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 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.

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.

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.

Effect of systemic delivery of Substance P on experimental tooth movement in rats.

INTRODUCTION: The purpose of this study was to investigate the effect of systemic delivery of Substance P (SP) on experimental tooth movement. METHODS: Forty-eight adult Sprague-Dawley rats were randomly divided into 2 groups and their maxillary first molars were mesially moved with the use of closed-coil springs. The experiment group received systemic injection of SP and the control group received phosphate-buffered saline solution. Transportation distances of first molars were measured. Hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and immunohistochemistry staining were performed to evaluate alveolar bone remodeling. Then the interferon (IFN) γ and tumor necrosis factor (TNF) α concentrations in peripheral blood and local periodontal tissue were measured. Finally, the effects of SP on bone marrow-derived stem cell (BMSC) proliferation and migration were tested in vitro. RESULTS: Systemic delivery of SP significantly increased the distance of tooth movement and stimulated both osteoclast and osteoblast activities. The concentrations of IFN-γ and TNF-α increased in peripheral blood at early phases of the experiment and decreased in periodontal tissue at late phases. In vitro, the proliferation and migration of BMSCs were promoted by SP. CONCLUSIONS: Systemic delivery of SP can accelerate orthodontic tooth movement and promote alveolar bone remodeling potentially through immunomodulation and mobilizing endogenous mesenchymal stem cells.

Application of Hydroxycholesterols for Alveolar Cleft Osteoplasty in a Rodent Model.

BACKGROUND: Bone morphogenetic proteins (BMPs) have played a central role in the regenerative therapies for bone reconstruction, including alveolar cleft and craniofacial surgery. However, the high cost and significant adverse effect of BMPs limit their broad application. Hydroxycholesterols, naturally occurring products of cholesterol oxidation, are a promising alternative to BMPs. The authors studied the osteogenic capability of hydroxycholesterols on human mesenchymal stem cells and the impact of hydroxycholesterols on a rodent alveolar cleft model. METHODS: Human mesenchymal stem cells were treated with control medium or osteogenic medium with or without hydroxycholesterols. Evaluation of cellular osteogenic activity was performed. A critical-size alveolar cleft was created and one of the following treatment options was assigned randomly to each defect: collagen sponge incorporated with hydroxycholesterols, BMP-2, or no treatment. Bone regeneration was assessed by means of radiologic and histologic analyses and local inflammation in the cleft evaluated. Moreover, the role of the hedgehog signaling pathway in hydroxycholesterol-mediated osteogenesis was examined. RESULTS: All cellular osteogenic activities were significantly increased on human mesenchymal stem cells treated with hydroxycholesterols relative to others. The alveolar cleft treated with collagen sponge with hydroxycholesterols and BMP-2 demonstrated robust bone regeneration. The hydroxycholesterol group revealed histologically complete bridging of the alveolar defect with architecturally mature new bone. The inflammatory responses were less in the hydroxycholesterol group compared with the BMP-2 group. Induction of hydroxycholesterol-mediated in vitro osteogenesis and in vivo bone regeneration were attenuated by hedgehog signaling inhibitor, implicating involvement of the hedgehog signaling pathway. CONCLUSION: Hydroxycholesterols may represent a viable alternative to BMP-2 in bone tissue engineering for alveolar cleft.

Mineralized nanofiber segments coupled with calcium-binding BMP-2 peptides for alveolar bone regeneration.

Bone loss around tooth extraction sites can occur, thus making future placement of dental implants difficult. Alveolar bone regeneration can be guided by the application of a nanofibrous bone graft coupled with osteoinductive proteins/peptides, following tooth loss or tooth extraction. In the present study, we demonstrate the potential of mineralized nanofiber segments coupled with calcium-binding bone morphogenetic protein 2 (BMP-2) mimicking peptides for periodontal bone regeneration. Thin electrospun nanofiber membranes of PLGA-collagen-gelatin (2:1:1 wt ratios) were mineralized in 10× modified simulated body fluid (10× mSBF) and cryocut to segments of 20 µm. For predetermined weights of the mineralized nanofiber segments, it was possible to load various amounts of heptaglutamate E7-domain-conjugated BMP-2 peptide. Mineralized short fiber grafts (2 mg), with and without E7-BMP-2 peptides, were implanted into 2 mm × 2 mm (diameter × depth) critical-sized socket defects created in rat maxillae, following extraction of the first molar teeth. A sustained release profile of E7-BMP-2 from the mineralized nanofiber segments was recorded over 4 weeks. X-ray microcomputed tomography (µ-CT) analysis of peptide-loaded nanofiber graft filled defects revealed ∼3 times greater new bone volume and bone mineral density over 4 weeks in comparison to unfilled control defects. Further, histopathology data confirmed the formation of greater new osseous tissue in the BMP2 peptide-loaded, mineralized nanofiber segment group than that of fibrous connective tissue in the unfilled defect group. Altogether, the mineralized nanofiber segments coupled with E7-BMP-2 peptides may be an effective treatment option for alveolar bone loss and defects. STATEMENT OF SIGNIFICANCE: With the high incidence of dental implants/fixtures for missing teeth, the success of the surgical procedures in restorative dentistry is dictated by the quality and quantity of the supporting alveolar bone. To address the problem of alveolar bone loss and defects due to tumor, periodontitis, or even postextraction remodeling, the present study is the first report on the application of mineralized nanofiber fragments coupled with calcium-binding osteoinductive BMP-2 peptides as a synthetic graft material for oral bone regeneration. The ease of fabrication and application of cryocut mineralized nanofiber fragments as maxillofacial bone defect fillers present a promising alternative to the current dental bone graft formulations. Furthermore, the nanofiber segments may also be utilized for several biomedical applications including hemostasis, soft tissue engineering, and wound healing.

