Supraphysiologic Testosterone Administration Impairs Late Osseointegration in Male Rats.

PURPOSE: To evaluate the effect of supraphysiologic administration of testosterone in an early and late model of implant osseointegration in rat tibiae. MATERIALS AND METHODS: A total of 40 rats were randomly allocated into four groups (n = 10/ group), which were divided according to the type of experiment and time of osseointegration: (1) vehicle (14 days), (2) testosterone (14 days), (3) vehicle (42 days), and (4) testosterone (42 days). Testosterone cypionate (7.5 mg/kg) administration started 4 weeks before implant placement, and the injections were performed daily until euthanasia. Machined-surface titanium implants (2.2 mm in diameter and 4 mm high) were placed bilaterally in the tibia of animals 28 days after the first testosterone injection. At days 14 and 42 after implant placement, euthanasia was performed and the tibiae were harvested to perform biomechanical evaluation and histomorphometric analysis of bone-to-implant contact (BIC%) and bone between the threads (BBT%). RESULTS: There was no statistical difference in the removal torque of the implants between the groups treated with the vehicle (control group) or testosterone (P > .05). At 14 days of osseointegration, the BIC% and BBT% did not differ between vehicle or testosterone groups (P > .05), while at 42 days, both the BIC% and BBT% were significantly reduced by testosterone compared to the vehicle group (P < .05). CONCLUSIONS: Testosterone cypionate in supraphysiologic dose impaired late-phase osseointegration in rat tibiae.

Beneficial Effects of Two Hydrogen Sulfide (H2S)-Releasing Derivatives of Dexamethasone with Antioxidant Activity on Atopic Dermatitis in Mice.

Hydrogen sulfide (H2S) is particularly produced in the skin, where it participates in the regulation of inflammation, pruritus, cytoprotection, scarring, and angiogenesis. In this study, we compared the effects of dexamethasone (Dex) with two H2S-releasing Dex derivatives in a murine model of atopic dermatitis (AD) induced by topical application of 2,4-dinitrochlorobenzene (DNCB). After sensitization with DNCB, the animals were topically treated for five consecutive days with either the H2S-releasing compounds 4-hydroxy-thiobenzamide (TBZ) and 5-(p-hydroxyphenyl)-1,2-dithione-3-thione (ADT-OH), Dex, or the derivatives Dex-TBZ or Dex-ADT. Topical treatment with equimolar doses of either Dex, Dex-TBZ, or Dex-ADT resulted in similar reductions in dermatitis score, scratching behavior, edema, eosinophilia, splenomegaly, and histological changes. In contrast with Dex, the H2S-releasing derivatives prevented IL-4 elevation and oxidative modification of skin proteins. On an equimolar dose basis, Dex-TBZ, but not Dex-ADT, promoted the elevation of endogenous H2S production and GPx activity. Neither Dex-TBZ nor Dex-ADT decreased GR activity or caused hyperglycemia, as observed with Dex treatment. We conclude that the presence of H2S-releasing moieties in the Dex structure does not interfere with the anti-inflammatory effects of this corticosteroid and adds beneficial therapeutical actions to the parent compound.

Effects of red and infrared laser on post extraction socket repair in rats subjected to alendronate therapy.

This study evaluated the effect of photobiomodulation therapy (PBMT) with a red or infrared laser on the repair of post extraction sockets in rats administered alendronate (ALN). Forty male rats were randomly allocated into four groups: Control Group (CTR): subcutaneous administration of saline solution throughout the experimental period; Alendronate Group (ALN): subcutaneous administration of alendronate during the entire experimental period; Alendronate/Red Laser Group (ALN/RL): administration of ALN and irradiation with a GaAlAs laser (λ 660 nm); and Alendronate/Infrared Laser Group (ALN/IRL): administration of ALN and irradiation with a GaAlAs laser (λ 830 nm). The first lower molars were extracted 60 days after the beginning of the administration of the drugs. The PBMT was applied after tooth extraction (7 sessions with intervals of 48 hours between sessions). Thirty days after tooth extraction, the animals were euthanized. Micro-CT and histometric analysis were performed to assess the bone healing and soft tissue repair of the tooth socket. The ALN group presented with more bone than the CTR; however, most of this bone was necrotic. ALN does not affect the bone microarchitecture. On the other hand, PBMT with IRL enhances the bone density due to the increase in the number and reduction in the spacing of the trabeculae. The amount of vital bone and connective tissue matrix was higher in the ALN/RL and ALN/IRL groups than in the ALN and CTR groups. PBMT enhanced the healing of the post extraction sockets in rats subjected to ALN administration. Furthermore, IRL improved the new bone microarchitecture.

