VISTA Blockade Aggravates Bone Loss in Experimental Murine Apical Periodontitis.

V-domain Ig suppressor of T cell activation (VISTA) is a novel coinhibitory immune checkpoint molecule that maintains immune homeostasis. The present study explored the role of VISTA in human and murine inflammatory tissues of apical periodontitis (AP). VISTA was upregulated in inflammatory tissues of human AP. In mice, the expression of VISTA gradually increased with the development of mouse experimental apical periodontitis (MAP), the CD3+ T cells, CD11b+ myeloid cells, and FOXP3+ regulatory T cells also gradually accumulated. Moreover, a blockade of VISTA using a mouse in vivo anti-VISTA antibody aggravated periapical bone loss and enhanced the infiltration of immune cells in an experimental mouse periapical periodontitis model. The collective results suggest that VISTA serves as a negative regulator of the development and bone loss of apical periodontitis.

The Glycoprotein/Cytokine Erythropoietin Promotes Rapid Alveolar Ridge Regeneration In Vivo by Promoting New Bone Extracellular Matrix Deposition in Conjunction with Coupled Angiogenesis/Osteogenesis.

The loss of bone following tooth extraction poses a significant clinical problem for maxillofacial esthetics, function, and future implant placement. In the present study, the efficacy of an erythropoietin-impregnated collagen scaffold as an alveolar ridge augmentation material versus a conventional collagen scaffold and a BioOss inorganic bovine bone xenograft was examined. The collagen/Erythropoietin (EPO) scaffold exhibited significantly more rapid and complete osseous regeneration of the alveolar defect when compared to bone xenograft and the collagen membrane alone. The new EPO induced extracellular matrix was rich in Collagen I, Collagen III, Fibronectin (Fn) and E-cadherin, and featured significantly increased levels of the osteogenic transcription factors Runt-related transcription factor 2 (Runx2) and Osterix (Osx). Histomorphometric evaluation revealed a significant two-fold increase in the number of capillaries between the EPO and the BioOss group. Moreover, there was a highly significant 3.5-fold higher level of vascular endothelial growth factor (VEGF) in the collagen/EPO-treated group compared to controls. The significant effect of EPO on VEGF, FN, and RUNX2 upregulation was confirmed in vitro, and VEGF pathway analysis using VEGF inhibitors confirmed that EPO modulated extracellular matrix protein expression through VEGF even in the absence of blood vessels. Together, these data demonstrate the effectiveness of an EPO-impregnated collagen scaffold for bone regeneration as it induces rapid matrix production and osseoinduction adjacent to new capillaries via VEGF.

The effect of high concentration of zoledronic acid on tooth induced movement and its repercussion on root, periodontal ligament and alveolar bone tissues in rats.

Zoledronic acid (ZA) is often prescribed for osteoporosis or resorptive metabolic bone disease. This study aims to evaluate the effect of ZA on orthodontic tooth movement (OTM) and root and bone resorption and its repercussion on root, periodontal ligament and alveolar bone tissues. The experimental group consisted of 72 Wistar rats divided in four subgroups: Naive, Saline and Zoledronic Acid groups at the concentration of 0.2 mg/kg [ZA (0.2)] or 1.0 mg/kg [ZA (1.0)]. The animals were subjected to i.v (dorsal penile vein) administrations of ZA or saline solution, on days 0, 7, 14 and 42. Under anesthesia, NiTi springs were installed in the first left maxillary molar with 50gf allowing the OTM, except for the negative control group (N) for mesial movement of the left first maxillary teeth. The animals were sacrificed and maxillae were removed for macroscopic and histopathological analyzes, scanning electron microscopy, computerized microtomography and confocal microscopy. Treatment with ZA decreased the OTM and the number of osteoclasts and loss of alveolar bone when compared to the naive and saline groups. Reduction of radicular resorption, increased necrotic areas and reduced vascularization in the periodontal ligament were observed in the ZA groups. ZA interferes with OTM and presents anti-resorptive effects on bone and dental tissues associated with a decreased vascularization, without osteonecrosis.

Thermoresponsive Hydrogel-Based Local Delivery of Simvastatin for the Treatment of Periodontitis.

