A Comparison of the Antiosteoporotic Effects of Cornelian Cherry (Cornus mas L.) Extracts from Red and Yellow Fruits Containing Different Constituents of Polyphenols and Iridoids in Osteoblasts and Osteoclasts.

Methods: Polyphenolic and iridoid constituents of extracts were analyzed qualitatively and quantitatively using the ultraperformance liquid chromatography system coupled with a quadrupole-time of flight mass spectrometry. Primary cultured osteoblasts isolated from mouse calvarias and osteoclast-lineage primary cultured monocytes isolated from mouse bone marrow were used for the assessment of osteoblast and osteoclast differentiation. In the osteoblast culture, cellular viability, alkaline phosphatase (ALP) activity, ALP staining, and mRNA expression of Alpl and Runx2 were examined. In the osteoclast culture, the examined parameters were cellular viability, tartrate-resistant acid phosphatase (TRAP) activity and staining, and mRNA expression of Nfatc1, Ctsk, and Acp. Results: A total of 41 main compounds of iridoids, anthocyanins, hydrolysable tannins, phenolic acids, and flavonols were identified in the three extracts. RED EXT1 contained most of the tested polyphenols and iridoids and was the only extract containing anthocyanins. YL EXT2 contained only one iridoid, loganic acid and gallic acid. YL EXT3 comprised a mixture of iridoids and polyphenols. RED EXT1, YL EXT 2, and to a lesser extent YL EXT3 promoted osteoblast differentiation increasing significantly ALP activity and the amount of ALP-positive stained cells. All extracts upregulated mRNA expression of Alpl and Runx2. RED EXT1 caused the most significant decrease in TRAP activity and the numbers of TRAP-positive multinucleated cells. RED EXT1 caused also the most significant downregulation of mRNA expression of osteoclast related genes Nfatc1, Ctsk, and Acp5. Extracts from yellow fruits, mostly YL EXT2 caused lower, but still significant inhibitory effect on TRAP and osteoclast related genes. Conclusions: The main conclusion of our study is that all three extracts, especially RED EXT1 from red cornelian cherry fruits, possess the antiosteoporotic potential and may be a promising phytomedicine candidate for the prevention and treatment of osteoporosis.

Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity.

Histochemical detection of tartrate-resistant acid phosphatase (TRAP) activity is a fundamental technique for visualizing osteoclastic bone resorption and assessing osteoclast activity status in tissues. This approach has mostly employed colorimetric detection, which has limited quantification of activity in situ and co-labelling with other skeletal markers. Here we report simple colorimetric and fluorescent TRAP assays in zebrafish and medaka, two important model organisms for investigating the pathogenesis of bone disorders. We show fluorescent TRAP staining, utilising the ELF97 substrate, is a rapid, robust and stable system to visualise and quantify osteoclast activity in zebrafish, and is compatible with other fluorescence stains, transgenic lines and antibody approaches. Using this approach, we show that TRAP activity is predominantly found around the base of the zebrafish pharyngeal teeth, where osteoclast activity state appears to be heterogeneous.

The clinical utility of TRACP-5b to monitor anti-resorptive treatments of osteoporosis.