Testosterone replacement relieves ligature-induced periodontitis by mitigating inflammation, increasing pro-resolving markers and promoting angiogenesis in rats: A preclinical study.

OBJECTIVES: This study aimed to evaluate the inflammatory profile as well as the resolution of inflammation in a ligature-induced periodontal inflammation in rats with depletion and/or supraphysiological testosterone replacement. DESIGN: Sixty male rats (Holtzman) were used in the present study. Study groups were created as following: (1) Sham (no testicle removal); (2) Orchiectomy (OCX), 3) OCX + Testosterone (OCX + T); (4) Sham + Ligature (SH + L); (5) OCX+L; and 6) OCX + T + L. The surgeries were performed on day 1, and testosterone was administered weekly since day 1. On day 15, a cotton ligature was placed around the lower first molars and maintained for 15 days. Morphological changes in periodontal tissues were determined by histopathological analysis. Immunohistochemistry (factor VIII) and immunoenzymatic assay were performed to evaluate angiogenesis process and (pro- and anti-) inflammatory markers, respectively. RESULTS: Ligature promoted a marked inflammatory gingival infiltrate and bone loss (P < 0.05). Supraphysiological testosterone treatment increased the percentage of blood vessels, extracellular matrix and fibroblasts in the presence and absence of periodontal inflammation (P < 0.05). A high dose of testosterone increased factor VIII+ blood vessels and IL-10 expression in inflamed gingival tissue, while PGE2, LXA4 and MPO were reduced as a result of supraphysiological testosterone administration (P < 0.05). CONCLUSIONS: These results, in our experimental model, suggest that supraphysiological testosterone treatment stimulated gingival tissue repair during ligature-induced periodontitis, and it seems to be related to an anti-inflammatory and pro-resolutive mechanism resulting by the modulatory effect on PGE2 and IL-10 related to an enhanced angiogenesis.

Effects of red and infrared laser on post extraction socket repair in rats subjected to alendronate therapy

Abstract This study evaluated the effect of photobiomodulation therapy (PBMT) with a red or infrared laser on the repair of post extraction sockets in rats administered alendronate (ALN). Forty male rats were randomly allocated into four groups: Control Group (CTR): subcutaneous administration of saline solution throughout the experimental period; Alendronate Group (ALN): subcutaneous administration of alendronate during the entire experimental period; Alendronate/Red Laser Group (ALN/RL): administration of ALN and irradiation with a GaAlAs laser (λ 660 nm); and Alendronate/Infrared Laser Group (ALN/IRL): administration of ALN and irradiation with a GaAlAs laser (λ 830 nm). The first lower molars were extracted 60 days after the beginning of the administration of the drugs. The PBMT was applied after tooth extraction (7 sessions with intervals of 48 hours between sessions). Thirty days after tooth extraction, the animals were euthanized. Micro-CT and histometric analysis were performed to assess the bone healing and soft tissue repair of the tooth socket. The ALN group presented with more bone than the CTR; however, most of this bone was necrotic. ALN does not affect the bone microarchitecture. On the other hand, PBMT with IRL enhances the bone density due to the increase in the number and reduction in the spacing of the trabeculae. The amount of vital bone and connective tissue matrix was higher in the ALN/RL and ALN/IRL groups than in the ALN and CTR groups. PBMT enhanced the healing of the post extraction sockets in rats subjected to ALN administration. Furthermore, IRL improved the new bone microarchitecture.

Systemic Dietary Hesperidin Modulation of Osteoclastogenesis, Bone Homeostasis and Periodontal Disease in Mice.