Except for routine scaling and root planing, there are few effective nonsurgical therapeutic interventions for periodontitis and associated alveolar bone loss. Simvastatin (SIM), one of the 3-hydroxy-3-methylglutaryl-cosenzyme A reductase inhibitors, which is known for its capacity as a lipid-lowering medication, has been proven to be an effective anti-inflammatory and bone anabolic agent that has shown promising benefits in mitigating periodontal bone loss. The local delivery of SIM into the periodontal pocket, however, has been challenging due to SIM's poor water solubility and its lack of osteotropicity. To overcome these issues, we report a novel SIM formulation of a thermoresponsive, osteotropic, injectable hydrogel (PF127) based on pyrophosphorolated pluronic F127 (F127-PPi). After mixing F127-PPi with F127 at a 1:1 ratio, the resulting PF127 was used to dissolve free SIM to generate the SIM-loaded formulation. The thermoresponsive hydrogel's rheologic behavior, erosion and SIM release kinetics, osteotropic property, and biocompatibility were evaluated in vitro. The therapeutic efficacy of SIM-loaded PF127 hydrogel on periodontal bone preservation and inflammation resolution was validated in a ligature-induced periodontitis rat model. Given that SIM is already an approved medication for hyperlipidemia, the data presented here support the translational potential of the SIM-loaded PF127 hydrogel for better clinical management of periodontitis and associated pathologies.

The Dual Effects of Reactive Oxygen Species on the Mandibular Alveolar Bone Formation in SOD1 Knockout Mice: Promotion or Inhibition.

The status of reactive oxygen species (ROS) correlates closely with the normal development of the oral and maxillofacial tissues. Oxidative stress caused by ROS accumulation not only affects the development of enamel and dentin but also causes pathological changes in periodontal tissues (periodontal ligament and alveolar bone) that surround the root of the tooth. Although previous studies have shown that ROS accumulation plays a pathologic role in some oral and maxillofacial tissues, the effects of ROS on alveolar bone development remain unclear. In this study, we focused on mandibular alveolar bone development of mice deficient in superoxide dismutase1 (SOD1). Analyses were performed using microcomputerized tomography (micro-CT), TRAP staining, immunohistochemical (IHC) staining, and enzyme-linked immunosorbent assay (ELISA). We found for the first time that slightly higher ROS in mandibular alveolar bone of SOD1(-/-) mice at early ages (2-4 months) caused a distinct enlargement in bone size and increased bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), and osteopontin (OPN). With ROS accumulation to oxidative stress level, increased trabecular bone separation (Tb.Sp) and decreased expression of ALP, Runx2, and OPN were found in SOD1(-/-) mice at 6 months. Additionally, dosing with N-acetylcysteine (NAC) effectively mitigated bone loss and normalized expression of ALP, Runx2, and OPN. These results indicate that redox imbalance caused by SOD1 deficiency has dual effects (promotion or inhibition) on mandibular alveolar bone development, which is closely related to the concentration of ROS and the stage of growth. We present a valuable model here for investigating the effects of ROS on mandibular alveolar bone formation and highlight important roles of ROS in regulating tissue development and pathological states, illustrating the complexity of the redox signal.

Macrophage Polarization and Alveolar Bone Healing Outcome: Despite a Significant M2 Polarizing Effect, VIP and PACAP Treatments Present a Minor Impact in Alveolar Bone Healing in Homeostatic Conditions.

Host inflammatory immune response comprises an essential element of the bone healing process, where M2 polarization allegedly contributes to a favorable healing outcome. In this context, immunoregulatory molecules that modulate host response, including macrophage polarization, are considered potential targets for improving bone healing. This study aims to evaluate the role of the immunoregulatory molecules VIP (Vasoactive intestinal peptide) and PACAP (Pituitary adenylate cyclase activating polypeptide), which was previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups were submitted to tooth extraction and maintained under control conditions or treated with VIP or PACAP were evaluated by microtomographic (µCT), histomorphometric, immunohistochemical, and molecular analysis at 0, 3, 7, and 14 days to quantify tissue healing and host response indicators at the healing site. Gene expression analysis demonstrates the effectiveness of VIP or PACAP in modulating host response, evidenced by the early dominance of an M2-type response, which was paralleled by a significant increase in M2 (CD206+) in treated groups. However, despite the marked effect of M1/M2 balance in the healing sites, the histomorphometric analysis does not reveal an equivalent/corresponding modulation of the healing process. µCT reveals a slight increase in bone matrix volume and the trabecular thickness number in the PACAP group, while histomorphometric analyzes reveal a slight increase in the VIP group, both at a 14-d time-point; despite the increased expression of osteogenic factors, osteoblastic differentiation, activity, and maturation markers in both VIP and PACAP groups. Interestingly, a lower number of VIP and PACAP immunolabeled cells were observed in the treated groups, suggesting a reduction in endogenous production. In conclusion, while both VIP and PACAP treatments presented a significant immunomodulatory effect with potential for increased healing, no major changes were observed in bone healing outcome, suggesting that the signals required for bone healing under homeostatic conditions are already optimal, and additional signals do not improve an already optimal process. Further studies are required to elucidate the role of macrophage polarization in the bone healing process.