TRACP-5b can be used to monitor the response of treatments in osteoporosis. We investigated the effect of feeding on levels of TRACP-5b and how these markers perform in a clinical setting. After feeding, there was no effect on levels TRACP-5b. It has similar diagnostic accuracy to CTX and PINP. INTRODUCTION: Bone turnover markers (BTMs) can be used to monitor response to osteoporosis treatment. However, some are affected by food intake and are not suitable to measure in a clinical setting. An assay is available which is capable of detecting the active isoform 5b of tartrate resistance acid phosphatase (TRACP-5b) and it may have minimal biological variation. Our aims were to investigate the effect of feeding on levels of TRACP-5b and compare this to CTX and PINP and then to compare the diagnostic accuracy of TRACP-5b to CTX and PINP in patients with osteoporosis given commonly used treatments. METHODS: Eighteen patients were recruited to investigate the effect of feeding on BTMs. Ninety-seven patients (74 females and 23 males) receiving 5 mg annual intra-venous zoledronate (mean age 70) and 97 patients receiving no treatment were recruited as group-matched controls. Sixteen patients receiving 60 mg subcutaneous denosumab every 6 months, (mean age 76) and 16 matched controls were recruited. Seventy-six patients were receiving oral bisphosphonates: 70 mg alendronate weekly, 35 mg risedronate and 150 mg monthly ibandronate (4%). Thirty of these patients had BMD measured at the total hip and lumbar spine. An estimate of compliance was not determined. Eighty patients receiving no treatment were recruited as group-matched controls. TRACP-5b (ELISA, Nittobo) and CTX and PINP were measured in serum in the non-fasting state between 0800 and 1700. RESULTS: After feeding, there was no effect on levels TRACP-5b and significant reductions in CTX and PINP, 29% and 10%, respectively (p < 0.001). In the zoledronate and denosumab groups, there were no differences in the areas under the curves (AUCs) between TRACP-5b, PINP and CTX. In the oral bisphosphonates group, the AUCs between TRACP-5b and PINP and TRACP-5b and CTX were significantly different, p < 0.01 and p = 0.001, respectively. TRACP-5b was negatively correlated with BMD. CONCLUSION: TRACP-5b is not affected by food intake, unlike CTX and PINP. All three BTMs correlate with change in BMD at the lumbar spine and total hip. TRACP-5b has similar diagnostic accuracy to CTX and PINP with commonly used treatments for osteoporosis with the exception of oral bisphosphonate therapy.

Analytical evaluation of the Nittobo Medical tartrate resistant acid phosphatase isoform 5b (TRACP-5b) EIA and comparison with IDS iSYS in different clinically defined populations.

OBJECTIVES: Tartrate-resistant acid phosphatase, isoform 5b (TRACP-5b) is a bone resorption marker not influenced by renal function or food intake. TRACP-5b can be measured with Nittobo Medical enzymatic-immunoassay and IDS-iSYS automated immunoassay. We evaluated the Nittobo assay and established reference ranges for a Western-European population. We compared Nittobo and IDS results in different well-defined clinical populations. METHODS: We established the limits of detection and quantification (LOD-LOQ), linearity, imprecision and the reference ranges in 119 males, 50 women (<45 years) and 120 women (>60 years) for TRACP-5b with the Nittobo assay. We compared both assays in 30 hemodialyzed (HD), and 40 stage 3-5 patients suffering from chronic kidney disease (CKD), 40 patients suffering from rheumatoid arthritis and osteoporosis and 80 post-menopausal women. We measured TRACP-5b, ß-crosslaps (ß-CTX), bone alkaline phosphatase (B-ALP) and PTH in 20 hemodialyzed (HD) and 40 CKD patients. RESULTS: LOD and LOQ were 0.02 and 0.35 U/L. CV ranged from 8.3 to 4.3% (2/5 samples presenting CV > desirable CV). Method was linear up to of 11.3 U/L. Upper and lower limits of normality were 0.8-7.6 U/L in men, 0.9-4.7 U/L in women <45 and 0.9-7.1 U/L in women >60. The regression equation between the 2 methods was Nittobo = 1.13 (95% CI: 1.09-1.16) × iSYS - 0.4 (95% CI: -0.5; -0.3). TRACP-5b and b-ALP were in their respective reference ranges for most of CKD and HD patients. That was not the case for ß-CTX, which increased with decreasing eGFR. CONCLUSIONS: Nittobo TRACP-5b presents interesting analytical features and a good concordance with IDS iSYS. These methods could thus potentially be harmonized.

A Novel Sandwich ELISA for Tartrate-Resistant Acid Phosphatase 5a and 5b Protein Reveals that Both Isoforms are Secreted by Differentiating Osteoclasts and Correlate to the Type I Collagen Degradation Marker CTX-I In Vivo and In Vitro.

