is matrix degradation. ECM components, more specifically PGs and GAGs, are likely to provide valuable information for a better understandi ng of periodontal disease activity.     

A biochemical and immunohistochemical study of the proteoglycans of alveolar bone

The purpose of this investigation was to study the proteoglycans in alveolar bone of three animal species. Alveolar bone was obtained from humans, pigs, and rabbits. Portions were fixed, sectioned, and stained with monoclonal antibodies against keratan sulfate and chondroitin sulfate. In other samples, biochemical analyses were performed. After removal of the organic matrix by 4 mol/L guanidinium HCl extraction in the presence of proteinase inhibitors, proteoglycans in the mineralized matrix were extracted with 4 mol/L guanidinium HCl/0.5 mol/L EDTA/proteinase inhibitors, and characterized on the basis of their glycosaminoglycan content (cellulose acetate membrane electrophoresis), charge (DEAE-Sephacel and hydroxylapatite chromatography), size (Sepharose CL-6B chromatography and agarose/polyacrylamide gel electrophoresis), and amino acid content. The results indicated that keratan sulfate could be detected immunohistochemically and biochemically in rabbit bone only. The predominant glycosaminoglycan in pig and human alveolar bone was chondroitin sulfate, although some hyaluronate, dermatan sulfate, and heparan sulfate were also detected. The proteoglycans were found to be slightly smaller than gingival proteoglycans, but similar to those in cementum, dentin, and other bones. In addition to intact proteoglycans, some free glycosaminoglycan chains were also extracted from the mineralized matrix. Amino acid analyses showed some subtle differences between alveolar bone proteoglycan and those of the soft tissues of the periodontium.     

On the mechanism of drug-induced gingival hyperplasia

Proposed mechanisms of the side effect of drug-induced gingival hyperplasia are reviewed. Hypotheses with regard to inflammation from bacterial plaque, increased sulfated glycosaminoglycans, immunoglobulins, gingival fibroblast phenotype population differences, epithelial growth factor, pharmacokinetics and tissuebinding, collagenase activation, disruption of fibroblast cellular sodium/calcium flux, folic acid and a combination hypothesis are evaluated.     

Synthesis of proteoglycans in rat mucosal keratinocytes

The synthesis of hyaluronic acid and proteoglycans by rat mucosal keratinocytes of an established cell line (CCL-10) has been investigated. Proliferating cultures at or near confluency were grown in the presence of [35S] sulfate or D-[1-3H] glucosamine for 24 h, and the glycosaminoglycan composition of cells and medium was determined. Characterization of the 35S-labelled glycosaminoglycans showed that heparan sulfate was the major component (approximately 90%) and that small amounts (approximately 10%) of galactosaminoglycans had also been synthesized. Analysis of cultures labelled with D-[1-3H] glucosamine demonstrated that hyaluronic acid was also present, most prominently in the medium where approximately one third of the radioactivity in the glycosaminoglycan pool was found in the hyaluronic acid fraction. [35S]-labelled proteoglycans extracted from the cell layer in the presence of protease inhibitors showed substantial heterogeneity upon chromatography on Sepharose CL-6B. In contrast, the proteoglycans in the medium gave a major peak which was eluted at a Kav of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.     

Glycosaminoglycans of temporomandibular articular discs

abstract — The glycosaminoglycans of the fibrous cartilage of temporomandibular articular discs from rat, rabbit, dog and monkey have been separated by means of cellulose acetate membrane electrophoresis on a micro scale. The total glycosaminoglycan amount present in these tissues was found to be 1.8, 1.8, 1.4 and 1.2 μg uronic acid, respectively, per mg tissue wet weight. Two major glycosaminoglycan fractions with the electrophoretic mobility of hyaluronate and dermatan sulfate were present, with the latter predominating.     

Glycosaminoglycans of human cementum

The glycosaminoglycans in human cementum have been studied. Following proteolytic digestion of guanidine/EDTA and collagenase extracts of cementum, glycosaminoglycans were isolated and then separated by cellulose acetate membrane electrophoresis. After specific elimination by enzymatic and chemical treatments the glycosaminoglycans were identified as hyaluronic acid, chondroitin sulfate and dermatan sulfate. Neither heparan sulfate nor keratan sulfate were observed. Quantitation of the glycosaminoglycans in both extracts revealed chondroitin sulfate to represent the major species present. Hyaluronic acid was observed predominantly in the guanidine/EDTA extract while dermatan sulfate was a quantitative minor component of both extracts.     