Glucagon-Like Peptide-1 Receptor Agonist Liraglutide Ameliorates the Development of Periodontitis.

Periodontitis is one of the diabetic complications due to its high morbidity and severity in patients with diabetes. The prevention of periodontitis is especially important in diabetic patients because the relationship between diabetes and periodontitis is bidirectional. Here, we evaluated the impacts of glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on the amelioration of periodontitis. Five-wk-old Male Sprague-Dawley (SD) rats (n = 30) were divided into 3 groups: normal, periodontitis, and periodontitis with liraglutide treatment groups. Periodontitis was induced by ligature around the maxillary second molar in SD rats. Half of the rats were administered liraglutide for 2 weeks. Periodontitis was evaluated by histological staining, gene expressions of inflammatory cytokines in gingiva, and microcomputed tomography. Periodontitis increased inflammatory cell infiltration, macrophage accumulation, and gene expressions of tumor necrosis factor-α and inducible nitric oxide synthase in the gingiva, all of which were ameliorated by liraglutide. Liraglutide decreased M1 macrophages but did not affect M2 macrophages in periodontitis. Moreover, ligature-induced alveolar bone resorption was ameliorated by liraglutide. Liraglutide treatment also reduced osteoclasts on the alveolar bone surface. These results highlight the beyond glucose-lowering effects of liraglutide on the treatment of periodontitis.

Functionalized Scaffold and Barrier Membrane with Anti-BMP-2 Monoclonal Antibodies for Alveolar Ridge Preservation in a Canine Model.

INTRODUCTION: The aim of this study was to investigate the ability of anti-bone morphogenetic protein 2 monoclonal antibody (anti-BMP-2 mAb) to functionalize scaffolds to mediate bone regeneration in a canine model. MATERIALS AND METHODS: The mandibular right premolar 4 (PM4) was extracted in eight beagle dogs and grafted with anti-BMP-2 mAb+anorganic bovine bone mineral with 10% collagen (ABBM-C) and porcine bilayer native collagen membrane (CM). The ABBM-C and CM were functionalized with either anti-BMP-2 mAb (test group) or an isotype matched control mAb (control group). Animals were euthanized at 12 weeks for radiographic, histologic, and histomorphometric analyses. Outcomes were compared between groups. RESULTS: 3D imaging using cone beam computed tomography (CBCT) revealed that sites treated with ABBM-C and CM functionalized with anti-BMP-2 mAb exhibited significantly more remaining bone width near the alveolar crest, as well as buccal bone height, compared with control groups. Histologic and histomorphometric analyses demonstrated that in anti-BMP-2 mAb-treated sites, total tissue volume was significantly higher in the coronal part of the alveolar bone crest compared with control sites. In anti-BMP-2 mAb-treated sites, bone formation was observed under the barrier membrane. CONCLUSION: Functionalization of the ABBM-C scaffold and CM appeared to have led to bone formation within healing alveolar bone sockets.

Impact of hyperglycemia and treatment with metformin on ligature-induced bone loss, bone repair and expression of bone metabolism transcription factors.