Tartrate-resistant acid phosphatase type 5 (TRAP) exists as two isoforms, 5a and 5b. 5b is a marker of osteoclast number and 5a of chronic inflammation; however, its association with bone resorption is unknown. In this study, a double-TRAP 5a/5b sandwich ELISA measuring 5a and 5b protein in the same sample was developed. TRAP 5a and 5b protein levels were evaluated as osteoclast differentiation/activity markers in serum and in culture, and their correlation to the resorption marker CTX-I was examined. Serum TRAP 5a and 5b concentrations in healthy men were 4.4 ± 0.6 ng/ml and 1.3 ± 0.2 ng/ml, respectively, and they correlated moderately to each other suggesting that their secretion is coupled under healthy conditions. A correlation was also observed between serum TRAP 5a and 5b with CTX-I, suggesting that both TRAP isoforms associate with osteoclast number. During osteoclast differentiation on plastic/bone, predominantly 5b increased in media/lysate from M-CSF/RANKL-stimulated CD14+ PBMCs. However, substantial levels of 5a were detected at later stages suggesting that both isoforms are secreted from differentiating OCs. More TRAP 5b was released on bone indicating a connection to osteoclast resorptive activity, and a peak in TRAP 5b/5a-ratio coincided with rapid CTX-I release. At the end of the culture period of M-CSF + RANKL-stimulated CD14+ PBMCs, there was a correlation between the secretion of TRAP 5a and 5b proteins with CTX-I. The correlation of not only 5b but also 5a with collagen degradation, both in serum and osteoclast cultures indicates that a considerable proportion of the TRAP 5a originates from osteoclasts and may reflect a hitherto undisclosed regulatory mechanism during bone resorption and bone remodeling.

Effect of avocado/soybean unsaponifiables on periodontal repair in rats with arthritis and induced periodontitis.

OBJECTIVE: This study aimed to evaluate the effect of avocado/soybean unsaponifiables (ASU) on periodontal repair in rats with induced periodontitis and arthritis. METHODOLOGY: Forty-five rats were submitted to periodontitis induction by insertion of ligatures into the upper second molars, maintained for 15 days. These animals were randomly allocated to 3 groups according to the presence of induced arthritis (ART) and the application of the ASU: Control (CTR) group-healthy animals, where saline solution was administered; ART-animals with induced arthritis, where saline solution was administered; ART/ASU-animals with induced arthritis, where ASU (0.6 mg/ kg) was administered. The drugs were administered daily by gavage and the animals were euthanized after 7, 15 and 30 days of the ligature removal. Bone resorption, inflammatory infiltrate composition and marker proteins expression of the differentiation and formation of osteoclasts (RANKL and TRAP) were assessed. RESULTS: The ART/ASU group presented higher bone volume than the ART group at 7 and 30 days after the ligature removal. Furthermore, the ART group presented higher quantity of inflammatory cells and expression of TRAP and RANKL than the other groups. CONCLUSION: ASU administration improves the repair of periodontal tissues in an experimental periodontitis model in rats with induced arthritis.

The effect of colchicine on alveolar bone loss in ligature-induced periodontitis.

Colchicine is widely used in the treatment of several inflammatory diseases due to its anti-inflammatory effect, but effects on bone metabolism are unclear. The aim of this study was to evaluate the effects of systemically-administered colchicine on healthy periodontium and experimentally-induced periodontitis. In total, 42 male Wistar rats were included in this study. A non-ligated group constituting the negative control group (Control, C, n = 6) and a ligature-only group forming the positive control group (LO, n = 12) were created separately. Twelve rats were treated with 0.4 mg/kg colchicine and another 12 with 1 mg/kg colchicine. In the colchicine-administered groups, right mandibles constituted the ligated groups (1 mgC-L or 0.4 mgC-L) and left mandibles formed the corresponding non-ligated controls (1mgC or 0.4mgC). Silk ligatures were placed at the gingival margin of the lower first molars. The animals were euthanized at different time-points of healing (11 or 30 days). Alveolar bone loss was clinically measured and TRAP+ osteoclasts, osteoblastic activity, and MMP-1 expression were examined histologically. There was no increase in alveolar bone loss with either colchicine dose in healthy periodontium (p > 0.05) and the highest level of alveolar bone loss, TRAP+ osteoclast number, and MMP-1 expression were measured in the LO group (p < 0.05). The 0.4 mgC-L group showed less alveolar bone loss at 11 days (p < 0.05), but greater loss at 30 days. The 1 mgC-L group showed higher osteoblast number than the other ligated groups (p < 0.05) at both time-points. In summary, colchicine did not increase alveolar bone loss in healthy periodontium and also may tend to reduce periodontitis progression. However, further extensive study is necessary to understand the mechanism of colchicine action on alveolar bone loss in periodontitis.

