The Multifarious Functions of Leukotrienes in Bone Metabolism.

Leukotrienes (LTs) are derived from arachidonic acid metabolism by the 5-lipoxygenase (5-LO) enzyme. The production of LTs is stimulated in the pathogenesis of rheumatoid arthritis (RA), osteoarthritis, and periodontitis, with a relevant contribution to bone resorption. However, its role in bone turnover, particularly the suppression of bone formation by modulating the function of osteoclasts and osteoblasts, remains unclear. We investigated the effects of LTs on bone metabolism and their impact on osteogenic differentiation and osteoclastogenesis using a 5-LO knockout (KO) mouse model. Results from micro-computed tomography (µCT) analysis of femur from 8-week-old 5-LO-deficient mice showed increased cortical bone and medullary region in females and males and decreased trabecular bone in females. In the vertebra, we observed increased marrow area in both females and males 5-LO KO and decreased trabecular bone only in females 5-LO KO. Immunohistochemistry (IHC) analysis showed higher levels of osteogenic markers tissue-nonspecific alkaline phosphatase (TNAP) and osteopontin (OPN) and lower expression of osteoclastogenic marker tartrate-resistant acid phosphatase (TRAP) in the femurs of 5-LO KO mice versus wild-type (WT). Alkaline phosphatase activity and mineralization assay results showed that the 5-LO absence enhances osteoblasts differentiation and mineralization but decreases the proliferation. Alkaline phosphatase (ALP), Bglap, and Sp7 gene expression were higher in 5-LO KO osteoblasts compared to WT cells. Eicosanoids production was higher in 5-LO KO osteoblasts except for thromboxane 2, which was lower in 5-LO-deficient mice. Proteomic analysis identified the downregulation of proteins related to adenosine triphosphate (ATP) metabolism in 5-LO KO osteoblasts, and the upregulation of transcription factors such as the adaptor-related protein complex 1 (AP-1 complex) in long bones from 5-LO KO mice leading to an increased bone formation pattern in 5-LO-deficient mice. We observed enormous differences in the morphology and function of osteoclasts with reduced bone resorption markers and impaired osteoclasts in 5-LO KO compared to WT osteoclasts. Altogether, these results demonstrate that the absence of 5-LO is related to the greater osteogenic profile. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Long-term dexamethasone treatment alters the histomorphology of acinar cells in rat parotid and submandibular glands.

Glucocorticoids (GCs) induce insulin resistance (IR), a condition known to alter oral homeostasis. This study investigated the effects of long-term dexamethasone administration on morphofunctional aspects of salivary glands. Male Wistar rats received daily injections of dexamethasone [0.1 mg/kg body weight (b.w.), intraperitoneally] for 10 days (DEX), whereas control rats received saline. Subsequently, glycaemia, insulinaemia, insulin secretion and salivary flow were analysed. The parotid and submandibular glands were collected for histomorphometric evaluation and Western blot experiments. The DEX rats were found to be normoglycaemic, hyperinsulinaemic, insulin resistant and glucose intolerant (P < 0.05). DEX rat islets secreted more insulin in response to glucose (P < 0.05). DEX rats had significant reductions in the masses of the parotid (29%) and submandibular (16%) glands (P < 0.05) that was associated with reduced salivary flux rate. The hypotrophy in both glands observed in the DEX group was associated with marked reduction in the volume of the acinar cells in these glands of 50% and 26% respectively (P < 0.05). The total number of acinar cells was increased in the submandibular glands of the DEX rats (P < 0.05) but not in the parotid glands. The levels of proteins related to insulin and survival signalling in both glands did not differ between the groups. In conclusion, the long-term administration of dexamethasone caused IR, which was associated with significant reductions in both mass and flux rate of the salivary glands. The parotid and submandibular glands exhibited reduced acinar cell volume; however, the submandibular glands displayed acinar hyperplasia, indicating a gland-specific response to GCs. Our data emphasize that GC-based therapies and insulin-resistant states have a negative impact on salivary gland homeostasis.

The impact of healing time before loading on orthodontic mini-implant stability: a histomorphometric study in minipigs.

OBJECTIVE: To evaluate healing time before loading, areas compression and tension and location of insertion on mini-implant stability. DESIGN: Six minipigs were used. Each animal received 3 mini-implants in each quadrant: 1 mini-implant was used as an unloaded control (G1, n=24); the other 2 were loaded with 150g-force at three time intervals (G2: immediate loading, G3: after 15 days and G4: after 30 days), with 16 mini-implant in each experimental group. After 120 days, tissue blocks of the areas of interest were harvested. Clinical analysis (exact Fisher test) determined the survival rate. Histological analysis (Kontron KS 300™, Zeiss) quantified the fractional bone-to-implant contact (%BIC) and bone area (%BA) at each healing time point, areas of interest, and insertion site (ANOVA and t tests for dependent and independent samples). RESULTS: The mini-implant survival rates were G1: 71%, G2: 50%, G3: 75% and G4: 63%, with no statistical differences between them. The groups presented similar %BIC and %BA. There were no differences between the compression and tension sides or maxillary and mandibular insertion sites. CONCLUSIONS: These results suggest that low-intensity immediate or early orthodontic loading does not affect mini-implant stability, because similar histomorphometric results were observed for all the groups, with partial osseointegration of the mini-implants present.