This study evaluated the influence of type 2 diabetes mellitus on bone loss, bone repair and cytokine production in hyperglycemic rats, treated or not with metformin. The animals were distributed as follow: Non-Hyperglycemic (NH), Non Hyperglycemic with Ligature (NH-L), Treated Non Hyperglycemic (TNH), Treated Non Hyperglycemic with Ligature Treated (TNH-L), Hyperglycemic (H), Treated Hyperglycemic (TH), Hyperglycemic with Ligature (H-L), Treated Hyperglycemic with Ligature (TH-L). At 40th day after induction of hyperglycemia, the groups NH-L, TNH-L, H-L, TH-L received a ligature to induce periodontitis. On the 69th, the TNH, TNH-L, TH, TH-L groups received metformin until the end of the study. Bone repair was evaluated at histometric and the expression levels of Sox9, RunX2 and Osterix. Analysis of the ex-vivo expression of TNF-α, IFN-γ, IL-12, IL-4, TGF-ß, IL-10, IL-6 and IL-17 were also evaluated. Metformin partially reverse induced bone loss in NH and H animals. Lower OPG/RANKL, increased OCN and TRAP expression were observed in hyperglycemic animals, and treatment with metformin partially reversed hyperglycemia on the OPG/RANKL, OPN and TRAP expression in the periodontitis. The expression of SOX9 and RunX2 were also decreased by hyperglycemia and metformin treatment. Increased ex vivo levels of TNF-α, IL-6, IL-4, IL-10 and IL-17 was observed. Hyperglycemia promoted increased IL-10 levels compared to non-hyperglycemic ones. Treatment of NH with metformin was able to mediate increased levels of TNF-α, IL-10 and IL-17, whereas for H an increase of TNF-α and IL-17 was detected in the 24- or 48-hour after stimulation with LPS. Ligature was able to induce increased levels of TNF-α and IL-17 in both NH and H. This study revealed the negative impact of hyperglycemia and/or treatment with metformin in the bone repair via inhibition of transcription factors associated with osteoblastic differentiation.

Asperosaponin VI Injection Enhances Orthodontic Tooth Movement in Rats.

BACKGROUND This study was performed to investigate the effect of local injection of asperosaponin VI (ASA VI) on the orthodontic tooth movement in rats. MATERIAL AND METHODS A total of 64 healthy female Sprague-Dawley rats were selected and divided into 2 groups randomly: the ASA VI group and the control group. For the ASA VI group, 10 mg/kg ASA VI solution was injected into buccal submucoperiosteal of bilaterally first maxillary molars, and the same volume of normal saline was given to the control group. The orthodontic force was applied to the maxillary first molars. All rats were sacrificed on days 3, 7, or 14. Tooth movement effects on the periodontium were analyzed through hematoxylin and eosin (H&E) staining, tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemistry analysis. Tooth movement measurements and alveolar bone volumetric changes were analyzed using a micro-computed tomography (CT) scan. Molecular changes were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. RESULTS The ASA VI group presented with a significant increase of tooth movement, osteoclast number, and the expression of osteoclast differentiation factor (ODF) compared with the control group. ASA VI also induced a significant decrease in bone volume and density and an increase in trabecular spacing and RANKL (receptor activator of nuclear factor kappa-B ligand) expression at the compression side. Furthermore, ASA VI stimulated bone formation on the tension side by enhancing OCN (osteocalcin) expression and RUNX2 (runt-related transcription factor 2) expression, increasing bone volume and density and decreasing in trabecular spacing. CONCLUSIONS Injection of ASA VI may accelerate tooth movement via increasing the activity of osteoclasts, stimulating bone resorption at the compression side. Furthermore, ASA VI has a positive effect on bone formation at the tension side.

Pam2CSK4 (TLR2 agonist) induces periodontal destruction in mice.

Lipoproteins are important bacterial immunostimulating molecules capable of inducing receptor activator of nuclear factor-κB (RANKL) and osteoclast formation in vitro and in vivo . Although these molecules are present in periodontopathogenic bacteria, their role in periodontitis is not known. In this study, we used Pam2CSK4 (PAM2), a synthetic molecule that mimics bacterial lipoprotein, to investigate the effects of lipoproteins on periodontitis in mice. C57BL/6 male mice were randomly divided into three experimental groups: 1) Negative control group: animals received vehicle injection; 2) Positive control group: animals received injection of Escherichia coli lipopolysaccharide (LPS); 3) PAM2 group: animals received PAM2 injection. All the injections were performed bilaterally every other day into the palatal mucosa between first and second molars. After twenty-four days, the animals were euthanized to assess alveolar bone volume (micro-CT), cellular and extracellular composition in the gingiva (stereometric analysis), and osteoclast numbers (TRAP staining). Treatment with either PAM2 or LPS induced gingival inflammation, as demonstrated by increased infiltration of inflammatory cells and enhanced angiogenesis, associated with a smaller number of fibroblasts and decreased extracellular matrix. Importantly, treatment not only with LPS but also with PAM2 resulted in a larger number of TRAP+ multinucleated osteoclasts and significant loss of alveolar bone. Collectively, our data demonstrate that PAM2 can induce gingival inflammation and bone loss in mice, broadening the avenues of investigation into the role of lipoproteins in the pathogenesis of periodontal disease.