Osteoclast Differentiation Assay.

Osteoclasts are highly specialized multinucleated cells derived from the monocyte/macrophage hematopoietic lineage that are uniquely capable of adhering to bone matrix and resorbing bone. The tartrate-resistant acid phosphatase (TRAP) assay is the most common method to detect osteoclasts population in vitro. Here we described a general protocol of inducing osteoclast differentiation from the murine macrophage cell line, RAW264.7, and identification of osteoclasts with the classical TRAP assay.

Effect of avocado/soybean unsaponifiables on periodontal repair in rats with arthritis and induced periodontitis

Abstract Objective: This study aimed to evaluate the effect of avocado/soybean unsaponifiables (ASU) on periodontal repair in rats with induced periodontitis and arthritis. Methodology: Forty-five rats were submitted to periodontitis induction by insertion of ligatures into the upper second molars, maintained for 15 days. These animals were randomly allocated to 3 groups according to the presence of induced arthritis (ART) and the application of the ASU: Control (CTR) group-healthy animals, where saline solution was administered; ART-animals with induced arthritis, where saline solution was administered; ART/ASU-animals with induced arthritis, where ASU (0.6 mg/ kg) was administered. The drugs were administered daily by gavage and the animals were euthanized after 7, 15 and 30 days of the ligature removal. Bone resorption, inflammatory infiltrate composition and marker proteins expression of the differentiation and formation of osteoclasts (RANKL and TRAP) were assessed. Results: The ART/ASU group presented higher bone volume than the ART group at 7 and 30 days after the ligature removal. Furthermore, the ART group presented higher quantity of inflammatory cells and expression of TRAP and RANKL than the other groups. Conclusion: ASU administration improves the repair of periodontal tissues in an experimental periodontitis model in rats with induced arthritis.

The effect of colchicine on alveolar bone loss in ligature-induced periodontitis

Abstract Colchicine is widely used in the treatment of several inflammatory diseases due to its anti-inflammatory effect, but effects on bone metabolism are unclear. The aim of this study was to evaluate the effects of systemically-administered colchicine on healthy periodontium and experimentally-induced periodontitis. In total, 42 male Wistar rats were included in this study. A non-ligated group constituting the negative control group (Control, C, n = 6) and a ligature-only group forming the positive control group (LO, n = 12) were created separately. Twelve rats were treated with 0.4 mg/kg colchicine and another 12 with 1 mg/kg colchicine. In the colchicine-administered groups, right mandibles constituted the ligated groups (1 mgC-L or 0.4 mgC-L) and left mandibles formed the corresponding non-ligated controls (1mgC or 0.4mgC). Silk ligatures were placed at the gingival margin of the lower first molars. The animals were euthanized at different time-points of healing (11 or 30 days). Alveolar bone loss was clinically measured and TRAP+ osteoclasts, osteoblastic activity, and MMP-1 expression were examined histologically. There was no increase in alveolar bone loss with either colchicine dose in healthy periodontium (p > 0.05) and the highest level of alveolar bone loss, TRAP+ osteoclast number, and MMP-1 expression were measured in the LO group (p < 0.05). The 0.4 mgC-L group showed less alveolar bone loss at 11 days (p < 0.05), but greater loss at 30 days. The 1 mgC-L group showed higher osteoblast number than the other ligated groups (p < 0.05) at both time-points. In summary, colchicine did not increase alveolar bone loss in healthy periodontium and also may tend to reduce periodontitis progression. However, further extensive study is necessary to understand the mechanism of colchicine action on alveolar bone loss in periodontitis.

