A preliminary study of the effects of laser radiation on collagen metabolism in cell culture

A low power Ga-As pulse laser was used to stimulate cultured human embryonic fibroblast cells. Energy fluencies varied from 0–1 J/cm² over a period of 1–4 days. Fibroblast procollagen production was monitored by the synthesis of [³H] hydroxy-proline, and DNA replication was assessed by [³H] thymidine incorporation. Following laser treatment, controlled pepsin digestion measured the increase in cell biostimulation. Maximum increase in collagen production and cell biostimulation occurred after 4 episodes of laser treatment at 24-hour intervals. Laser doses between 0.099 and 0.522 J/cm² had the most significant stimulatory effects on fibroblast function. Clinical efficacy of the low power Ga-As pulse laser may be related to enhanced connective tissue repair.     

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-laballed
glycosaminoglycans showed that heparan sulfate was the major component (∼90%) and that small amounts (∼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 K_av of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.     

Effect of macrophage depletion on growth and neovascularization of hamster buccal pouch carcinomas

The effect of macrophage depletion on growth and neovascularization of 7, 12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinomas (HBPC) was evaluated by quantitating tritiated thymidine (³[H]TdR) incorporation by tumor cells and microvascular endothelium in light microscopic autoradiographs. Tumors that were depleted of macrophages with systemic hydrocortisone acetate (HA) and intratumor injections of antimacrophage serum (AMS) were examined 7 days after treatment. In control animals 30% of infiltrating host cells were esterase-positive tumor-associated macrophages (TAM) and 28.14% of tumor cells and 14.45% of endothelial cells were ³[H]TdR labelled. Hamsters treated with HA or HA and control serum showed no significant reduction in either the number of TAM or proportion of [³H]TdR labelled tumor of endothelial cells. AMS administered alone had no effect on either the content of TAM or ³[H]TdR labelling. In contrast hamsters treated with HA and AMS showed a 54% decrease in TAM and a 50% and 63% reduction in tumor and endothelial cell labelling respectively. These results suggest that growth and neovascularization of these tumors is mediated in part by macrophages.     

Effect of calcium, given before or after a fluoride insult, on hamster secretory amelogenesis in vitro

We tested the hypothesis that high-calcium medium given prior to or immediately after exposure to fluoride (F) reduces the negative effects of F on secretory amelogenesis. Hamster molar tooth germs were grown in organ culture in media with different calcium levels. Deposition of enamel matrix and matrix mineralization were monitored by incorporation of [³H]proline and uptake of ⁴⁵Ca and acid-soluble ³²PO₄. Ameloblast structure and the occurrence of a fluorotic enamel matrix were examined by light and electron microscopy. A preculture of explants in high-calcium medium partially prevented the formation of fluorotic (non-mineralizing) enamel matrix, increased matrix secretion but could not prevent F-induced hypermineralization of the pre-exposure enamel. High-calcium medium, applied after F insult, accelerated the recovery of fluorotic matrix, improved ameloblast structure, enhanced amelogenin secretion, and increased enamel thickness. The data indicate that it might be the balance between the amount of mineral deposition and that of matrix secretion which is critical for the mineralization of newly secreted enamel. Exposure to F disturbs this balance by enhancing mineralization of the pre-exposure enamel, probably generating an excess of protons. High calcium may protect against F exposure by enhancing amelogenin secretion into the enamel space, thereby increasing the local buffering capacity at the mineralization front.     

Effects of epidermal growth factor and nerve growth factor on isoproterenol-induced DNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

When epidermal (EFG) (10 ng/kg body wt) or nerve growth factor (NGF) (1 ng/kg body wt) was given intraperitoneally to sialadenectomized young rats (submandibular-sublingual (SM-SL) glands removed) prior to injection of isoproterenol (ISO) (50 mg/kg body wt), the inhibition of ISO-induced thymidine incorporation into DNA of parotid gland and pancreas caused by removal SM-SL glands was reversed, and thymidine incorporation of sialadenectomized ISO-treated organs was as high as that of parotid and pancreas of surgically intact animals given ISO. EOF alone caused an increase in [³H] thymidine incorporation into DNA of parotid (63%) and pancreas (59%); removal of the SM-SL glands caused a decrease of 57–70% in thymidine incorporation into DNA of parotid, pancreas, liver, lung, kidney, and spleen. A growth effect attributable to the EGF and NGF of the submandibular gland was thus apparent for all organs examined, but even if they had large complements of β₁, adrenoceptors, only the exocrine organs showed the ISO-induced β₁, adrenoceptor response to EOF and NGF. EGF and NGF thus interact only with β₁ adrenoceptors of exocrine organs to cause marked increase in [³H] thymidine incorporation of these organs.     