Chloroquine and 3-Methyladenine Attenuates Periodontal Inflammation and Bone Loss in Experimental Periodontitis.

Periodontitis is an inflammation characterized by alveolar bone resorption caused by imbalance in bone homeostasis. It is known that autophagy is related to inflammation and bone metabolism. However, whether autophagy inhibitors could be used for periodontitis in animal models remains unknown. We investigated the role of two classical autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), on the development of rat experimental periodontitis in terms of the bone loss (micro-CT), the number of inflammatory cells (hematoxylin and eosin staining), and the osteoclastic activity (tartrate-resistant acid phosphatase staining). Expression of autophagy-related genes and nuclear factor kappa B p65 (NF-κB p65) were assessed by immunohistochemistry. Expression of Beclin-1 and microtubule-associated proteins 1A/1B light chain 3 (LC3) were analyzed by Western blot. To further observe the effect of autophagy inhibitors on osteoclasts (OCs) in vitro, bone marrow-derived mononuclear macrophages were used. Together, these findings indicated that topical administration of 3-MA or CQ reduced the infiltration of inflammatory cells and alveolar bone resorption in experimental periodontitis. Furthermore, 3-MA and CQ may attenuate activation of OCs by autophagy. Therefore, 3MA and CQ may have prophylactic and therapeutic potential for inflammation and alveolar bone resorption in periodontitis in the future.

High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis.

The inhibition of high glucose on the proliferation and differentiation of osteoblast in alveolar bone are well documented. However, a comprehensive study focused on the molecular mechanisms is still unknown. Recent studies have revealed that caspase-1 participates in the pathological processes of hepatic injury, cancers and diabetes related complications. However, the relationship between pyroptosis and proliferation and differentiation of osteoblasts has not been investigated. This study aimed to explore the possible pyroptosis participating in the inhibition of high glucose on the proliferation and differentiation of osteoblast in alveolar bone. The diabetes model was constructed both in vitro and in vivo to detect the expression of pyroptosis related factors. These results show that high glucose inhibits proliferation and differentiation of osteoblast in alveolar bone through pyroptosis pathway. Furthermore, caspase-1 inhibitor was co-administered with high glucose in ME3T3-E1 cells, which shows that caspase-1 inhibitor could repress effect of high glucose on the proliferation and differentiation of osteoblast. In conclusion, High glucose could activate the pyroptosis through the caspase-1/GSDMD/IL-1ß pathway to inhibit the proliferation and differentiation of osteoblast in alveolar bone, which provides a theoretical basis for clinical treatment of alveolar bone disease in diabetic patients.

Pam2CSK4 (TLR2 agonist) induces periodontal destruction in mice

Abstract Lipoproteins are important bacterial immunostimulating molecules capable of inducing receptor activator of nuclear factor-κB (RANKL) and osteoclast formation in vitro and in vivo . Although these molecules are present in periodontopathogenic bacteria, their role in periodontitis is not known. In this study, we used Pam2CSK4 (PAM2), a synthetic molecule that mimics bacterial lipoprotein, to investigate the effects of lipoproteins on periodontitis in mice. C57BL/6 male mice were randomly divided into three experimental groups: 1) Negative control group: animals received vehicle injection; 2) Positive control group: animals received injection of Escherichia coli lipopolysaccharide (LPS); 3) PAM2 group: animals received PAM2 injection. All the injections were performed bilaterally every other day into the palatal mucosa between first and second molars. After twenty-four days, the animals were euthanized to assess alveolar bone volume (micro-CT), cellular and extracellular composition in the gingiva (stereometric analysis), and osteoclast numbers (TRAP staining). Treatment with either PAM2 or LPS induced gingival inflammation, as demonstrated by increased infiltration of inflammatory cells and enhanced angiogenesis, associated with a smaller number of fibroblasts and decreased extracellular matrix. Importantly, treatment not only with LPS but also with PAM2 resulted in a larger number of TRAP+ multinucleated osteoclasts and significant loss of alveolar bone. Collectively, our data demonstrate that PAM2 can induce gingival inflammation and bone loss in mice, broadening the avenues of investigation into the role of lipoproteins in the pathogenesis of periodontal disease.