High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets.

Activation of insulin signaling and cell cycle intermediates is required for adult beta-cell proliferation. Here, we report a model to study beta-cell proliferation in living rats by administering three different doses of dexamethasone (0.1, 0.5, and 1.0 mg/kg ip, DEX 0.1, DEX 0.5, and DEX 1.0, respectively) for 5 days. Insulin sensitivity, insulin secretion, and histomorphometric data were investigated. Western blotting was used to analyze the levels of proteins related to the control of beta-cell growth. DEX 1.0 rats, which present moderate hyperglycemia and marked hyperinsulinemia, exhibited a 5.1-fold increase in beta-cell proliferation and an increase (17%) in beta-cell size, with significant increase in beta-cell mass, compared with control rats. The hyperinsulinemic but euglycemic DEX 0.5 rats also showed a significant 3.6-fold increase in beta-cell proliferation. However, DEX 0.1 rats, which exhibited the lowest degree of insulin resistance, compensate for insulin demand by improving only islet function. Activation of the insulin receptor substrate 2/phosphatidylinositol 3-kinase/serine-threonine kinase/ribosomal protein S6 kinase pathway, as well as protein retinoblastoma in islets from DEX 1.0 and DEX 0.5, but not in DEX 0.1, rats was also observed. Therefore, increasing doses of dexamethasone induce three different degrees of insulin requirement in living rats, serving as a model to investigate compensatory beta-cell alterations. Augmented beta-cell mass involves beta-cell hyperplasia and, to a lower extent, beta-cell hypertrophy. We suggest that alterations in circulating insulin and, to a lesser extent, glucose levels could be the major stimuli for beta-cell proliferation in the dexamethasone-induced insulin resistance.

[Assessment of bovine biomaterials containing bone morphogenetic proteins bound to absorbable hydroxyapatite in rabbit segmental bone defects]. / Avaliação de biomateriais bovinos contendo proteínas morfogenéticas ósseas absorvidas a hidroxiapatita em defeitos ósseos segmentares em coelhos.

PURPOSE: To evaluate the osteo-regenerative capacity of two proprietary bone grafting materials, using a segmental defect model in both radial diaphyses of rabbits. METHODS: The right defect was filled with pooled bone morphogenetic proteins (pBMPs) bound to absorbable ultrathin powdered hydroxyapatite (HA) mixed with inorganic and demineralized bone matrix and bone-derived collagen, derived from bovine bone (Group A). The left defect was filled with bovine demineralized bone matrix and pBMPs bound to absorbable ultrathin powdered HA (Group B). In both groups, an absorbable membrane of demineralized bovine cortical was used to retain the biomaterials in the bone defects, and to guide the tissue regeneration. The rabbits were euthanized 30, 90 and 150 days after surgery. Radiographic, tomographic and histologic evaluations were carried out on all specimens. RESULTS: At 30 days, the demineralized cortical bone cover was totally resorbed in both groups. HA was totally resorbed from Group A defects, whereas HA persisted in Group B defects. A prominent foreign body reaction was evident with both products, more pronounced in sections from Group B. At 90 days, the defects in Group B exhibited more new bone than Group A. However, at 150 days after surgery, neither treatment had stimulated complete repair of the defect. CONCLUSION: The partial bone healing of the segmental defect occurred with low or none performance of the biomaterials tested.

Assessment of bovine biomaterials containing bone morphogenetic proteins bound to absorbable hydroxyapatite in rabbit segmental bone defects

PURPOSE: To evaluate the osteo-regenerative capacity of two proprietary bone grafting materials, using a segmental defect model in both radial diaphyses of rabbits. METHODS: The right defect was filled with pooled bone morphogenetic proteins (pBMPs) bound to absorbable ultrathin powdered hydroxyapatite (HA) mixed with inorganic and demineralized bone matrix and bone-derived collagen, derived from bovine bone (Group A). The left defect was filled with bovine demineralized bone matrix and pBMPs bound to absorbable ultrathin powdered HA (Group B). In both groups, an absorbable membrane of demineralized bovine cortical was used to retain the biomaterials in the bone defects, and to guide the tissue regeneration. The rabbits were euthanized 30, 90 and 150 days after surgery. Radiographic, tomographic and histologic evaluations were carried out on all specimens. RESULTS: At 30 days, the demineralized cortical bone cover was totally resorbed in both groups. HA was totally resorbed from Group A defects, whereas HA persisted in Group B defects. A prominent foreign body reaction was evident with both products, more pronounced in sections from Group B. At 90 days, the defects in Group B exhibited more new bone than Group A. However, at 150 days after surgery, neither treatment had stimulated complete repair of the defect. CONCLUSION: The partial bone healing of the segmental defect occurred with low or none performance of the biomaterials tested.