Antimicrobial photodynamic therapy improves the alveolar repair process and prevents the occurrence of osteonecrosis of the jaws after tooth extraction in senile rats treated with zoledronate.

This study evaluated the effects of antimicrobial photodynamic therapy (aPDT) in the alveolar repair of rats with major risk factors for bisphosphonate-related osteonecrosis of the jaws (BRONJ). Senile rats received 0.45 ml of vehicle (VEH and VEH/aPDT) or 0.45 ml of zoledronate (ZOL and ZOL/aPDT) every three days for seven weeks. After three weeks of treatment, the first lower left molar was extracted. VEH/aPDT and ZOL/aPDT were submitted to aPDT on the extraction site at 0, 2 and 4 days postoperatively. Euthanasia was performed 28 days postoperatively and the extraction site was evaluated by clinical, histological, histometric, histochemical and immunohistochemical analysis. ZOL showed tissue repair impairment; lower percentage of newly formed bone tissue (NFBT); higher percentage of non-vital bone tissue (NVBT); fewer mature collagen fibers and increased immunolabeling for tumor necrosis factor (TNFα), interleukin (IL)-1ß and IL-6. ZOL/aPDT showed clinical and histological characteristics of the extraction site, percentage of NFBT and percentage of mature collagen fiber similar to VEH. Percentage of NVBT and immunolabeling for inflammatory cytokines in ZOL/aPDT was lower than in ZOL. Immunolabeling for tartarato-resistant acid phosphatase (TRAP) was lower in ZOL and ZOL/aPDT. aPDT in the dental extraction site improves tissue repair process and prevents the occurrence of BRONJ-like lesions after tooth extraction.

Influence of omega-3 fatty acid on orthodontic tooth movement in rats: A biochemical, histological, immunohistochemical and gene expression study.

OBJECTIVE: The aim of this study was to investigate the effects of omega-3 fatty acids on orthodontic tooth movement. SETTING AND SAMPLE POPULATION: For this study, 56 12-week-old adult male Wistar albino rats from the Animal Laboratory at Adnan Menderes University, Faculty of Medicine, were used. MATERIAL AND METHODS: Rats were randomly divided into seven groups (n = 8 each): control group (without any treatment), tooth movement groups (three groups of animals with only tooth movement) and omega groups (three groups of animals with tooth movement and omega-3 administration). Omega-3 fatty acids were administered to the rats systemically during the tooth movement period. On the 3rd, 7th and 14th days after the orthodontic tooth movement, the rats were sacrificed and biochemical, histological, immunohistochemical andgene expression examinations were performed. RESULTS: On the 14th experimental day, the amount of tooth movement in the omega groups was significantly lower than the tooth movement groups (P = 0.012). Biochemical experimentsshowed that the omega groups had significantly lower total oxidant levels and higher total antioxidant levels compared to the tooth movement group on the 14th experimental day (P = 0.001). The levels of RANKL, IL-6 and IL-1ß in the omega groups were significantly lower than the tooth movement groups on all experimental days (P < 0.05). CONCLUSION: Systemic administration of omega-3 fatty acids showed antioxidant and antiinflammatory effects and decelerate the orthodontic tooth movement.

Effects of formaldehyde on vascular endothelial growth factor, matrix metallopeptidase 2 and osteonectin levels in periodontal membrane and alveolar bone in rats.

