G. Cimasoni

Functional Characteristics of Gingival and Periodontal Ligament Fibroblasts

In periodontal surgery, healing after Guided Tissue Regeneration (GTR) may be explained by differences in functional activities of gingival and periodontal ligament fibroblasts (GF and PDLF). Several studies in vitro have supported this hypothesis, but much remains to be defined. In the present work, gingival and periodontal ligament fibroblasts derived from five healthy subjects were isolated and compared in vitro. The morphology of the cells was observed under scanning electron microscopy (SEM). Several extracellular matrix components (ECM) were studied to compare the effects on fibroblast attachment, proliferation, and protein synthesis. Several biochemical markers were examined in both cellular extract (CE) and conditioned medium (CM). We also examined the muscle differentiation markers alpha-smooth muscle actin, desmin, and smooth-muscle myosin. Finally, we studied the effects of epithelial cells on the proliferation and protein synthesis of the two types of fibroblasts. GF and PDLF appeared identical under the SEM. All ECM components enhanced attachment; however, while collagen types I and IV promoted the attachment of GF, gelatin, laminin, and vitronectin promoted that of PDLF. Most ECM components increased the proliferation rate of GF and the biosynthetic activity of PDLF. The biochemical markers were similarly distributed between the two cell types, except for alkaline phosphatase, which was detected only in the CE of PDLF. Both GF and PDLF strongly expressed alpha-smooth-muscle actin and were negative for desmin; only PDLF were positive for smooth-muscle myosin. Epithelial cells increased the proliferation of both GF and PDLF but had no effect on their biosynthetic activity. These in vitro results may better explain the in vivo functional differences between GF and PDLF.

Increase of free collagenase and neutral protease activities in the gingival crevice during experimental gingivitis in man

Abstract Free activities of collagenase and neutral protease were measured in the gingival washings collected with individual appliances before, during and after a 21-day period of experimental gingivitis in 8 male subjects who refrained from tooth-brushing. Collagenase was determined by measuring the release of soluble radioactivity from3H-labelled, reconstituted collagen fibrils and neutral protease by measuring the breakdown of haemoglobin. The concentration of collagenase and neutral protease increased significantly during abstention from tooth-brushing, as did the specific activities of the two enzymes (free enzyme activity/number of polymorphonuclear (leukocytes). SDS-polyacrylamide gel electrophoresis of the digestion products of native collagen mixed with concentrated gingival washings showed that the collagenase in the gingival crevice is partly of mammalian origin. The inhibition study with serum and EDTA suggested a minor contribution of bacterial collagenase.

Neutrophil Elastase and its Inhibitors in Human Gingival Crevicular Fluid during Experimental Gingivitis

The relative concentrations and absolute amounts of neutrophil elastase and its two inhibitors, α2-macroglobulin (α2-M) and alantitrypsin (α1-AT), were determined in gingival crevicular fluid (GCF) collected from six dental students who refrained from brushing the upper left or right quadrant during three weeks. Plaque and gingival indices and flow of GCF were measured before, during, and after the three weeks of no brushing. Functional elastase, representing the enzyme complexed with a2-M, was measured by use of a low-molecular-weight fluorogenic substrate. Elastolytic activity in GCF was also assayed by use of elastin as substrate. Antigenic elastase, representing the enzyme complexed with a 1-AT, as well as the inhibitors a2-M and al-AT were measured by ELISA. After three weeks of plaque accumulation, the concentrations of both functional and antigenic elastase increased by a factor of about 3, whereas the concentrations of the inhibitors increased in a much higher proportion. No free elastase could be detected in GCF, as evidenced by the Sephadex G-75 elution profile of GCF, by the negative results obtained when elastin was used as substrate, and by the demonstration that pure enzyme kept its activity against the low-molecular-weight substrate after being saturated by a2-M.

Human Gingival Crevicular Fluid Contains MRP8 (S100A8) and MRP14 (S100A9), Two Calcium-binding Proteins of the S100 Family

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (S100A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.

Beta-glucuronidase correlated with inflammation in the exudate from human gingiva

Abstract Gingival exudate and serum were collected from 23 patients with extensive periodontitis. The depths of the periodontal pockets were measured on the upper canine and incisor teeth, while the amount of bone loss in the same regions was evaluated on radiographs. Assays of β-glucuronidase activity have shown very high levels of the enzyme in gingival fluid, as compared to the enzyme concentrations found in serum. A positive, statistically significant correlation was found between the concentrations of β-glucuronidase in gingival exudate and the depths of the periodontal pockets, as well as the amount of bone loss. A positive, statistically significant correlation was also noticed between the activities of the enzyme in gingival fluid and those of serum, but the serum enzyme values remained within normal limits.