This study aimed to evaluate the effects of hesperidin (HE) on in vitro osteoclastogenesis and dietary supplementation on mouse periodontal disease and femoral bone phenotype. RAW 264.7 cells were stimulated with RANKL in the presence or absence of HE (1, 100 or 500 µM) for 5 days, and evaluated by TRAP, TUNEL and Western Blot (WB) analyses. In vivo, C57BL/6 mice were given HE via oral gavage (125, 250 and 500 mg/kg) for 4 weeks. A sterile silk ligature was placed between the first and second right maxillary molars for 10 days and microcomputed tomography (µCT), histopathological and immunohistochemical evaluation were performed. Femoral bones subjected or not to dietary HE (500 mg/kg) for 6 and 12 weeks were evaluated using µCT. In vitro, HE 500 µM reduced formation of RANKL-stimulated TRAP-positive(+) multinucleated cells (500 µM) as well as c-Fos and NFATc1 protein expression (p < 0.05), markers of osteoclasts. In vivo, dietary HE 500 mg/kg increased the alveolar bone resorption in ligated teeth (p < 0.05) and resulted in a significant increase in TRAP+ cells (p < 0.05). Gingival inflammatory infiltrate was greater in the HE 500 mg/kg group even in the absence of ligature. In femurs, HE 500 mg/kg protected trabecular and cortical bone mass at 6 weeks of treatment. In conclusion, HE impaired in vitro osteoclastogenesis, but on the contrary, oral administration of a high concentration of dietary HE increased osteoclast numbers and promoted inflammation-induced alveolar bone loss. However, HE at 500 mg/kg can promote a bone-sparing effect on skeletal bone under physiological conditions.

Supraphysiological testosterone supplementation improves granulation tissue maturation through angiogenesis in the early phase of a cutaneous wound healing model in rats.

OBJECTIVE: The aim of this study was to evaluate the effects of both testosterone depletion and supraphysiological testosterone supplementation in the early phase of an animal cutaneous wound healing model in comparison with the physiological hormonal condition. MATERIAL AND METHODS: Forty rats were distributed into the following four groups: Control, Orchiectomy (OCX), Durateston (Dura) and OCX+Dura. On day 1, the testicles were removed (OCX group) and the rats (Dura group) received a supraphysiological dose (250 mg/kg) of exogenous testosterone weekly. After 15 days a full-thickness excisional skin wound was created in all animals, which was healed by the second intention for 7 days. On day 22, the rats were euthanatized and the wounds were harvested for histopathological evaluation, immunohistochemistry analyses and multiplex immunoassay. One-way ANOVA and post-hoc Tukey tests were performed. RESULTS: It was found that the supraphysiological testosterone level increased extracellular matrix deposition, promoted higher blood vessel formation and reduced wound contraction (p < 0.05). Additionally, it also stimulated PCNA-positive fibroblasts and KGF-positive cells (p < 0.05), while orchiectomy reduced the expression of IL-6 and TNF-α and increased VEGF and PDGF (p < 0.05) . CONCLUSION: In conclusion, the results provide evidence that supraphysiological testosterone supplementation plays a positive role in the early phase of cutaneous wound healing, thus improving granulation tissue maturation through the enhancement of angiogenesis.

Strontium ranelate improves post-extraction socket healing in rats submitted to the administration of bisphosphonates.

The aim of this study was to evaluate the effect of strontium ranelate (Sr) on post-extraction socket healing in rats submitted to the administration of bisphosphonates. Sixty rats were submitted to the tooth extraction of the first lower molar after 60 days of the daily administration of saline solution (SS) or alendronate (ALN). Then, the animals were allocated into six groups namely CTR: administration of SS during the whole experiment, ALN: administration of ALN during the whole experiment, ALN/SS: application of SS for 30 days after extraction in animals previously treated with ALN, ALN/Sr: application of Sr for 30 days after extraction in animals previously treated with ALN, ALN/S60: ALN therapy interruption 30 days before the extraction followed by the application of SS for 60 days, and ALN/Sr60: ALN therapy interruption 30 days before the tooth extraction followed by the application of Sr for 60 days. The healing of the post-extraction sockets was evaluated by microCT and histomorphometry. The use of ALN induced partial bone necrosis, inflammatory infiltration, and a delay in soft tissue healing; the use of Sr improved the connective tissue organization. Sr has subtle positive effects on the post-extraction healing in animals submitted to the administration of bisphosphonate.