Ⅱ°根分叉病变患者引导组织再生治疗术前术后龈沟液中糖氨多糖水平的变化

目的　观察Ⅱ°根分叉病变患者引导组织再生治疗术 (guidedtissueregeneration ,GTR)前后龈沟液(gingivalcrevicularfluid ,GCF)中糖氨多糖 (glycosaminoglycans ,GAG)水平变化的同时 ,探讨GCF中GAG能否作为判断GTR术后组织成熟的指标。方法　对 6例Ⅱ°根分叉病变的患牙采用GTR治疗 ,并于手术前 ,手术后 1、2、3、4、5、6周收集GCF。用 0 .1%阿尔辛兰 (Alcianblue)染色 ,分光光度法测定GCF中总的硫酸化GAG及硫酸软骨素 (chondroitinsulfate ,CS)的水平。结果　GTR术后 1～ 2周 ,GCF中CS明显降低 (P <0 .0 5 ) ,然后逐渐升高 ,第 6周恢复到基线水平。而GCF中总的硫酸化GAG则在术后 1周明显升高 (P <0 .0 5 ) ,然后下降 ,到第 6周升高并超过基线水平。结论　GCF中总的硫酸化GAG ,尤其是CS可用作监测牙周伤口愈合和组织再生的一个潜在指标 ,但是否可以用作GTR术后组织成熟的指标 ,还需加大样本并结合病理进行进一步的纵向观察     

Studies on matrix components relevant to structure and function of the temporomandibular joint]

Proteoglycans (PGs), or their component glycosaminoglycan (GAG) chains, have long been recognized as small in quantity but as significant components for many connective tissues. The temporomandibular joint (TMJ) disc and synovial fluid were rarely investigated in this regard. This research was aimed at the biochemical analysis of the composition of collagen, GAGs and PGs in the normal human and porcine TMJ discs, and of PGs in the synovial fluid. 1. The collagen type analysis by gel electrophoresis revealed that the human and porcine TMJ discs were composed only of type I collagen. 2. The human TMJ disc contained 69.9% chondroitin sulfate, 24.5% dermatan sulfate, 5.6% hyaluronic acid and a trace amount of keratan sulfate, and the porcine TMJ disc, 5.8%, 91.4%, 2.8% and a trace amount, respectively. Immunohistochemical staining of the functional parts of the TMJ disc showed a universal distribution of these GAGs. 3. PGs were extracted from the TMJ disc with 4 M guanidine chloride and separated by ion-exchange and gel-filtration chromatography. A high molecular weight PG resembling the cartilage PG and two low molecular weight PGs having a core protein of Mr 40 K were isolated. 4. PGs suggested to be derived from the matrix of other tissues were found in the abnormal human synovial fluid. From these results it was suggested that the TMJ disc was thought not to be fibrocartilage but a fibrous tissue adapted against to the compressive and tensile force.     

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.     

Qualitative and quantitative analyses of bovine gingival glycosaminoglaycans.

Bovine gingival glycosaminoglycans have been analysed qualitatively and quantitatively by two-dimensional electrophoresis on a cellulose acetate strip. The four spots observed were identified as chondroitin 4-sulphate, dermatan sulphate, hyaluronic acid and heparan sulphate. Neither chondroitin 6-sulphate nor heparin and keratan sulphate were observed.The major components of bovine gingival glycosaminoglycans were chondroitin 4-sulphate, 32–40 per cent; dermatan sulphate, 33–37 per cent; hyaluronic acid, 17–27 per cent. Heparan sulphate was present only in a limited amount. The total uronic acid content of bovine gingiva, however, decreased with age, especially during the first three years of life, possibly due to the marked decrease of both chondroitin 4-sulphate and dermatan sulphate. After 3 years of age, the decrease of these glycosaminoglycans slowed down considerably. Hyaluronic acid decreased rather slowly from the time of birth to 10 years of age, and heparan sulphate decreased initially but increased later.     

