Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism.

AIMS/HYPOTHESIS: Diabetes interferes with bone formation and impairs fracture healing, an important complication in humans and animal models. The aim of this study was to examine the impact of diabetes on mesenchymal stem cells (MSCs) during fracture repair. METHODS: Fracture of the long bones was induced in a streptozotocin-induced type 1 diabetic mouse model with or without insulin or a specific TNFα inhibitor, pegsunercept. MSCs were detected with cluster designation-271 (also known as p75 neurotrophin receptor) or stem cell antigen-1 (Sca-1) antibodies in areas of new endochondral bone formation in the calluses. MSC apoptosis was measured by TUNEL assay and proliferation was measured by Ki67 antibody. In vitro apoptosis and proliferation were examined in C3H10T1/2 and human-bone-marrow-derived MSCs following transfection with FOXO1 small interfering (si)RNA. RESULTS: Diabetes significantly increased TNFα levels and reduced MSC numbers in new bone area. MSC numbers were restored to normal levels with insulin or pegsunercept treatment. Inhibition of TNFα significantly reduced MSC loss by increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established that TNFα alone was sufficient to induce apoptosis and inhibit proliferation of MSCs. Furthermore, silencing forkhead box protein O1 (FOXO1) prevented TNFα-induced MSC apoptosis and reduced proliferation by regulating apoptotic and cell cycle genes. CONCLUSIONS/INTERPRETATION: Diabetes-enhanced TNFα significantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNFα reduced MSC proliferation and increased MSC apoptosis. Reducing the activity of TNFα in vivo may help to preserve endogenous MSCs and maximise regenerative potential in diabetic patients.

Clopidogrel enhances periodontal repair in rats through decreased inflammation.

AIM: We hypothesized that platelet inactivation induced by drugs might interfere with periodontal repair in experimental periodontitis by suppressing the release of biological mediators from platelets at the site of injury. MATERIAL AND METHODS: Sixty rats were randomly assigned to six groups (n = 10) and ligatures were placed around lower first molars of three groups. The other three groups were used as negative controls. Ligatures were removed after 10 days of periodontitis induction and all groups were submitted to treatment with aspirin (Asp) (30 mg/kg), clopidogrel (Clop) (75 mg/kg) or NaCl 0.9% intra-gastrically once daily for 3 days. Periodontal tissue was assessed by the measurement of CXCL12, CXCL4, CCL5 and platelet-derived growth factor (PDGF) by enzyme-linked immunosorbent assay; histomorphometrical analysis of polymorphonuclear (PMN) infiltration, attachment loss, bone loss and osteoclast numbers and quantification of blood vessels by imunnohistochemistry. RESULTS: During periodontal repair and treatment with NaCl 0.9%, CCL5 was decreased and CXCL12 increased when compared with negative control groups. Asp and Clop did not affect CCL5 expression, decreased CXCL12 but only Clop decreased CXCL4 and PDGF content compared with saline-treated animals. Clop increased blood vessel number, reduced PMN count and decreased attachment and bone loss, also decreased osteoclast number in animals submitted or not to periodontal repair. CONCLUSION: Systemic administration of Clop for 3 days improved the repair process associated with experimental periodontal disease, suggesting that it may have therapeutic value under situations where tissues undergo a transition from inflammation to repair.

Intermittent therapy with 1,25 vitamin D and calcitonin prevents cyclosporin-induced alveolar bone loss in rats.

Bone loss associated with cyclosporin A (CsA) therapy can result in serious morbidity to patients. Intermittent administration of 1,25 Vitamin D and calcitonin reduces osteopenia in a murine model of postmenopausal osteoporosis. The purpose of this study was to evaluate the effects of this therapeutic approach on CsA-induced alveolar bone loss in rats. Forty male Wistar rats were allocated to four experimental groups according to the treatment received during 8 weeks: (1) CsA (10 mg/kg/day, s.c.); (2) 1,25 Vitamin D (2 microg/kg, p.o.; in weeks 1, 3, 5, and 7) plus calcitonin (2 microg/kg, i.p.; in weeks 2, 4, 6, and 8); (3) CsA concurrently with intermittent 1,25 Vitamin D and calcitonin administration; and (4) the control treatment group (vehicle). At the end of the 8-week treatment period, serum concentrations of bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase (TRAP-5b), osteocalcin, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were measured and an analysis of bone volume, bone surface, number of osteoblasts, and osteoclasts was performed. CsA administration resulted in significant alveolar bone resorption, as assessed by a lower bone volume and an increased number of osteoclasts, and increased serum bone-specific alkaline phosphatase, TRAP-5b, IL-1 beta, IL-6, and TNF-alpha concentrations. The intermittent administration of calcitriol and calcitonin prevented the CsA-induced osteopenic changes and the increased serum concentrations of TRAP-5b and inflammatory cytokines. Intermittent calcitriol/calcitonin therapy prevents CsA-induced alveolar bone loss in rats and normalizes the production of associated inflammatory mediators.