Physiological testosterone replacement effects on male aged rats with orchiectomy-induced osteoporosis in advanced stage: a tomographic and biomechanical pilot study.

Aim: This study aimed to evaluate the effect of physiological testosterone replacement on male aged rats with orchiectomy-induced osteoporosis in advanced stage.Methods: Thirty male rats (Rattus norvegicus albinus, Holtzman lineage) were randomly distributed into 3 groups (n = 10): 1-sham, 2-orchiectomy (OCX), 3-OCX + testosterone replacement (OCX + T). On day 0, a sham or orchiectomy surgery was performed according to the groups. Thirty and sixty days after surgeries, the animals from OCX + T group received testosterone intramuscularly, and the rats in all groups were euthanized on day 77. The femurs were removed for micro-CT scanning and biomechanical test.Results: Orchiectomy resulted in a marked trabecular bone damage (p < 0.05), which was not reversed with testosterone treatment (OCX + T group). The femoral strength was lower in orchiectomized animals (p < 0.05), while the bone strength in OCX + T group was similar to that observed in the sham animals (p > 0.05) and correlated to this parameter the deformation of rupture was smaller in OCX + T group.Conclusion: In conclusion, testosterone depletion induced by orchiectomy established an osteoporotic environment, mainly affecting the trabecular bone. Moreover, even though testosterone treatment did not enhance these variables, the hormonal replacement improved the femoral fracture strength and promoted beneficial effects on the biomechanical parameters compromised by castration in femoral bone.

Impact of citrus flavonoid supplementation on inflammation in lipopolysaccharide-induced periodontal disease in mice.

In general, the consumption of flavonoid-rich foods may influence the control/dysregulation of the magnitude and duration of inflammation and oxidative stress, which are known to contribute to multiple pathologies. Information regarding the impact of citrus flavonoid dietary supplementation on periodontal disease is still scarce. Herein, we investigated whether a diet supplemented with eriocitrin and eriodictyol could alter the course of the inflammatory response associated with LPS-induced periodontal disease in mice. Sixty BALB/c mice received a standard diet or a diet supplemented with different concentrations of eriocitrin or eriodictyol. After 30 days of food supplementation, a solution containing LPS from Escherichia coli was injected into the gingival tissues three times per week for four weeks. Neutrophils, mononuclear cells and eosinophils were assessed using a severity analysis system in H&E-stained sections and modified picrosirius red. The activities of myeloperoxidase (MPO), a marker of granulocyte infiltration, and eosinophil peroxidase (EPO) were determined spectrophotometrically. The oxidative damage was determined by measuring the malondialdehyde (MDA) content and anti-oxidative activity through the assessment of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Interleukin (IL)-1ß, TNF-α, and IL-10 were quantified by multiplex immunoassay. Periodontal inflammation was significantly inhibited by citrus flavonoid supplementation, including reduced flatness of the gingival epithelium and chronic and acute inflammatory cell infiltration, as well as loss of connective tissue in the gingival papillae. Both eriocitrin and eriodictyol inhibited gingival IL-1ß and TNF-α and increased IL-10 secondary to periodontitis. Significant protection and decreased MPO and EPO activity were detected in the periodontal tissue of citrus flavonoid-treated animals. In comparison with the LPS group, SOD, CAT and GPx activities were increased, while the MDA content was reduced, indicating decreased oxidative damage. These results suggest that a diet supplemented with the citrus flavonoids eriocitrin or eriodictyol may aid in the prevention of periodontitis, representing a potential method to enhance local immunity and host defense.

Leukotriene receptor antagonist reduces inflammation and alveolar bone loss in a rat model of experimental periodontitis.