Dentin formation in formocresol pulpotomized primary monkey teeth studied by tetracycline and 3H-proline incorporation

Abstract – The influence of formocresol treatment on the pulp tissue of 24 primary monkey teeth was studied using tetracycline and ³H-proline labeling techniques. Six untreated monkey teeth served as controls. The tetracycline labeled teeth were examined between 352 and 600 d, following treatment. The ³H-proline labeled teeth were observed over a period of 22–607 d, the isotope being administered 15 d before extraction. The labeling was evaluated in the coronal, middle and apical area of the roots. Dentin formation as indicated by tetracycline labeling was observed in both control teeth and successfully treated teeth, as well as in some of the unsuccessfully treated teeth. The average dentin formation rate per day varied from 1 μm in the control teeth to 0.14 μm in the pulpotomized teeth ( P <0.01). Success of treatment was of significant importance for the amount of dentin formation ( P <0.001). Labeling with ³H-proline, indicating collagen synthesis, could be observed in the pulp and predentin of the majority of areas judged to be normal, degenerated or inflamed. Labeling was not observed in fixed or necrotic tissue. In degenerated pulp tissue the proline labeling was clearly reduced. The findings indicate that dentin formation and collagen synthesis may take place in formaldehyde influenced pulp tissue although at a decreased rate.     

Transmission electron microscopic study of giant tubules in bovine dentin

Giant tubules in the axiomesiodistally extended incisal dentin of unerupted permanent bovine incisors from 1/2-1 1/2-yr-old calves were studied by TEM. The pulpal portion of each giant tubule contained two capillaries forming a loop incisally. The capillaries consisted of a delicate, continuous, or fenestrated endothelium and often had a pericyte coating. Cells with a rich RER, mitochondria, and Golgi apparatus dominated along and just incisal to the vessels. Extravasated erythrocytes and occasional macrophages were also seen in the latter location. It was suggested that the pulpal retraction of the giant tubule vessels during primary dentinogenesis might be the result of an intermittent sequestration of the most incisal portion of the loop, concomitant with endothelial proliferation in the pulpal portion of the capillaries. The major portion of the giant tubules, incisal to the vessels, contained a dense matrix of more or less axially oriented unmineralized 0.07-0.1-micron-thick collagen fibrils and single cells showing gradually increasing degenerative and necrotic changes in an incisal direction. Longitudinal sections of the mineralized tubular walls revealed transversely cut collagen fibrils. The incisally located giant tubule origins were circumvented by dentinal tubules and contained 0.10-0.22-micron-thick, densely packed collagen fibrils with an electron dense perifibrillar zone.     

Autoradiography of 90Sr in developing rats

Abstract— The distribution patterns of ⁹⁰Sr in five littermate. 8-day-old Wistar rats were studied by whole body autoradiography. Rats were killed 15 min, I, 4, 24, and 72 h after a single intraperitoneal injection of the isotope. Immediately after administration, ⁹⁰Sr was distributed throughout most of the soft tissues of the body. The soft tissue deposits had practically disappeared after 4 h. In the hard tissues of the body, ⁹⁰Sr accumulated up to 24–72 h. Fifteen minutes after injection the uptake of ⁹⁰Sr in the enamel of the teeth was highest in the occlusal and incisal regions. ⁹⁰Sr gradually accumulated throughout the enamel and after 72 h its distribution in this layer was fairly uniform. Immediately after injection a narrow zone of radioactivity appeared in the dentin near the pulp. This zone broadened with time towards the dentinoenamel junction and included the entire dentin layer 72 h after injection. Initially, the uptake of ⁹⁰Sr was higher in the dentin than in the enamel, particularly in the cervical areas of the crown. This difference became less apparent with time. There was good correlation between the uptake in teeth and bones, supporting the use of teeth as indicators of the ⁹⁰Sr body burden.     