The long-term administration of calcineurin inhibitors decreases antioxidant enzyme activity in the rat parotid and submandibular salivary glands.

AIMS: Calcineurin inhibitors are widely used for prevention of graft rejection and treatment of autoimmune disorders, which result in increased longevity and enhanced quality of life for patients. Unfortunately, the toxic side effects of these drugs (mainly renal, hepatic and cardiac) limit their use. In this work, we studied the effects of long-term treatment of rats with the immunosuppressant cyclosporin (CsA) or tacrolimus (Tac) on salivation, saliva composition and on the major salivary glands (parotid and submandibular) in terms of histological alterations and oxidative stress, evaluated as lipoperoxidation (thiobarbituric acid reactive species--TBARS) and antioxidant enzyme activity contents (superoxide dismutase--SOD, catalase--CAT and glutathione peroxidase--GPx). MAIN METHODS: Male adult rats were treated with either CsA (10 mg/kg/day) or Tac (1 mg/kg/day) subcutaneously for 30 or 60 days. At the end of the experimental periods, pilocarpine-stimulated salivary flow rate was measured, saliva samples were collected and the salivary glands were dissected for morphological and biochemical analyses. KEY FINDINGS: After a 60-day treatment with any of the immunosuppressants, the total protein, Ca(2+) and Na(+) saliva concentrations were decreased but salivary flow rates were unaffected. In addition, both parotid and submandibular glands showed decreased SOD, CAT and GPx activities, increased TBARS contents and histomorphological alterations involving the epithelium and acini. SIGNIFICANCE: Based on these results, we suggest that the systemic long-term administration of the calcineurin inhibitor CsA or Tac induces an impairment of the antioxidant enzymatic defense in the rat major salivary glands, which may, in turn, lead to altered saliva composition.

Peripheral blood mononuclear phagocytes from patients with chronic periodontitis are primed for osteoclast formation.

BACKGROUND: During inflammatory periodontal disease, peripheral blood mononuclear cells (PBMCs) are attracted to bone and differentiate into active bone-resorbing osteoclasts (OCs), thus providing evidence that the impact of chronic periodontitis (CP) on the activity of circulating mononuclear cells is of central importance. The authors test the hypothesis that peripheral blood mononuclear phagocytes (PBMPs) from patients with CP are activated and more susceptible to differentiation into OCs, which in turn would lead to more intense bone resorption. METHODS: In vitro cytokine production by both unstimulated and lipopolysaccharide-stimulated PBMCs from individuals with (n = 10) or without (n = 12) periodontitis was determined by cytokine array. OC differentiation from CD14(+) PBMCs was induced by receptor activator of nuclear factor-kappa B ligand (RANKL), either alone or in the presence of macrophage colony-stimulating factor (M-CSF). PBMC differentiation to OCs was confirmed by tartrate-resistant acid phosphatase staining; bone resorbing activity was assessed by using an osteologic plate assay (bone resorption pit formation). RESULTS: PBMCs from patients with CP produced tumor necrosis factor-α and higher amounts of interferon-γ, interleukin (IL)-1α, IL-1ß, IL-1rα, CXC motif chemokine 10, macrophage migration inhibitory factor, macrophage inflammatory protein (MIP)-1α, and MIP-1ß than the control cells. OC differentiation was induced by RANKL alone in PBMCs from patients with CP, but not in PBMCs from the healthy controls, which required the addition of M-CSF. In addition, PBMC-derived OCs from patients with CP showed significantly higher resorption activity than that observed in the healthy controls. Also, the circulating concentrations of M-CSF were significantly higher in patients with CP than in the control participants. CONCLUSIONS: These data indicate that in patients with CP, circulating PBMCs are primed for increased proinflammatory activity and that M-CSF plays a central role in this process by increasing OC formation and the consequent bone resorption activity.

Curcumin modulates the immune response associated with LPS-induced periodontal disease in rats.

Curcumin is a plant-derived dietary spice ascribed various biological activities. Curcumin therapeutic applications have been studied in a variety of conditions, but not on periodontal disease. Periodontal disease is a chronic inflammatory condition initiated by an immune response to micro-organisms of the dental biofilm. Experimental periodontal disease was induced in rats by injecting LPS in the gingival tissues on the palatal aspect of upper first molars (30 µg LPS, 3 times/week for 2 weeks). Curcumin was administered to rats daily via oral gavage at 30 and 100 mg/kg body weight. Reverse transcriptase-qPCR and ELISA were used to determine the expression of IL-6, TNF-α and prostaglandin E(2) synthase on the gingival tissues. The inflammatory status was evaluated by stereometric and descriptive analysis on hematoxylin/eosin-stained sections, whereas modulation of p38 MAPK and NK-κB signaling was assessed by Western blot. Curcumin effectively inhibited cytokine gene expression at mRNA and protein levels, but NF-κB was inhibited only with the lower dose of curcumin, whereas p38 MAPK activation was not affected. Curcumin produced a significant reduction on the inflammatory infiltrate and increased collagen content and fibroblastic cell numbers. Curcumin potently inhibits innate immune responses associated with periodontal disease, suggesting a therapeutic potential in this chronic inflammatory condition.