BACKGROUND: Leukotrienes (LTs) participate in the process of tissue damage in periodontal disease by leukocyte chemotaxis and osteoclastic activation. The activation of Cysteinyl-LT receptor is associated with increased expression of proinflammatory molecules and osteoclastogenesis. However, its implications on periodontal disease progression have not been studied. The present study evaluated the effect of the cysteinyl-LT receptor antagonist (montelukast [MT]) on ligature-induced experimental periodontitis (EP) in rats. METHODS: Adult male Wistar rats were subjected to bilateral ligature-induced periodontitis and orally treated with MT (at doses of 10 or 30 mg/kg/d, MT10, and MT30, respectively). Sham animals had the ligatures immediately removed and received placebo treatment. Sets of animals were euthanized 7, 14, or 21 days after ligature placement and the mandibles were removed for macroscopic evaluation of alveolar bone loss (ABL). In addition, histological analysis of periodontal tissues, myeloperoxidase (MPO) activity of gingival tissues, and periodontal tissue expression of collagen type I, RUNX2, RANK, RANKL, OPG, BLT1, Cys-LTR1, LTA4H, and LTC4S were also analyzed. RESULTS: MT significantly reduced ABL at 14 (MT10 and MT30) and 21 days (MT10) (P < 0.05), gingival MPO at 7 (MT10) and 14 days (MT30) (P < 0.05), LTA4H, BLT1 and LTC4S gene expression on day 14 day (MT30, P < 0.05) and increased RUNX2 expression on day 14 (MT30, P < 0.05). CONCLUSION: Systemic therapy with MT decreases periodontal inflammation and ABL in ligature-induced periodontitis in rats.

Periodontal clinical status, microbial profile, and expression of interleukin-1ß in men under androgenic anabolic steroids abuse.

OBJECTIVES: Androgenic anabolic steroids (AAS) abuse is a serious health problem associated to several systemic complications. Here, we evaluated the periodontal clinical status, microbial profile, and expression of total protein (TP) and interleukin (IL)-1ß in men using AAS. MATERIALS AND METHODS: Men using AAS were recruited (case group) and matched for age with men who had never used AAS (control group) but also performed physical activities. Plaque index (PI), marginal bleeding (MB), probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BoP) were evaluated. Crevicular fluid and subgingival biofilm were collected from healthy and diseased sites (PD ≥ 4 mm with CAL ≥ 1 mm and BoP) and evaluated for TP, IL-1ß, and proportions of 40 bacterial species. RESULTS: Thirty patients were included (n = 15/group). AAS consumers had significantly higher mean PD and higher percentage of diseased sites; sites with PD ≥ 4 mm or with CAL ≥ 1 mm than non-consumers. Also, AAS users showed a more dysbiotic biofilm containing lower proportions of host-compatible species and higher proportions of pathogens. IL-1ß expression was statistically higher in diseased than in healthy sites only in the control group. A statistically positive correlation was detected between periodontal pathogens and IL-1ß expression. The number of AAS cycles was positively associated with higher percentages of periodontal pathogens, but not with IL-1ß or total protein concentrations. CONCLUSIONS: AAS intake can worsen clinical and immunological periodontal conditions and the biofilm composition in healthy sites. CLINICAL RELEVANCE: Dental care professionals should perform full mouth periodontal screening and schedule regular follow-up appointments for patients under AAS use.

Silencing matrix metalloproteinase-13 (Mmp-13) reduces inflammatory bone resorption associated with LPS-induced periodontal disease in vivo.

OBJECTIVES: The aim of this study was to evaluate the effect of specific inhibition of MMP-13 on inflammation and inflammatory bone resorption in a murine model of lipopolysaccharide (LPS)-induced periodontitis. MATERIALS AND METHODS: Periodontitis was induced in mice by micro-injections of LPS into the gingival tissues adjacent to the palatal surfaces of maxillary molars twice a week for 15 days. Matrix metalloproteinase-13 (Mmp-13) shRNA or a specific biochemical inhibitor were also injected into the same sites in alternating days with the LPS injections. Efficacy of shRNA-mediated silencing of Mmp-13 was verified by quantitative real-time polymerase chain reaction (qPCR) and immunoblot. Bone resorption was assessed by microcomputed tomography (uCT). Histological sections stained with hematoxylin/eosin (H/E) were used in the stereometric analysis of the inflammatory infiltrate. Gingival tissues were used to evaluate expression of Mmp-13, Il-6, Tnf-α, Ptgs2, and Rankl (qPCR). Protein levels of TGF-ß and IL-10 in the tissues were determined by enzyme-linked immunosorbent assays (ELISA) or by MMP-13 and p38 immunoblot. RESULTS: Silencing Mmp-13 expression reduced bone resorption significantly. Expression of Mmp-13, Il-6, and Tnf-α, as well as the protein levels of IL-6 and TNF-α, was reduced in the animals treated with adenovirus-delivered shRNA; however, these effects were not associated with modulation of p38 MAPK signaling. Interestingly, inhibition Mmp-13 did not affect the severity of inflammatory infiltrate. CONCLUSIONS: Site-specific inhibition of MMP-13 reduced bone resorption and production of inflammatory mediators associated with periodontal disease. CLINICAL RELEVANCE: The results suggest that site-specific inhibition of MMP-13 may be an interesting strategy to modulate inflammation and reduce bone resorption in osteolytic inflammatory diseases.