Transmission electron microscopic study of giant tubules in bovine dentin

Abstract – Giant tubules in the axiomesiodistally extended incisal dentin of unerupted permanent bovine incisors from 1/2-11/2-yr-old calves were studied by TEM. The pulpal portion of each giant tubule contained two capillaries forming a loop incisally. The capillaries consisted of a delicate, continuous, or fenestrated endothelium and often had a pericyte coating. Cells with a rich RER, mitochondria, and Golgi apparatus dominated along and just incisal to the vessels. Extravasated erythrocytes and occasional macrophages were also seen in the latter location. It was suggested that the pulpal retraction of the giant tubule vessels during primary dentinogenesis might be the result of an intermittent sequestration of the most incisal portion of the loop, concomitant with endothelial proliferation in the pulpal portion of the capillaries. The major portion of the giant tubules, incisal to the vessels, contained a dense matrix of more or less axially oriented unmineralized 0.07–0.1-um-thick collagen fibrils and single cells showing gradually increasing degenerative and necrotic changes in an incisal direction. Longitudinal sections of the mineralized tubular walls revealed transversely cut collagen fibrils. The incisally located giant tubule origins were circumvented by dentinal tubules and contained 0.10–0.22-um-thick, densely packed collagen fibrils with an electron dense perifibrillar zone.     

Mucopolysaccharide localization in gingival epithelium

Short incubations, at 37°C, of small freshly excised pieces of healthy human gingivae iancorporated [³⁵S]-sulphate and [³H]-acetate into macromolecular material which could be precipitated intercellularly in the epithelium. Following radioactively pulse-chased incubations, such localization was observed by autoradiography on cryostat histological sections fixed in cetylpyridinium chloride. Critical electrolyte salt concentration elution indicated that most of the intercellular material was soluble in 0.63M-MgCl₂, and any material which remained was intracellular. In vitro and in vivo incorporation studies were compared. These data corroborate biochemical studies (Wiebkin, Bartold & Thonard 1979) together with other histochemical observations that proteoglycans (mucopolysaccharides) are a major intercellular component of human gingival epithelium. Molecular conformation and the relatively rapid synthesis and secretion rate for this class of epithelial macromolecule may explain the lack of susceptibility of this material in the intercellular site, both to degradation by some specific enzymes previously reported and to elution with critical salt concentrations from cationic detergent precipitates.
The method described, together with in vivo incorporation studies, provides a useful technique for studying direct effects of some microenvironmental influences on gingival epithelium.     

Effect of methyl mercaptan on synthesis and degradation of collagen

Measurements of the conversion of [¹⁴C]-proline to [¹⁴C]-hydroxyproline were employed to assess the effect of methyl mercaptan on intra- and extracellular metabolism of collagenous proteins in human gingival fibroblast cultures. Following a 30-min pulse, 10 ng of methyl mercaptan per ml of 95% air/5% CO₂, headspace suppressed collagen synthesis by 39% and increased the intracellular degradation of newly synthesized collagen from 26% to 42%. Parallel cultures assayed for proline transport demonstrated a 29% inhibition of [¹⁴C]-proline uptake. A similar analysis of cultures exposed to methyl mercaptan for 12 h revealed an increase in intracellular degradation (20% control vs. 30% test) and a marked increase in extracellular collagenolysis (4% control vs. 55% test). While pulsing, collagen synthesis was decreased by 39%. Slab gel electrophoresis also demonstrated that treatment with methyl mercaptan caused reductions both in mature a, and α₂ chains of type I collagen and in type III procollagen. Identities of the procollagen species were confirmed by pepsin digestion. Reverse transcribed polymerase chain reaction was utilized to compare expression of a1 chains of type I procollagen with type III procollagen and indicated suppression of mRNA synthesis for type III procollagen in cultures exposed to methyl mercaptan.     

Cyclosporin A upregulates prostaglandin E2 production in human gingival fibroblasts challenged with tumor necrosis factor alpha in vitro

The effect of Cyclosporin A (CsA) on prostaglandin E₂ (PGE₂) production in human gingival fibroblasts challenged with tumor necrosis factor alpha (TNF-α) was studied. TNF-α (1-100 ng/ml) dose-dependently stimulated PGE₂; formation in 24 h cultures. CsA (1-100 ng/ml) did not induce PGE₂; formation itself but potentiated TNF-α induced PGE; formation in gingival fibroblasts in a manner dependent on the concentrations of both CsA and TNF-α. TNF-α (10 ng/ml) stimulated the release of [³H]-arachidonic acid (A.A) from prelabelled fibroblasts that was potentiated by CsA (100 ng/ml). Addition of exogenous unlabelled AA (5-20 μM/ml) to the cells resulted in enhanced PGE₂: formation that was not potentiated by CsA (100 ng/mi). Furthermore. CsA (100 ng/ml) did not further increase the level of cyclooxygenase-2 mRNA induced by TNF-α (10 ng/ml). although PGE₂ formation was enhanced. The results indicate that CsA and TNF-α act in concert on PGE₂ formation in gingival fibroblasts. which may be of importance in the pathogenesis of gingival overgrowth induced by the drug.     