The effect of supra- and subphysiologic testosterone levels on ligature-induced bone loss in rats--a radiographic and histologic pilot study.

BACKGROUND: Testosterone is the primary male sexual hormone, and varying concentrations of the hormone mediated by physiologic, pathologic, or pharmacologic mechanisms may induce large variations in the body. Data regarding the general role of testosterone in mediating inflammation are still inconclusive. Therefore, the purpose of this study is to assess the consequences of supra- and subphysiologic levels of testosterone on ligature-induced bone loss in rats. METHODS: Three male adult Holtzman rats were used to observe the course of serum testosterone concentration following orchiectomy (Ocx) and testosterone injections. Another 60 rats were randomly assigned to the following groups: (1) sham-operation controls (n = 10); (2) sham-operation and ligature-induced bone loss (n = 10); (3) orchiectomy without ligature (Ocx; n = 10); (4) Ocx and ligature (n = 10); (5) Ocx plus 250 mg/kg body weight intramuscular testosterone esters injection without ligature (Ocx+T; n = 10); and (6) Ocx, T, and ligature (n = 10). The ligatures were placed 30 days postorchiectomy (or sham-operation) and maintained for 15 days. Thereafter, the rats were sacrificed, and their hemimandibles were used for radiographic evaluation of bone loss along with histologic and histometric analyses of gingival tissue. RESULTS: The results indicated a significant increase in bone loss in the Ocx and Ocx+T groups in the presence and absence of inflammation, respectively. In addition, the Ocx and Ocx+T groups presented increased gingival area accompanying ligature-induced bone loss. CONCLUSIONS: Both sub- and supraphysiologic testosterone levels may influence bone metabolism, but only subphysiologic levels significantly increase ligature-induced bone loss. Moreover, testosterone has a regulatory effect on the gingival area.

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells.

OBJECTIVE: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. DESIGN: Cell line SAOS-2 (1×10(5)cells/mL) were grown in culture medium α-MEM with 10% FBS for 24h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10g/mL) and IL-1ß (1mg/mL) for 24h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO(2)(-) with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. RESULT: There were no significant differences amongst the groups in terms of NO(2)(-), protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p<0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1ß caused up-regulation of this cytokine. CONCLUSIONS: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation.

Influence of antiplatelet drugs in the pathogenesis of experimental periodontitis and periodontal repair in rats.

BACKGROUND: Platelets contain an array of biologic mediators that can modulate inflammation and repair processes including proinflammatory mediators and growth factors. Previous studies have shown that periodontitis and periodontal repair are associated with platelet activation. We hypothesized that drug-induced platelet inactivation may interfere in the processes of inflammation and repair in experimental periodontitis in rats by suppressing the release of biologic mediators from platelets to the site of injury. METHODS: To measure the effects on periodontitis, ligatures were placed around first molars, and aspirin (Asp, 30 mg/kg) or clopidogrel (Clo, 75 mg/kg) was given intragastrically once daily for 15 days. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and thromboxane A(2) levels were measured by enzyme-linked immunosorbent assay. To evaluate the effects of antiplatelet drugs on periodontal repair, ligatures were removed after 15 days of periodontitis induction, and Asp or Clo were administered beginning the following day for 15 days. Periodontal repair was assessed by microcomputed tomography. RESULTS: On periodontitis phase, Asp and Clo significantly reduced levels of TNF-α and Il-6 (P <0.05), but only Asp decreased thromboxane A(2) (P <0.05). Asp and Clo decreased inflammatory infiltration; however, this reduction was more pronounced with Clo treatment (P <0.05). Histometric analysis showed that Asp and Clo impaired alveolar bone resorption. During the repair phase and after removal of the ligatures, microcomputed tomography analysis demonstrated that treatment with Asp and Clo did not impair alveolar bone repair. CONCLUSION: Systemic administration of Asp and Clo attenuates the inflammation associated with periodontitis without affecting the repair process when stimulus is removed.

Invasão microbiana: infecção focal e a relação com aterosclerose / Microbial Invasion: focal infection and relationship with atherosclerosis

Os extraordinários avanços no desempenho de técnicas laboratoriais corroboram na evolução do entendimento dos mecanismos das doenças de modo geral, facilitando o tratamento e/ou a prevenção. De fato, informações equilibradas, curadas e atuais estão colaborando de forma estimulante no estudo da relação entre doença periodontal e periapical crônica com o desenvolvimento ou estabelecimento de algumas doenças cardiovasculares. Dessa forma, o objetivo da presente trabalho foi descrever de forma sucinta e objetiva a relação entre doença periodontal crônica e doença periapical como fatores de risco para o desenvolvimento ou estabeleciomento da aterosclerose. Estudos epidemiológicos têm procurado demonstrar que indivíduos com doença periodontal ou periapical poderiam apresentar um aumento significativo do risco de desenvolver algumas doenças sistêmicas incluindo a aterosclerose.