Pam2CSK4 (TLR2 agonist) induces periodontal destruction in mice

Abstract Lipoproteins are important bacterial immunostimulating molecules capable of inducing receptor activator of nuclear factor-κB (RANKL) and osteoclast formation in vitro and in vivo . Although these molecules are present in periodontopathogenic bacteria, their role in periodontitis is not known. In this study, we used Pam2CSK4 (PAM2), a synthetic molecule that mimics bacterial lipoprotein, to investigate the effects of lipoproteins on periodontitis in mice. C57BL/6 male mice were randomly divided into three experimental groups: 1) Negative control group: animals received vehicle injection; 2) Positive control group: animals received injection of Escherichia coli lipopolysaccharide (LPS); 3) PAM2 group: animals received PAM2 injection. All the injections were performed bilaterally every other day into the palatal mucosa between first and second molars. After twenty-four days, the animals were euthanized to assess alveolar bone volume (micro-CT), cellular and extracellular composition in the gingiva (stereometric analysis), and osteoclast numbers (TRAP staining). Treatment with either PAM2 or LPS induced gingival inflammation, as demonstrated by increased infiltration of inflammatory cells and enhanced angiogenesis, associated with a smaller number of fibroblasts and decreased extracellular matrix. Importantly, treatment not only with LPS but also with PAM2 resulted in a larger number of TRAP+ multinucleated osteoclasts and significant loss of alveolar bone. Collectively, our data demonstrate that PAM2 can induce gingival inflammation and bone loss in mice, broadening the avenues of investigation into the role of lipoproteins in the pathogenesis of periodontal disease.

H2S in periodontal immune-inflammatory response and bone loss: a study in rats / Influência do H2S na resposta imunoinflamatória e perda óssea periodontal: um estudo em ratos

ABSTRACT Halitosis is highly prevalent in periodontitis and attributed mainly to the presence of volatile sulfur compounds (VSC), where hydrogen sulfide (H2S) is the chief culprit in the characteristic malodor of periodontitis and thus may play an active role in its pathogenesis. The aim of this study was to evaluate the effect of H2S in the acute, intermediate and chronic immuneinflammatory host response and alveolar bone loss in vivo by using an animal model of induced periodontal disease. Thirtysix rats were divided into 2 groups: test group (n = 18), rats exposed to H2S (NaHS H2S donor molecule) and control group (n = 18), rats treated with saline only (Ctrl). All animals had one of their lower second molars ligated to induce periodontal disease (PD). The sound contralateral molar was used as control (H). Each group was subdivided into 3 (n = 6), according to followup time (3h, 5 days and 14 days). The gingival tissue was used for mRNA expression analysis (IL1, IL6, RANKL, OPG and SOFAT) by realtime PCR and the mandibles were analyzed morphometrically. Data analysis showed that the ligature promoted alveolar bone loss, observed mainly at 14 days, both in the group exposed to H2S and in the Ctrl group. H2S administration did not result in additional bone loss. Gene expression showed a significant increase in IL1, IL6, RANKL and SOFAT only in the CtrlPD group (p<0.05). A significant downregulation in OPG expression was observed over time in the CtrlPD group (p<0.05). In conclusion, H2S had no effect on alveolar bone loss in the absence of a ligature. In the presence of a ligature, however, exposure to H2S had an immunoregulatory effect on the expression of proinflammatory and proresorptive cytokines.