Evidence for excessive osteoclast activation in SIRT6 null mice.

SIRT6 is a NAD-dependent histone 3 deacetylase. SIRT6 null mice have been reported suffering osteopenia. However, the role of SIRT6 in bone resorption is still not well understood. In this study, we focused on the role of SIRT6 in osteoclast. We performed histological analysis on the femur, spine, alveolar bone and even tail of mutant mice, and found the bone mass is sharply decreased while the osteoclast activity is significantly increased. These phenotypes were further demonstrated by the osteoclast differentiation in cell-cultures with TRAP staining and Pit Resorption Assay. We next found the proliferation activity of mutant osteoclast precursors was increased, which might account for the enhanced osteoclast formation. The concentration of tartrate-resistant acid phosphatase 5b, a marker of osteoclast differentiation, was significantly higher in the mutant mice than control. Besides, the osteoclastogenic and NF-κB signaling related genes were significantly up-regulated. Moreover, osteoblast/osteoclast co-culture demonstrated that SIRT6 regulated osteoclast mainly through osteoblast paracrine manner, rather than osteoclast-autonomous behavior. Together, the enhanced osteoclast activation in SIRT6 null mice might be regulated by the hyperactive NF-κB signaling and the enhanced proliferation activity of osteoclast precursors through osteoblast paracrine manner at the cellular level.

Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis.

INTRODUCTION: Alveolar bone healing after upper incisor extraction in rats is a classical model of preclinical studies. The underlying morphometric, cellular and molecular mechanism, however, remains imprecise in a unique study. OBJECTIVES: The aim of this study was therefore to characterize the alveolar bone healing after upper incisor extraction in rats by micro computed tomographic (Micro-CT), immunohistochemical and real-time polymerase chain reaction (RT-PCR) analysis. MATERIAL AND METHODS: Thirty animals (Rattus norvegicus, Albinus Wistar) were divided into three groups after upper incisors extraction at 7, 14, and 28 days. Micro-CT was evaluated based on the morphometric parameters. Subsequently, the histological analyses and immunostaining of osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL) and tartrate resistant acid phosphate (TRAP) was performed. In addition, RT-PCR analyses of OPG, RANKL, the runt-related transcription factor 2 (RUNX2), osteocalcin (OC), osteopontin (OPN), osterix (OST) and receptor activator of nuclear kappa B (RANK) were performed to determine the expression of these proteins in the alveolar bone healing. RESULTS: Micro-CT: The morphometric parameters of bone volume and trabecular thickness progressively increased over time. Consequently, a gradual decrease in trabecular separation, trabecular space and total bone porosity was observed. Immunohistochemical: There were no differences statistically significant between the positive labeling for OPG, RANKL and TRAP in the different periods. RT-PCR: At 28 days, there was a significant increase in OPG expression, while RANKL expression and the RANKL/OPG ratio both decreased over time. CONCLUSION: Micro-CT showed the newly formed bone had favorable morphometric characteristics of quality and quantity. Beyond the RUNX2, OC, OPN, OST, and RANK proteins expressed in the alveolar bone healing, OPG and RANKL activity showed to be essential for activation of basic multicellular units during the alveolar bone healing.

Morphometric and histopathological evaluation of the effect of grape seed proanthocyanidin on alveolar bone loss in experimental diabetes and periodontitis.