Pentoxifylline mitigates renal glycoxidative stress in obese mice by inhibiting AGE/RAGE signaling and increasing glyoxalase levels.

AIM: The pharmacological properties of pentoxifylline have been re-evaluated, particularly in chronic kidney disease in diabetes, favored by its anti-inflammatory action. Definitive evidences of renal outcomes are lacking, which indicates the need for investigation of novel mechanisms of action of pentoxifylline. We postulated that components associated with the metabolism of advanced glycation end products (AGEs) may be modulated by pentoxifylline, which consequently decreases the detrimental effects of obesity on kidneys. MAIN METHODS: C57BL-6J mice were fed a high-fat diet for 14 weeks and treated with 50 mg/kg pentoxifylline during the last 7 weeks. Changes in the renal levels of AGE metabolism-associated components were investigated, with particular focus on the receptor for AGEs (RAGE), its downstream components, and components related to AGE detoxification, including glyoxalase 1 (GLO 1). KEY FINDINGS: Pentoxifylline reduced body weight gain, improved insulin sensitivity and glucose tolerance, downregulated biomarkers of glycoxidative stress, and enhanced plasma paraoxonase 1 activity. In the kidneys, pentoxifylline inhibited glomerular expansion, lipid deposition, reduced pro-inflammatory cytokine levels, and induced the activation of AMP-activated protein kinase. Pentoxifylline inhibited the renal accumulation of AGEs and reduced the levels of RAGE and its downstream components, and consequently mitigated oxidative stress and apoptosis. Pentoxifylline also increased the renal levels of GLO 1 and the activities of antioxidant enzymes. Urinary albumin levels were observed to be lowered, which reconfirmed the antialbuminuric effects of pentoxifylline. SIGNIFICANCE: The novel mechanisms of action help explain the renoprotective effects of pentoxifylline and the attenuation of obesity-associated renal complications related to glycoxidative stress.

Testosterone Increases Fibroblast Proliferation in vitro Through Androgen and Estrogen Receptor Activation.

BACKGROUND: Skin-related disorders and periodontitis are distinct diseases that have been associated with altered levels of testosterone. Understanding the mechanisms through which testosterone mediates gingival enlargement in animals and humans is crucial for preventing or treating this condition. In this study, we investigated the impact of different doses of androgens, the role of aromatase inhibition, and the effects of testosterone association with sex hormone receptor antagonists or aromatase inhibitors on human gingival fibroblast proliferation and migration in vitro. METHODS: Fibroblasts were cultivated in Dulbecco's Modified Eagle's Medium in a humidified atmosphere and treated with different doses of testosterone or dihydrotestosterone, and testosterone in association with: aromatase inhibitor - anastrozole; antagonist of androgen receptors - flutamide; and antagonist of estrogen receptors - fulvestrant. RESULTS: Low (1nM) and high (1µM) doses of testosterone significantly increased cell migration, but the higher dose did not alter cell proliferation. Those effects were related to both androgen and estrogen receptors activation, as evidenced by the dihydrotestosterone and drug interaction groups. CONCLUSIONS: Testosterone association with sex hormone receptor antagonists flutamide and fulvestrant suggests that not only androgen receptors, but also estrogen receptors, may take part in fibroblast cell proliferation and migration in vitro.

Topical application of lectin Artin M improves wound healing in defects created in the palatal mucosa: an in vivo study in dogs.