OBJETIVO: Avaliar a capacidade osteo-regenerativa de dois biomateriais utilizando um modelo de defeito segmentar efetuado nas diáfises do rádio de coelhos. MÉTODOS: O defeito direito foi preenchido com pool de proteínas morfogenéticas ósseas (pBMPs) e hidroxiapatita em pó ultrafina absorvível (HA) combinada com matriz óssea inorgânica desmineralizada e colágeno, derivados do osso bovino (Grupo A). O defeito esquerdo foi preenchido com matriz óssea desmineralizada bovina com pBMPs e hidroxiapatita em pó ultrafina absorvível (Grupo B). Em ambos os defeitos utilizou-se membrana reabsorvível de cortical bovina desmineralizada para reter os biomateriais no defeito ósseo e guiar a regeneração tecidual. Os coelhos foram submetidos à eutanásia aos 30, 90 e 150 dias após a cirurgia. Foram efetuados exames radiográficos, tomográficos e histológicos em todos os espécimes. RESULTADOS: Aos 30 dias de pós-cirúrgico, o osso cortical desmineralizado foi totalmente reabsorvido em ambos os grupos. A HA tinha reabsorvido nos defeitos do Grupo A, mas persistiu nos do Grupo B. Uma reação de corpo estranho foi evidente com ambos os produtos, porém mais pronunciada no Grupo B. Aos 90 dias os defeitos do grupo B tinham mais formação óssea que os do Grupo A. Entretanto, aos 150 dias após a cirurgia, nenhum tratamento havia promovido o completo reparo do defeito. CONCLUSÃO: Os biomateriais testados contribuíram pouco ou quase nada para a reconstituição do defeito segmentar.

Ultrastructural morphometric analysis of ameloblasts exposed to fluoride during tooth development.

Since a considerable amount of the world population is exposed to high doses of fluoride, it is of special concern to investigate its action mechanisms during dental enamel development. In this study, the toxicity of fluoride in ameloblasts during enamel development was evaluated by means of ultrastructural morphometric analysis. A total of 18 male Wistar rats were distributed into three groups. In Group I, the animals received deionized drinking water ad libitum (negative control) and in Groups II and III, they received sodium fluorided (NaF) drinking water at doses of 7 and 100 ppm ad libitum, respectively, for 6 weeks. Morphometric data were expressed as volume density of the most significant organelles present in the secretory and maturation phases of amelogenesis such as RER, granules, lysosomes, phagic vacuoles, microfilaments and mitochondria. The results showed that the volume density of mitochondria in the 100 ppm experimental group was 29% (P < 0.05) higher than the control group in secretory ameloblasts. No remarkable differences were found in maturation ameloblasts for all organelles evaluated. Taken together, these data indicate that NaF at high doses is able to induce cellular damage in secretory ameloblasts, whereas no noxious effect was observed during maturation stage of amelogenesis as depicted by ultrastructural analysis.

Exfoliative cytology of the oral mucosa in type II diabetic patients: morphology and cytomorphometry.

BACKGROUND: In recent years, important advances have occurred in the determination of diagnostic criteria for the disease diabetes mellitus and in new strategies for its treatment. The purpose of this research was to develop a new method for diabetes diagnosis by microscopic and cytomorphometric analyses of the oral epithelium. METHODS: The smears were obtained from three distinct oral sites: buccal mucosa (cheek), tongue dorsum, and floor of the mouth in 10 control individuals and 10 type II diabetic patients. The oral smears were stained with Papanicolaou EA-36 solution. The nuclear (NA) and cytoplasmic (CA) areas were evaluated from 50 integral cells predominant in each oral site by the use of the KS 300 image analysis system (Carl Zeiss, Germany), by which the cytoplasmic/nuclear ratio (C/N) was calculated. RESULTS: The results showed that: (i) the epithelial cells of the diabetic group exhibited figures of binucleation and occasional karyorrhexis in all layers; (ii) the NA was markedly higher (P < 0.05) in the diabetic group; (iii) the CA did not exhibit a statistically significant difference (P > 0.05) between these two groups; and (iv) the C/N mean was 37.4% lower in the type II diabetic group. CONCLUSIONS: These results associated with clinical observations suggest that diabetes mellitus can produce alterations in oral epithelial cells, detectable by microscopy and cytomorphometry, which can be used in the diagnosis of this disease.