OBJECTIVE: Grape seed proanthocyanidine extract (GSPE) is a strong antioxidant derived from the grape seeds (Vitis vinifera, Terral J.F.) and has a polyphenolic structure with a wide range of biological activity. The aim of the present study was to evaluate the effects of GSPE on alveolar bone loss and histopathological changes in rats with diabetes mellitus and ligature-induced periodontitis. MATERIAL AND METHODS: Forty rats were divided into 6 study groups. Control (C, 6 rats) group, periodontitis (P, 6 rats) group, diabetes (D, 6 rats) group, diabetes and periodontitis (D+P, 6 rats) group, diabetes, periodontitis and 100 mg/kg/day GSPE (GSPE-100, 8 rats), and diabetes, periodontitis and 200 mg/kg/day GSPE (GSPE-200, 8 rats) group. Diabetes mellitus was induced by intraperitoneal injection of a single dose of streptozotocin (60 mg/kg). Periodontitis was induced via ligation method. Silk ligatures were placed at the mandibular right first molars. GSPE was administered by oral gavage. After 30 days, all rats were killed. Alveolar bone loss was measured morphometrically via a stereomicroscope. For histopathological analyses, Alizarin red staining, and matrix metalloproteinase (MMP)-8, vascular endothelial growth factor and hypoxia inducible factor (HIF)-1α immunohistochemistry were performed. Tartrate-resistant acid phosphatase-positive osteoclast cells and relative total inflammatory cells were also determined. RESULTS: The highest alveolar bone loss was observed in the D+P group (P < .05). GSP-200 group decreased alveolar bone loss (P < .05). The D+P group had the highest osteoclast counts, but the difference was not significant compared to the P, GSPE-100 and GSPE-200 groups (P > .05). The inflammation in the D+P group was also higher than the other groups (P < .05). The osteoblast numbers increased in the GSPE-100 and GSPE-200 groups compared to the P and D+P groups (P < .05). MMP-8 and HIF-1α levels were highest in the D+P group and GSPE significantly decreased these levels (P < .05). CONCLUSION: Within the limits of this animal study, it can be suggested that GSPE administration may decrease periodontal inflammation and alveolar bone loss via decreasing MMP-8 and HIF-1α levels and increase osteoblastic activity in diabetic rats with experimental periodontitis.

Bone repair access of BoneCeramic™ in 5-mm defects: study on rat calvaria.

OBJECTIVE: The aim of this study was to evaluate the osteoconductive potential of BoneCeramic™ on bone healing in rat calvaria 5-mm defects. MATERIAL AND METHODS: A 5-mm calvaria bone defect was induced in three groups and the defect was not filled with biomaterial [Clot Group (CG)], autogenous bone (AG), or Bone Ceramic Group (BCG). Animals were euthanized after 14 or 28 days and the bone tissue within the central area of the bone defect was evaluated. Results were compared using ANOVA and Tukey test (p<0.05). Immunohistochemistry was performed using primary antibodies against osteocalcin, RUNX-2, TRAP, VEGF proteins, and 3-dimensional images of the defects in µCT were obtained to calculate bone mineral density (BMD). RESULTS: In BCG, the defect was completely filled with biomaterial and new bone formation, which was statistically superior to that in the GC group, at both time-points (p<0.001 for 14 days; p=0.002 for 28 days). TRAP protein showed weak, RUNX-2 showed a greater immunolabeling when compared with other groups, VEGF showed moderate immunostaining, while osteocalcin was present at all time-points analyzed. The µCT images showed filling defect by BCG (BMD= 1337 HU at 28 days). CONCLUSION: Therefore, the biomaterial tested was found to be favorable to fill bone defects for the reporting period analyzed.

The effects of storage time and temperature on the stability of salivary phosphatases, transaminases and dehydrogenase.

OBJECTIVES: To investigate the influence of temperature and storage time on salivary acid phosphatase (ACP), tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and lactate dehydrogenase (LDH). DESIGN: Unstimulated whole expectorated saliva was collected from healthy men and women subjects (n=26) between 8 and 10a.m. The saliva samples were centrifuged, and the supernatants were measured for ACP, TRAP, ALP, AST, ALT and LDH activities immediately (without freezing) [baseline values] and after time intervals of 3, 7, 14 and 28days (d) of storage at -20°C and -80°C using spectrophotometric methods The influence of storage time was analyzed by one-way ANOVA followed by the Dunnett post-test, while the paired Student's-t-test was used to compare the differences between the temperature (p<0.05). RESULTS: There was significant decline in the activities of all enzymes at -20°C with increasing storage time. This decrease was relevant from day 14 onward for the majority of the enzymes, with the exception of AST. After day 28, the more sensitive enzymes were ALP and LDH, which showed residual activity of 39% and 16%, respectively, compared with baseline values. There were considerable, but insignificant changes, in the activities of all enzymes after storage at -80°C for 28days. CONCLUSIONS: Frozen samples should be kept at -80°C to preserve these activities, but there are restrictions for the enzymes ALP, ALT and LDH. Storage of samples at -20°C could introduce high error variance in measured activities.

Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis

Abstract Alveolar bone healing after upper incisor extraction in rats is a classical model of preclinical studies. The underlying morphometric, cellular and molecular mechanism, however, remains imprecise in a unique study. Objectives The aim of this study was therefore to characterize the alveolar bone healing after upper incisor extraction in rats by micro computed tomographic (Micro-CT), immunohistochemical and real-time polymerase chain reaction (RT-PCR) analysis. Material and Methods Thirty animals (Rattus norvegicus, Albinus Wistar) were divided into three groups after upper incisors extraction at 7, 14, and 28 days. Micro-CT was evaluated based on the morphometric parameters. Subsequently, the histological analyses and immunostaining of osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL) and tartrate resistant acid phosphate (TRAP) was performed. In addition, RT-PCR analyses of OPG, RANKL, the runt-related transcription factor 2 (RUNX2), osteocalcin (OC), osteopontin (OPN), osterix (OST) and receptor activator of nuclear kappa B (RANK) were performed to determine the expression of these proteins in the alveolar bone healing. Results Micro-CT: The morphometric parameters of bone volume and trabecular thickness progressively increased over time. Consequently, a gradual decrease in trabecular separation, trabecular space and total bone porosity was observed. Immunohistochemical: There were no differences statistically significant between the positive labeling for OPG, RANKL and TRAP in the different periods. RT-PCR: At 28 days, there was a significant increase in OPG expression, while RANKL expression and the RANKL/OPG ratio both decreased over time. Conclusion Micro-CT showed the newly formed bone had favorable morphometric characteristics of quality and quantity. Beyond the RUNX2, OC, OPN, OST, and RANK proteins expressed in the alveolar bone healing, OPG and RANKL activity showed to be essential for activation of basic multicellular units during the alveolar bone healing.

Bone repair access of BoneCeramic™ in 5-mm defects: study on rat calvaria

Abstract Objective: The aim of this study was to evaluate the osteoconductive potential of BoneCeramic™ on bone healing in rat calvaria 5-mm defects. Material and Methods: A 5-mm calvaria bone defect was induced in three groups and the defect was not filled with biomaterial [Clot Group (CG)], autogenous bone (AG), or Bone Ceramic Group (BCG). Animals were euthanized after 14 or 28 days and the bone tissue within the central area of the bone defect was evaluated. Results were compared using ANOVA and Tukey test (p<0.05). Immunohistochemistry was performed using primary antibodies against osteocalcin, RUNX-2, TRAP, VEGF proteins, and 3-dimensional images of the defects in μCT were obtained to calculate bone mineral density (BMD). Results: In BCG, the defect was completely filled with biomaterial and new bone formation, which was statistically superior to that in the GC group, at both time-points (p<0.001 for 14 days; p=0.002 for 28 days). TRAP protein showed weak, RUNX-2 showed a greater immunolabeling when compared with other groups, VEGF showed moderate immunostaining, while osteocalcin was present at all time-points analyzed. The μCT images showed filling defect by BCG (BMD= 1337 HU at 28 days). Conclusion: Therefore, the biomaterial tested was found to be favorable to fill bone defects for the reporting period analyzed.

[Overexpression of Klotho inhibits differentiation of RAW264.7 cells into osteoclasts].