Human cytomegalovirus in peripheral giant cell granuloma

Background:  The peripheral giant cell granuloma is a relatively common non-neoplastic inflammatory lesion of gingiva, but the etiopathogeny remains unknown. This study aimed to evaluate the importance of human cytomegalovirus and Epstein–Barr virus in a peripheral giant cell granuloma of a 47-year-old female.
Methods:  The lesion was studied clinically, histopathologically, immunologically and virologically using established procedures.
Results:  The gingival growth was located at the mesial surface of the maxillary left canine having a vital pulp. The mass was 12 × 21 mm in size and exhibited a smooth surface with no evidence of fluctuation on palpation. An excisional biopsy revealed giant cells in a fibrohistiocytic stroma with areas of haemorrhage. Serum protein levels and lymphocyte subsets were within normal limits, except CD3⁺ and CD4⁺ cells were below normal ranges. Polymorphonuclear leukocytes expressed p150,95 (CD11c/CD18) and CXCR-2 receptors within normal ranges, but the CXCR1 receptor showed decreased density, and CD15 were below normal range. A virological sample of the tooth surface adjacent to the gingival swelling yielded 7.6 × 10³ copy-counts of cytomegalovirus and 4.3 × 10³ copy-counts of Epstein–Barr virus.
Conclusions:  The clinical and histological findings were consistent with the diagnosis of peripheral giant cell granuloma. Cytomegalovirus has the potential to induce multinucleated giant cells, and the possibility that the virus contribute to the development of peripheral giant cell granuloma merits further study.     

Synergistic enhancement of collagenous protein synthesis by human gingival fibroblasts exposed to nifedipine and interleukin-1-beta in vitro

Abstract: Gingival overgrowth commonly occurs coincident to therapy with calcium channel blockers. The biologic mechanism for this condition is unknown; however, many clinicians suggest that poor oral hygiene may contribute to development of the overgrowth. This study tests the hypothesis that collagenous protein synthesis by gingival fibroblasts is synergistically enhanced when they are exposed to both nifedipine (N) and the pro-inflammatory cytokine, interleukin-1-beta, a cytokine expressed in inflamed gingiva. Human gingival fibroblasts were isolated from biopsies of normal gingiva and cells separated into two groups. Group 1 was exposed to media containing 0, 5, 50, or 500 pg/ml IL-1-beta, or 10⁻⁷ M N for 7 days; Group 2 was exposed to those concentrations of IL-1-beta +10⁻⁷ M N. [³H]-proline was added to the medium for the final 24 h. Cells and matrix were harvested and radioactivity determined by liquid scintillation analysis. Means (d.p.m./10³ cells) were compared by factorial ANOVA and Scheffè comparisons. Collagenous protein synthesis was significantly reduced by 5 pg/ml IL-1-beta +10⁻⁷ M N and enhanced by 500 pg/ml IL-1-beta +10⁻⁷ M N as compared to N or IL-1-beta alone. Thus, patients may be more susceptible to gingival overgrowth coincident to nifedipine therapy as a result of the synergistic enhancement of connective tissue synthesis by these agents.     

Increased number of CD25+ FoxP3+ regulatory T cells in oral squamous cell carcinomas detected by chromogenic immunohistochemical double staining

Background:  The role of tumor-infiltrating regulatory T cells (Treg) compromising antitumor effects of immune cells in oral squamous cell carcinoma (OSCC) is largely unknown.
Purpose:  The presence of CD25⁺ FoxP3⁺ Treg as well as of CD3⁺ FoxP3⁺ and of CD8⁺ FoxP3⁺ tumor-infiltrating lymphocytes (TIL) was verified in OSCC and compared with non-cancerous lymphoepithelial tissue.
Method:  Three double stainings (CD3/FoxP3, CD8/FoxP3 and CD25/FoxP3) were performed on tissue sections of 15 OSCC and compared with 15 human tonsils.
Results:  OSCC biopsy samples provide evidence for a strong infiltration of TIL, in particular, naturally occurring CD25⁺ FoxP3⁺ Treg. Whereas a comparison of OSCC and control tissue did not show significant changes in the number of CD3⁺ FoxP3⁺ TIL and of CD8⁺ FoxP3⁺ TIL, a significantly higher frequency of CD25⁺ FoxP3⁺ TIL (Treg) could be observed in OSCC ( P  < 0.001, two-sided t -test). Given the small number of specimens, a significant correlation with tumor stage could not be verified.
Conclusion:  Chromogenic double staining of CD4/FoxP3 is a promising tool for the detection of Treg in paraffin-embedded tissue of OSCC.     