RESUMO A halitose é altamente prevalente na periodontite e é atribuída principalmente à presença de compostos sulforosos voláteis (CSV), sendo o sulfeto de hidrogênio (H2S) o principal gás relacionado ao mau odor e que pode estar envolvido na patogênese da doença periodontal. O objetivo deste estudo foi avaliar o efeito agudo, intermediário e crônico do H2S na resposta imunoinflamatória e na perda óssea alveolar em ratos, com e sem doença periodontal induzida. Trinta e seis ratos foram divididos em 2 grupos: teste (n = 18), ratos expostos ao H2S (NaHS molécula doadora de H2S) e grupo controle (n = 18), ratos tratados apenas com solução salina (Ctrl). Todos os animais tiveram um dos seus segundos molares inferiores submetidos à colocação de uma ligadura para o desenvolvimento da doença periodontal (DP), em comparação com o dente contralateral saudável (H). Cada grupo foi subdividido em 3 (n = 6), de acordo com o tempo de eutanásia (3h, 5 dias e 14 dias). Os tecidos gengivais foram utilizados para a análise da expressão gênica (IL1, IL6, RANKL, OPG e SOFAT) por PCR em tempo real e as mandíbulas foram analisadas morfometricamente. Análise dos dados demonstrou que a ligadura promoveu perda óssea alveolar, observada principalmente aos 14 dias, tanto no grupo exposto ao H2S quanto no grupo Ctrl. A administração de H2S não resultou em perda óssea adicional. A expressão gênica demonstrou aumento significativo de IL1, IL6, RANKL e SOFAT apenas no grupo CtrlPD (p <0,05). Uma significativa regulação negativa na expressão de OPG foi observada ao longo do tempo no grupo CtrlPD (p <0,05). Podese concluir que o H2S não teve efeito adicional na perda óssea alveolar, na ausência de ligadura. Entretanto, na presença de ligadura, a exposição ao H2S teve um efeito imunorregulatório na expressão de citocinas próinflamatórias e próreabsortivas.

In Situ Endoscopic Analysis of Vascular Supply and Regenerated Alveolar Bone in ß-TCP Grafted and Ungrafted Postextraction Sites before Implant Placement: A Prospective Case Control Study.

BACKGROUND: Endoscopy has seen a significant development over recent years in various medical fields with its application expanding from the support of minimal invasive surgery to in situ imaging. In this context, the application of endoscopic techniques to assess the quality of the regenerated bone in situ in the drill hole before implant placement is an appealing approach. AIM: The aim of this study was to use short distance support immersion endoscopy (SD-SIE) to compare the quality of regenerated bone in healed postextraction sites, which are grafted with an in situ hardening ß-TCP, against ungrafted sites, before implant placement. This assessment was based on microscopic bone analysis in combination with the blood vessel count. METHOD: 13 spontaneously healed and 13 grafted postextraction sites in 3 men and 6 women, aged 26-83 years, were evaluated using SD-SIE after 4-6 months. SD-SIE was applied in drill holes before implant placement, and videos were taken from representative central buccal areas. The video recordings were analyzed using Image J software for (1) number of blood vessels per area (NBV), (2) relative area of vessels (VA), (3) relative area of mineralized bone (MBA), (4) relative area of unmineralized bone (UMBA), and (5) relative area of bone substitute (BSA). RESULTS: The grafted sites showed more (1) NBV as well as (2) VA (8.6 ± 1.1; 2.03 ± 0.28%) than the ungrafted sites (2.5 ± 0.6; 1.18 ± 0.36%) (independent t-test; p < 0.05); (3) MBA and (4) UMBA were similar to those in the grafted sites (86.3 ± 2.2 %; 13.7 ± 2.2 %) and to the ungrafted sites (89.5 ± 3.7%; 10.5 ± 3.6%) (independent t-test; p > 0.05); and (5) BSA in the grafted sites was 18.2 ± 5.4%. CONCLUSION: SD-SIE is an interesting new approach for in situ assessment of bone quality and blood supply before implant placement. The regenerated bone in ß-TCP grafted extraction sockets showed an increased vascularization compared to ungrafted sites providing a vital support for subsequent implant placement.

Prevention of Bone Resorption by HA/ß-TCP + Collagen Composite after Tooth Extraction: A Case Series.

After tooth extraction, alveolar ridge loss due to resorption is almost inevitable. Most of this bone loss occurs during the first six months after the extraction procedure. Many studies have indicated that applying socket-filling biomaterials after extraction can effectively reduce the resorption rate of the alveolar ridge. The purpose of this study was to investigate the clinical efficacy of the application of a hydroxyapatite/ß-tricalcium plus collagen (HA/ß-TCP + collagen) dental bone graft in dental sockets immediately after tooth extraction, so as to prevent socket resorption. The study was conducted on 57 extraction sockets located in the mandible and maxilla posterior regions in 51 patients. HA/ß-TCP + collagen was inserted into all of the dental sockets immediately after extraction, and was covered with a flap. Follow-up was performed for three months after extraction, using radiographs and stents for the vertical and horizontal alveolar ridge measurements. A minimal alveolar bone width reduction of 1.03 ± 2.43 mm (p < 0.05) was observed. The height reduction showed a slight decrease to 0.62 ± 1.46 mm (p < 0.05). Radiographically, the bone height was maintained after three months, indicating a good HA/ß-TCP + collagen graft performance in preserving alveolar bone. In conclusion, the HA/ß-TCP + collagen graft demonstrated adequate safety and efficacy in dental socket preservation following tooth extraction.