Objective To explore the effect of overexpression of Klotho (KL) on the differentiation of RAW264.7 cells intoosteoclasts induced by soluble receptor activator of nuclear factor-κB ligand ( sRANKL). Methods The RAW264.7 cells were divided into Ad-KL group, Ad-GFP group and control group. Inverted microscope was used to observe KL transfected cells. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to detect mRNA and protein expression level of KL, respectively. Tartrate-resistant acidphosphatase (TRAP) staining was applied to observe the osteoclast differentiation and maturation. The qRT-PCR and Western blotting were performed to assess mRNA or protein expression levels of TRAP and cathepsin K (CTSK). Results Klotho gene was transfected into RAW264.7 cells successfully. Compared with Ad-GFP group and control group, KL mRNA and protein expression levels of Ad-KL group increased significantly. TRAP staining showed that the number and volume of TRAP-positive cells in Ad-KL group were less than those in Ad-GFP group and control group. Compared with Ad-GFP group and control group, TRAP and CTSK mRNA and protein expression levels were reduced remarkably in Ad-KL group. Conclusion Overexpression of Klotho inhibits the differentiation of RAW264.7 cells into osteoclasts.

Effects of avocado/soybean unsaponifiables (ASU) on the treatment of ligature-induced periodontitis in rats

The purpose of this study is to evaluate the effect of the avocado/soybean unsaponifiables (ASU) on the treatment of induced periodontitis in rats. Periodontitis was induced in 84 rats via ligature placement around the second upper molar, which was removed after 7 days, and scaling and root planning (SRP) was performed at this time. Subsequently, the rats were randomly allocated to four groups with 21 animals each: One SRP group in which saline solution was administered (SS), and three groups in which ASU was administered (0.6 g/kg/day), beginning either 7 days before the induction of periodontitis (SRP/ASU-7), on the day of periodontitis induction (SRP/ASU0), or on the day of treatment (SRP/ASU+7). ASU and SS were administered daily by gavage until the sacrifice of the animals (7, 15, and 30 days after SRP). The % bone in the furcation area was evaluated by histomorphometry and micro-CT. The expression of proteins (TRAP, RANKL, and alkaline phosphatase) and mRNA (IL-1ß, TNF-α, IL-6, RANKL, and alkaline phosphatase) were evaluated by immunohistochemistry and qPCR. The SRP/ASU+7 group presented a higher percentage of bone fill in the furcation area and higher expression of alkaline phosphatase than in the SRP group (at 7 and 30 days, respectively). The SRP/ASU0 and SRP/ASU+7 groups presented lower expression levels of RANKL mRNA than the SRP and SRP/ASU-7 groups at 15 days. ASU administration on the day of the SRP treatment of the ligature-induced periodontitis promoted subtle beneficial effects on periodontal repair following the treatment of induced periodontitis within the experimental period of 7 days.

Bone resorptive activity in symptomatic and asymptomatic apical lesions of endodontic origin.

OBJECTIVES: The aim of this study is to assess the levels and diagnostic accuracy of a set of bone resorption biomarkers, including TRAP-5, RANKL, and OPG in symptomatic and asymptomatic apical lesions and controls. MATERIALS AND METHODS: Apical tissues from symptomatic and asymptomatic apical periodontitis patients and periodontal ligaments from healthy teeth extracted for orthodontic reasons were processed for tissue homogenization and the levels of TRAP-5, RANKL, and OPG were determined by multiplex assay. Marker levels were analyzed by Kruskal Wallis test, and diagnostic accuracy was analyzed with ROC curves. RESULTS: Higher levels of RANKL, OPG, and RANKL/OPG ratio were determined in both types of apical lesions compared to healthy periodontal ligament, whereas higher TRAP-5 levels were found only in symptomatic apical lesions (p < 0.05). OPG, RANKL, and RANKL/OPG ratio showed diagnostic potential to identify apical lesions versus healthy controls (AUC = 0.69, p < 0.05); while TRAP-5 showed a potential to discriminate symptomatic versus asymptomatic apical periodontitis (AUC = 0.71, p < 0.05) and healthy controls (AUC = 0.83, p < 0.05). CONCLUSIONS: Apical lesions showed higher RANKL and OPG levels than healthy tissues. TRAP-5 levels were the highest in symptomatic apical lesions, suggesting that these represent a progressive state, and showed diagnostic potential. CLINICAL RELEVANCE: Clinically symptomatic apical periodontitis might represent biologically progressive apical lesions based on TRAP5 levels. TRAP5 has diagnostic potential to identify these lesions, representing a candidate prognostic biomarker.