Scanning electron microscopic study of giant tubule content in bovine dentin

Incisal segments of unerupted permanent incisors from 1/2-1 1/2-yr-old calves were fractured along an axiomesiodistal plane exposing the organic components within the giant tubule lumina situated in this plane. Along the lining wall of the pulpal vascularized giant tubule portion, large flattened cells and a few odontoblasts were situated in shallow depressions. Just incisal to the vascular loop numerous cells were seen, both along the giant tubule wall and enclosed within a loosely textured collagenous matrix. Further incisally, the number and size of the cells decreased, and they were embedded in a compact unmineralized collagenous matrix that completely filled out the giant tubule lumina. This matrix consisted of fibrils regularly arranged in separate bundles whose orientation was mainly longitudinal. The incisal origins of the giant tubules were filled with coarse fibrils being about three times thicker than those of the luminal matrix and those of the circumpulpal dentin proper.     

The uptake of H3 proline in the guinea pig gingiva and palate

Keratinization is a complex process and at present only incompletely understood. This series of experiments demonstrates the uptake of H³ proline and subsequent appearance of a radioautographic label six to twelve hours later in the keratin of the guinea pig gingiva and palate. Furthermore, the label is lost between day four and day five. The negative result of externally applied H³ proline on the palate rules out external diffusion confirming that at least a portion of the keratin is proline derived.     

Binding of 3H-Methyltrienolone (3H-R1881) to androgen receptors in human gingiva

Binding of ³H-Methyltrienolone (³H-R1881) to cytoplasmic androgen receptor protein and its translocation into nuclei in human gingiva was demonstrated. ³H-R1881 binds to androgen receptors with high affinity (Kd= 1.7−1.9 × 10⁻⁹ M) and low capacity (10–16 fmol/mg protein). The binding was specific for DHT binding sites and was destroyed by proteolytic enzymes (pronase and trypsin). These results suggest that ³H-R1881 can be used as a ligand in combination with nonradioactive DHT to study androgen receptors in the gingiva.     

Whole cell agglutination and dextran binding in oral Actinomyces species

Dextran-induced agglutinability was studied with whole cells of 36 strains of oral Actinomyces species, including Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Rothia dentocariosa . Cells of seven A. viscosus strains of rodent origin were agglutinated upon addition of dextran T2000 (mean molecular weight, 2 × 10⁶) and bound more [¹⁴C]-dextran, which was synthesized by Leuconostoc mesenteroides dextransucrase from [¹⁴C]-sucrose, than cells of A. viscosus of human origin.     

The response of the fibroblast population in the periodontal ligament of rat incisors to altered eruption rates

The fibroblast population of the periodontal ligament (PDL) in impeded and unimpeded rat incisors was investigated. The material examined was limited to the functional compartment of the cementum-bordering tooth-related PDL (t-PDL) located between 7 mm and 15 mm from the tooth origin. In five albino rats, weight 200g, the lower left incisor was left to erupt unrestrainedly for three weeks (900μ/day) and in five other rats, eruption of the incisors was imeded (450μm/day). The left mandibles were dissected and fixed: the incisors were demineralized, embedded in glycolmethacrylate, and cut into 2-μ transverse sections. The sections were used to produce a three-dimensional reconstruction of the PDL by means of computerized histomorphomety and to count the fibroblast population. The obtained data was combined, thus providing information on cell kinetics. The size of the tooth-related PDL remained uniform along the impeded teeth, while in the unimpeded incisors it increased gradually in incisal direction, the total volumes estimated as 2.65 mm³ and 2.07 mm³, respectively. The calculated mean cell living space was 1,327 μm¹ in the impeded teeth, rising in the unimpeded ones from 503μm³ to 858μm³ in the ineisal direction. The estimated total number of fibroblasts in the PDL of the impeded teeth was 2,000X10³, the mean daily production rate amounting to 115x10³ cells, while in the unimpeded incisors these numbers totaled 2,957x10³ and 336x10³ cells, respectively.