Previous studies have shown that topical application of lectin Artin-M accelerates wound healing in the rat oral mucosa. The aim of this study was to evaluate, by means of histology and immunohistochemistry (IHC) the effects of Artin-M on wound healing in the palatal mucosa in dogs. Three full thickness wounds of 6 mm diameter were surgically created in the palatal mucosa of twenty dogs and randomly divided into three groups according to one of the treatment assigned: Group C-Control (coagulum); Group A-Artin-M gel; Group V-Vehicle (carboxymethylcellulose 3%). Each animal received all the three experimental treatments. Afterwards, four animals were killed at 2, 4, 7, 14 and 21 days post-surgery. Wounded areas were photographed and scored for macroscopic evaluation. Biopsies were harvested and used for descriptive histological analysis, proliferating cell nuclear antigen IHC and measurement of myeloperoxidase activity. The results demonstrated faster wound closure in group A in comparison to the other groups in all the periods evaluated. Histological analyses exhibited improved re-epithelialization and collagen fiber formation resulting in faster maturation of granulation tissue in group A compared to the other groups by day 14. Treatment with Artin-M gel significantly induced cell proliferation and increased volumetric density of fibroblasts at day 2 and 4 (p < 0.05). Neutrophil infiltration in group A was significantly higher than the other groups (p < 0.05) at the same time points. Collectively, our findings demonstrated that Artin-M may potentially favor wound healing on palatal mucosa lesions via recruitment of neutrophils and promotion of cell proliferation.

Role of testosterone and androgen receptor in periodontal disease progression in female rats.

BACKGROUND: Sex hormone therapy has strict recommendations in the treatment of postmenopausal symptoms, in which testosterone (TES) replacement may play a potential role. However, it remains unclear whether TES affects the course of chronic inflammation and alveolar bone loss in females. Herein, we investigated the role of androgen receptor and TES on the inflammatory response and alveolar bone resorption associated with ligature-induced periodontal disease in female rats. METHODS: Fifty female Holtzman rats were divided in five groups (n = 10/group): androgen receptor antagonist (flutamide); estrogen receptor antagonist (fulvestrant); TES supplementation; aromatase inhibitor (anastrozole); and TES plus anastrozole. Periodontitis was induced by ligatures around the lower first molars for 2 weeks. Twenty animals (n = 10/group) were used as untreated ligated or non-ligated controls. Bone loss and the number of osteoclasts were measured through radiographic and immunohistochemical analysis, respectively. Inflammatory cytokines, chemokines and bone markers were measured by multiplex immunoassay and ELISA in serum samples and periodontal tissues. RESULTS: The blockage of androgen receptor significantly increased radiographic bone loss and tissue levels of IL-1α (P <0.05), IL-1ß (P <0.001) and IL-10 (P <0.01) compared with the periodontitis group. Testosterone supplementation significantly increased EGF levels in tissue samples, whereas when combined with aromatase inhibitor anastrozole significantly increased both EGF and VEGF (P <0.05). None of the treatment conditions significantly impacted the number of osteoclasts compared with the periodontitis group. CONCLUSIONS: Androgen receptor activation is an important factor in the regulation of several inflammatory markers, and its blockage significantly increases bone loss.

Trigonelline and curcumin alone, but not in combination, counteract oxidative stress and inflammation and increase glycation product detoxification in the liver and kidney of mice with high-fat diet-induced obesity.