Possible role of lysosomal enzymes in the pathogenesis of periodontitis: A study on cathepsin D in human gingival fluid

Abstract Gingival exudate and serum were collected from sixteen patients with extensive periodontitis. The depths of the periodontal pockets were measured on the upper canine and incisor teeth, while the amount of bone loss in the same region was measured on radiographs. Cathepsin and protease were measured at increasing pH in gingival fluid, in serum and in an extract of bacteria grown from gingival fluid. Cathepsin was much higher in gingival fluid than in serum and showed a peak of optimum activity at pH 3.5 (cathepsin D). It was absent from the bacterial extract. A positive, statistically significant correlation was found between the concentrations of cathepsin D in gingival exudate and the depths of the periodontal pockets, as well as the mean percentage of bone loss. The results further substantiate a possible role of lysosomal enzymes in the pathogenesis of periodontitis.

Total Protein in Human Crevicular Fluid

Periodontal inflammation is accompanied by fluid in the crevicular sulcus in amounts that vary according to the severity of inflammation. Brill and Bronnestam (Acta Odont Scand 18: 95-100, 1960) have characterized, by means of immunoelectrophoresis, various protein fractions in crevicular fluid collected on filter paper strips; they have shown that the high molecular weight, a 2 macroglobulin can pass into the crevicular sulcus. Protein components also have been studied with paper electrophoresis by Mann and Stoffer (Periodontics 2:263-266, 1964), who found that the relative percentages of albumin and the various globulins are the same in gingival exudate as in serum. The difficulty encountered in collecting a sufficient amount of exudate for quantitative analysis is probably the reason why there is a lack of data in the literature on the total protein content of this fluid. Recently, a technique was standardized in our laboratory (T. SUEDA, J. BANG, and G. CIMASONI, J Dent Res 48:159, 1969) whereby 20 to 40 R1 of crevicular exudate can be collected in patients with moderate to severe gingivitis and periodontitis. This technique has made it possible to investigate quantitatively certain bacterial or lysosomal hydrolytic enzymes in gingival fluid (J. BANG, G. CIMASONI, and A. J. HELD, Arch Oral Biol 15:445-451, 1970). In 20 patients, analyses of total protein concentrations were carried out by the technique of Lowry, Rosenbrough, Farr, and Randall (I Biol Chem 193: 265-275, 1951). The gingival fluid collected from each patient was centrifuged for ten minutes in plastic microtubes, and 1 ml of supernatant was used for protein determination. The PMA index, the depths of the periodontal pockets, and the amount of bone loss were measured in the maxillary incisor and canine region with procedures reported previously (J. BANG, G.

Flow and albumin content of early (pre-inflammatory) gingival crevicular fluid from human subjects.

Gingival fluid was collected with glass capillaries tubing from the upper premolar area in a group of 7 volunteers, after allowing dental plaque to accumulate for 12-36 h, and in a group of patients with gingivitis. Whereas no fluid could be collected in the absence of plaque, increasing amounts were recovered during plaque accumulation, in the absence of clinical signs of gingival inflammation. The ratios of albumin concentrations in gingival fluid and plasma also increased significantly with increasing time of plaque accumulation. These fluid:plasma ratios of albumin concentrations were significantly lower than the ratios found for the inflamed sites in the second group of patients. These results support the hypothesis that, in an early inflammatory response, the fluid is not a typical inflammatory exudate and is probably modulated by an osmotic gradient.

Histopathology of the temporomandibular joint following bilateral extractions of molars in the rat: A preliminary report

Abstract Twenty-two female rats were divided into three groups. In each group some of the animals were kept as controls, while the upper molars of the other animals were extracted. Rats of the three groups were killed at the ages of 240 days, 330 days, and 480 days, respectively. The length of time that the animals lived with impaired occlusion increased from 200 days in Group 1 to 300 days in Group 2. One temporomandibular joint from each rat was prepared for histologic study. In the rats of Group 1, temporomandibular joint sections did not show any abnormal signs, whereas in most of the experimental rats of Groups 2 and 3 the following lesions were observed: 1. 1. Perichondrocytal calcifications (glenoid fossa) 2. 2. Necrosis of the cartilage of the fossa 3. 3. Structural alterations of the meniscus (repair?) 4. 4. Pannus The lesions were more frequent in older rats. All of the control animals had histologically normal temporomandibular joints.

Increase of extracellular cathepsin D activity in gingival washings during experimental gingivitis in man.

Abstract The free and total activities of cathepsin D were measured at regular intervals in the gingival washings, collected with individual appliances, in five subjects, during a period of experimental gingivitis. Counts of intact neutrophils and epithelial cells were made. The concentration of both free and total enzyme followed the changes in the clinical signs of gingivitis. The estimated amount of intracellular cathepsin D activity decreased in polymorphonuclear cells during the time of increasing inflammation. The specific activities of enzyme related to the number of neutrophils tended to decrease during the same period.