Losartan improves alveolar bone dynamics in normotensive rats but not in hypertensive rats.

OBJECTIVES: Hypertension is one of the main causes of premature death in the world; also, it is associated with several bone alterations. Preclinical studies have demonstrated delayed alveolar bone healing in hypertensive rats. However, losartan has been favorable for consolidation of bone grafts and reduction in active periodontitis. Therefore, losartan is suggested to be effective in bone formation stages, as well as in the synthesis of matrix proteins and mineralization. To evaluate the alveolar bone dynamics in hypertensive rats treated with losartan by laser confocal microscopy and histological analysis. METHODOLOGY: Thirty-two rats, 16 spontaneously hypertensive rats (SHR) and 16 Wistar albinus rats, treated or not with losartan (30 mg/kg/day) were used. Calcein fluorochrome at 21 days and alizarin red fluorochrome at 49 days were injected in rats (both 20 mg/kg). The animals were submitted to euthanasia 67 days after treatment, and then the right maxilla was removed for laser confocal microscopy analysis and the left maxilla for histological analysis. RESULTS: This study showed a greater calcium marking in normotensive animals treated with losartan in relation to the other groups. Laser confocal microscopy parameters showed higher values of bone volume formed, mineralized surface, active surface of mineralization and bone formation rate in normotensive animals treated with losartan. However, a smaller mineralized surface was observed in all hypertensive animals. CONCLUSION: Losartan can improve bone mineralization parameters under normal physiological conditions, but the same anabolic effect does not occur under hypertension.

Regeneration of a Pediatric Alveolar Cleft Model Using Three-Dimensionally Printed Bioceramic Scaffolds and Osteogenic Agents: Comparison of Dipyridamole and rhBMP-2.

BACKGROUND: Alveolar clefts are traditionally treated with secondary bone grafting, but this is associated with morbidity and graft resorption. Although recombinant human bone morphogenetic protein-2 (rhBMP-2) is under investigation for alveolar cleft repair, safety concerns remain. Dipyridamole is an adenosine receptor indirect agonist with known osteogenic potential. This study compared dipyridamole to rhBMP-2 at alveolar cleft defects delivered using bioceramic scaffolds. METHODS: Skeletally immature New Zealand White rabbits underwent unilateral, 3.5 × 3.5-mm alveolar resection adjacent to the growing suture. Five served as negative controls. The remaining defects were reconstructed with three-dimensionally printed bioceramic scaffolds coated with 1000 µm of dipyridamole (n = 6), 10,000 µm of dipyridamole (n = 7), or 0.2 mg/ml of rhBMP-2 (n = 5). At 8 weeks, new bone was quantified. Nondecalcified histologic evaluation was performed, and new bone was evaluated mechanically. Statistical analysis was performed using a generalized linear mixed model and the Wilcoxon rank sum test. RESULTS: Negative controls did not heal, whereas new bone formation bridged all three-dimensionally printed bioceramic treatment groups. The 1000-µm dipyridamole scaffolds regenerated 28.03 ± 7.38 percent, 10,000-µm dipyridamole scaffolds regenerated 36.18 ± 6.83 percent (1000 µm versus 10,000 µm dipyridamole; p = 0.104), and rhBMP-2-coated scaffolds regenerated 37.17 ± 16.69 percent bone (p = 0.124 versus 1000 µm dipyridamole, and p = 0.938 versus 10,000 µm dipyridamole). On histology/electron microscopy, no changes in suture biology were evident for dipyridamole, whereas rhBMP-2 demonstrated early signs of suture fusion. Healing was highly cellular and vascularized across all groups. No statistical differences in mechanical properties were observed between either dipyridamole or rhBMP-2 compared with native bone. CONCLUSION: Dipyridamole generates new bone without osteolysis and early suture fusion associated with rhBMP-2 in skeletally immature bone defects.