The development of obesity-associated complications is related to various pathogenic events including chronic inflammation, oxidative stress and generation of advanced glycation end products (AGEs). Due to their antioxidant, anti-inflammatory and antiglycation properties, trigonelline and curcumin are interesting candidates to counteract complications of obesity and diabetes mellitus. The current study aimed to investigate the effects of treatment with curcumin or trigonelline mixed into yoghurt, alone or in combination, on mice fed high-fat diet (HFD); the focus was mainly on the potential of these phytochemicals to counteract oxidative and glycative stress. Yoghurt alone improved glucose tolerance and reduced proinflammatory cytokine levels in HFD mice; however, it did not affect the antioxidant status. Trigonelline-enriched yoghurt prevented fat accumulation in adipose tissue, improved both insulin sensitivity and glucose tolerance and exerted anti-inflammatory and antiglycation activities (reduced AGEs and AGE receptor levels and increased the levels of components related to AGE detoxification) in liver and kidney of HFD mice. Curcumin-enriched yoghurt exerted anti-inflammatory and potent antioxidant properties (increased antioxidant enzyme activities and decreased lipid peroxidation) in liver and kidney of HFD mice. However, several beneficial effects were nullified when trigonelline and curcumin were administered in combination. Trigonelline and curcumin have emerged as promising complementary therapy candidates for liver and kidney complications associated with obesity. However, the administration of these phytochemicals in combination, at least in HFD mice, was not effective; inhibition of biotransformation processes and/or the reaching of toxic doses during combined treatment may be prevailing over the individual pharmacodynamic actions of these phytochemicals.

Effects of Selective Versus Non-Selective COX-2 Inhibition on Experimental Periodontitis.

In the present study we compared the effects of the selective COX-2 inhibitor etoricoxib with those of the classical non-selective NSAID diclofenac on the inflammatory process and alveolar bone loss in an experimental model of periodontitis in rats. Ninety male Holtzman rats (250 g) were randomly sorted into four experimental groups: Sham+CMC and Ligature+CMC (control) groups which received 0.5% carboxymethylcellulose sodium (CMC) solution; Ligature+Diclofenac and Ligature+Etoricoxib groups which received Potassium Diclofenac and Etoricoxib, respectively, suspended in 0.5% CMC (10 mg/kg/day). At 7, 14 and 21 days after placing ligatures in the cervical region of both the lower right and left first molars, the animals were euthanized. At the end of each period, the mandibles were collected for radiographic examination of alveolar bone loss. In addition, alveolar bone and periodontal ligament tissue samples were collected for COX-2 expression analysis and gingival tissues were collected for measurement of PGE2 contents. Animals with ligature-induced periodontal disease showed significant increased COX-2 gene expression at days 7, 14 and 21 (p<0.05) on alveolar bone and periodontal ligament. However, both treatments resulted in significantly reduced alveolar bone loss when compared to the untreated Ligature group (p<0.05), with no statistical difference between Etoricoxib and Diclofenac Potassium groups. This study shows that both drugs were able to reduce alveolar bone loss after periodontal disease induction.

Dose-response assessment of chemically modified curcumin in experimental periodontitis.

BACKGROUND: CMC2.24, a novel tri-ketonic chemically modified compound based on natural di-ketonic curcumin, has been shown to reduce bone loss and inflammatory mediators in experimental periodontitis, however, a potential dose-response relationship was not determined. The purpose of this study was to assess the effects of different doses of CMC2.24 on inflammation and bone resorption in vivo and also to describe on the effects of CMC2.24 on macrophage response. METHODS: CMC2.24 was administered daily to animals for 28 days by oral gavage, at the following doses: 0 (control), 1, 3, 10, and 30 mg/kg of body weight. Experimental periodontitis was induced by injections of lipopolysaccharide (LPS) into the gingival tissues. Outcomes assessed were bone resorption, detection of tartrate-resistant acid phosphatase, and determination of gene expression. In vitro, macrophages (RAW264.7) were treated with different concentrations of CMC2.24: 1, 3, 10, and 30 µM and then subjected to different activation stimuli. Gene expression, phagocytic activity, production of reactive oxygen species (ROS) and cytokine production were evaluated. RESULTS: CMC2.24 inhibited bone resorption, osteoclastogenesis, and tumor necrosis factor (TNF)-α expression in vivo. These beneficial responses reached maximum levels at a dose of 1 mg/kg, i.e. no dose-dependent effect. In vitro, CMC2.24 reduced the production of TNF-α and interleukin-10, inhibited phagocytic activity and stimulated production of ROS. A dose-dependent effect was observed only for ROS production. CONCLUSION: Low doses of CMC2.24 (1 mg/kg/day) administered orally were sufficient to significantly inhibit alveolar bone resorption associated with the experimental periodontal disease; whereas in vitro macrophage inflammatory gene expression and phagocytosis were reduced, whereas production of ROS was stimulated.