Gingival crevicular fluid glycosaminoglycan levels in patients with chronic adult periodontitis

Abstract This study investigated levels of hyaluronan and chondroitin-4-sulphate in the crevicular fluid of patients with chronic adult periodontitis at diseased and healthy sites before and after treatment. The relationship between clinical diagnostic parameters and levels of glycosaminoglycans in gingival crevicular fluid were also analysed. Within each patient. 4 sites either mesial or distal and on single rooted teeth were classified as diseased or healthy using a modified gingival index, pocket depth and attachment loss. Crevicular fluid was collected from each site using glass micropipettes and analysed for glycosaminoglycan content by cellulose acetate electrophoresis. Significantly higher levels of chondroitin-4-sulphate were detected at diseased sites prior to treatment correlating with increased pocket depth or attachment levels. Following a period of treatment consisting of oral hygiene instruction and root planing, the patients were reassessed for their response to treatment by measuring the modified gingival index, pocket depth, attachment loss and levels of glycosaminoglycans. Analysis of glycosaminoglycan levels at diseased sites that demonstrated a poor response to treatment also demonstrated significantly higher levels of chondroitin-4-sulphate than those sites that responded well to treatment. Hyaluronan levels were less significantly associated with clinically succesful treatment. This study confirmed the use of the sulphated glycosaminoglycan chondroitin-4–sulphate as a potential diagnostic aid of periodontal tissue destruction; however, further longitudinal studies are required to assess their performance.     

Glycosaminoglycans of gingival epithelium and connective tissue during experimental periodontitis in dogs

The purpose of this study was to investigate the changes in concentration of glycosaminoglycans (CAGs) and to investigate the incorporation of 3H-glucosamine into GAGs in vitro in the epithelium and sub-epithelium connective tissue separated from the gingiva during a period of experimental periodontitis. Periodontitis was induced by placement of a silk ligature below the gingival margin in dog molars. The GAGs extracted from gingival samples obtained 0, 7, 21, 60 and 90 days before and after the ligature placement were separated by cellulose acetate membrane electrophoresis for both qualitative and quantitative analysis. Hyaluronic acid content of the epithelium was decreased significantly at the acute phase of inflammation. In the connective tissue, the amounts of dermatan sulfate and hyaluronic acid were higher, but chondroitin sulfate and heparan sulfate levels lower than in the control. The incorporation of 3H-glucosamine into GAGs in the epithelium was greater than that in connective tissue at the acute phase. The greatest incorporation of 3H-glucosamine was found in chondroitin sulfate at the acute phase, and did not return to the basal level at the chronic phase. These findings suggest that the biochemical response of GAGs in the epithelium to inflammation might be different from that in connective tissue.     

Glycosaminoglycans in normal and osteoarthrotic human temporomandibular joint disks.

Glycosaminoglycans in normal and osteoarthrotic temporomandibular joint disks were studied by means of high-performance liquid chromatography methods. Normal disk tissue contains galactosaminoglycans (chondroitin sulfate and dermatan sulfate) as the main polysaccharides and with smaller amounts of hyaluronate and heparan sulfate. The galactosaminoglycans are mainly sulfated in 6-position, and some of the disaccharides contain iduronic acid. There was a slight general variation in glycosaminoglycan concentration with increasing age. In the severely arthrotic disks the content of glycosaminoglycans was considerably lower than in normal disk tissue. This decrease was far more extensive than that observed in relation to age in normal tissue. The 4/6-sulfate ratio of the galactosaminoglycans was increased, whereas the proportion of iduronic acid was markedly decreased.     

Occurrence of multiple dentigerous cysts in a patient with the Maroteaux-Lamy syndrome (mucopolysaccharidosis, type VI)

The mucopolysaccharidoses (MPS) are a group of genetic lysosomal storage diseases. These diseases result from a defect in specific lysosomal enzymes required for the degradation of specific mucopolysaccharides. These incompletely degraded saccharides accumulate in tissues and are excreted in the urine. A general characteristic of these diseases is dysostosis multiplex. Dental complications can be severe and include unerupted dentition, dentigerous cystlike follicles, malocclusions, condylar defects, and gingival hyperplasia. This report examines multiple dentigerous cysts in a patient with a deficiency in N-acetylgalactosamine-4-sulfatase, Maroteaux-Lamy syndrome (MPS VI). The inability to hydrolyze the sulfate group from N-acetylgalactosamine-4-sulfate residue of dermatan sulfate due to a deficiency in this enzyme results in the accumulation of dermatan sulfate in tissues and its excretion in the urine. Examination of dentigerous cyst fluid revealed glycosaminoglycan content of 397 microgram per milliliter. Compositional analyses revealed 60% hyaluronic acid, 30% chondroitin 4- and -6-sulfate, and only 10% dermatan sulfate. This was consistent with dentigerous cyst fluid derived from persons without mucopolysaccharide-storage disorders but distinctly different from glycosaminoglycans assayed from other body fluids of this patient.