BACKGROUND: The objective of this study was to investigate whether long term formaldehyde inhalation may affect periodontal membrane and alveolar bone loss leading to periodontitis. The negative effects of formaldehyde were described using vascular endothelial growth factor (VEGF), matrix metallopeptidase 2 (MMP-2) and osteonectin antibodies involved in the extracellular matrix and angiogenetic development. MATERIALS AND METHODS: Thirty adult Wistar albino rats were used in this study. Rats were divided into two groups: a control group (n = 15) and formaldehyde administered group (n = 15). Formaldehyde group was exposed to inhalation of 10 ppm formaldehyde 8 hours a day, 5 days a week for 5 weeks. Maxillary bone regions were dissected under anaesthesia. After fixation in 10% formaldehyde solution, tissues were passed through graded ethanol series to obtain paraffin blocks. Five-micrometre histological sections were cut with RM2265 rotary microtome stained with Masson trichrome and VEGF, MMP-2 and osteonectin antibodies for examination under Olympus BH-2 light microscopy. RESULTS: The present study revealed that congestion in blood vessels, degeneration of collagen fibres and alveolar matrix around alveolar bone were observed to be more significant in formaldehyde group than the control group (p ≤ 0.001). Interestingly, VEGF expression in the formaldehyde group was the most significant finding between the two groups (p < 0.001). When compared inflammation, MMP-2 and osteonectin expressions were significant (p < 0.01) in the formaldehyde group. CONCLUSIONS: It was suggested that formaldehyde toxicity decreased the expression of MMP-2 and in osteoblasts as well as affecting the retention of MMP levels in tooth cavity, which is very low in collagen fibres. But, vice versa for the expression of VEGF in dilated vascular endothelial cells and osteocytes in alveolar bone. As a conclusion, formaldehyde disrupts the periodontal membrane and may cause collagen fibres degeneration by affecting the alveolar bone matrix.

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

Abstract 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. Objectives: 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.

Effect of acute administration of nicotine and ethanol on tooth movement in rats.

The aim of this study was to evaluate the effect of acute administration of nicotine and ethanol on tooth movement in rats. Two hundred rats were divided into eight groups: S: saline; N: nicotine; E: ethanol; NE: nicotine and ethanol; SM: saline with tooth movement; NM: nicotine with tooth movement; EM: ethanol with tooth movement; and NEM: nicotine and ethanol with tooth movement. All the solutions were applied for 32, 44, or 58 days, according to the subgroup. Orthodontic movement (25 cN) was initiated 30 days after solution administration in the groups with tooth movement. The rats were euthanized 2, 14, or 28 days after initiation of tooth movement. Tooth sections were stained using picrosirius and tartrate-resistant acid phosphatase (TRAP). The data were compared by ANOVA using Tukey's HSD and Games-Howell. On day 28 of tooth movement, the NEM group had a lower percentage of type I collagen compared to the SM group (p = 0.0448), and the S group had a higher number of osteoclasts/µm2 compared to the N group (p = 0.0405). Nicotine and ethanol did not affect the tooth movement rate, regardless of induction of orthodontic movement. Nicotine influenced the number of osteoclasts by decreasing their quantity when dental movement was not induced. When nicotine was associated with ethanol, it interfered in the maturation of collagen fibers during orthodontic movement.

Evaluation of Periodontal Space Widening in Patients with Antiresorptive Drug-related Osteonecrosis of the Jaws (ARONJ) on Panoramic Radiographs.

BACKGROUND/AIM: The purpose of this study was to evaluate a potential widening of the periodontal space as an initial measurable imaging criterium on panoramic radiographs in patients with the diagnosis of antiresorptive drug related osteonecrosis of the jaw (ARONJ). PATIENTS AND METHODS: A retrospective analysis of panoramic radiographs of 16 patients (12 females and 4 males; mean age is 70.5 years, standard deviation is 14 years) was performed, over a period of 12 months with the diagnosis of ARONJ. Panoramic radiographs of 16 healthy patients (12 females and 4 males; mean age was 70.6 years, standard deviation was 13.8 years) without diagnosis of ARONJ served as controls. All images were taken with the same device and were evaluated by two experienced maxillofacial surgeons and one dentist. RESULTS: Compared to the control group, a mean widening of the periodontal space of 0.06 mm (confidence interval (CI)=0.05-0.17 mm) was found in the study group. However, this difference was not statistically significant. CONCLUSION: Although a very extensive and sophisticated interindividual comparison was performed to evaluate for slight changes of the periodontal space in patients with antiresorptive drug therapy, our results demonstrate that PDS widening is not a predictive parameter for ARONJ. Therefore, our results indicate that panoramic radiographs are not sufficient enough to allow assessment of stages and disease progress in ARONJ patients regarding to periodontal space widening.