Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism.@*METHODS@#MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells.@*RESULTS@#The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05).@*CONCLUSION@#Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.

Melatonin has a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/secretion / Melatonina tem efeito estimulador em osteoblastos, pela regulação positiva da expressão/secreção de COL-I e OPN

ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.

RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Effects of NSAIDs and environmental oxygen pressure on bone regeneration / Efecto de los AINEs y la presión de oxígeno ambiental sobre la regeneración ósea

Objective: To evaluate the effects of administering diclofenac and ketoprofen, as well as the effects of environmental oxygen pressure variation on mandibular bone regeneration. Methods: Thirty-six guinea pigs were distributed into two equal groups. Mandibular bone defects were performed on both groups. Group A was monitored under oxygen pressure at altitude (3320msl, 107mm Hg). Group B was monitored at sea level oxygen pressure (150msl, 157mm Hg). Each group was subdivided into 3 equal groups (A1, A2, A3 and B1, B2, B3). Subgroups A1 and B1 were given diclofenac; subgroups A2 and B2 ketoprofen; subgroups A3 and B3 NaCl. Bone regeneration was evaluated histologically on days 15 and 30. Results: After 15 days in the group controlled at sea level, the level of osteoblasts presented by the control subgroup was significantly higher (28.00±2.65) compared to the diclofenac subgroup (16.00±6.25) and to the ketoprofen subgroup (18.00±4.36); (p=0.041). After 15 days in the group controlled at altitude, the level of osteoblasts was significantly higher in the control subgroup (38.00±5.29) compared to the diclofenac subgroup (21.67±6.35) and to the ketoprofen subgroup (19.33±2.52); p=0.007. After 30 days in the group at sea level there was no difference found in the cell counting; p>0.05. After 30 days in the group controlled at altitude, the level of osteoblast was significantly higher in the control subgroup (58.00±4.58) compared to the diclofenac subgroup (34.33±4.73) and the ketoprofen subgroup (34.00±11.14); (p=0.003). Conclusion: The administration of diclofenac and ketoprofen produced lower mandibular bone regeneration, the effect being significantly more negative at sea level.

Objetivo: Evaluar el efecto de la administración de diclofenaco y ketoprofeno y de la variación de la presión de oxígeno ambiental sobre la regeneración ósea mandibular. Métodos: Participaron 36 cobayos distribuidos en dos grupos iguales. A ambos grupos se les realizaron defectos óseos mandibulares. El Grupo A fue controlado bajo presión de oxígeno en altura (3320msnm, 107mm Hg). El Grupo B fue controlado bajo presión de oxígeno a nivel del mar (150msnm, 157mm Hg). Cada grupo fue dividido en 3 subgrupos iguales (A1, A2, A3 y B1, B2, B3). Los subgrupos A1 y B1 recibieron diclofenaco; A2 y B2, ketoprofeno; A3 y B3, NaCl. La regeneración ósea fue evaluada histológicamente a los 15 y 30 días. Resultados: A nivel del mar, a los 15 días, hubo una significativa mayor cantidad de osteoblastos en el subgrupo control (28,00±2,65) comparado con el subgrupo diclofenaco (16,00±6.25) y ketoprofeno (18,00±4.36); (p=0,041). En altura, a los 15 días, hubo una significativa mayor cantidad de osteblastos en el subgrupo control (38,00±5,29) comparado con el subgrupo diclofenaco (21,67±6,35) y ketoprofeno (19,33±2,52); p=0,007. A nivel del mar, a los 30 días, no se encontró diferencia en el conteo celular; p>0,05. En altura, a los 30 días, se encontró una significativa mayor cantidad de osteoblastos en el subgrupo control (58,00±4,58) comparado con el subgrupo diclofenaco (34,33±4,73) y ketoprofeno (34,00±11,14); (p=0,003). Conclusión: La administración de diclofenaco y ketoprofeno produjeron una menor regeneración ósea mandibular, siendo este efecto significativamente más negativo a nivel del mar.

Regulation of ghrelin receptor by microbial and inflammatory signals in human osteoblasts

Abstract: Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1β and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1β and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1β-induced GHS-R1a upregulation. IL-1β and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1β and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1β and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1β and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.

Short-term use of resveratrol in alloplastic graft material applied with calvarial bone defects in rats

Abstract Purpose: The effects of resveratrol administration on calvarial bone defects with alloplastic graft material was investigated for osteoinductive reaction and bone development in rats. Methods: Healthy male rats were randomly divided into 3 groups consisting of 10 rats. Groups were as follows: control (defect) group, defect + graft group, and defect + graft + resveratrol group. A calvarial bone defect was created in all groups, alloplastic bone grafts were applied to the defect in the 2nd and 3rd group, resveratrol (5 mg/kg/day) was added to the drinking water of the animals following graft application for 28 days in the 3rd group. Results: Increase in osteoclasts and necrotic changes were observed histopathologically in the control group. In the 2nd group, reduction of inflammation, congestion of blood vessels, increased osteblastic activity, osteoinductive effect, progression of osteocyte development and increased collagen fibers in connective tissue were observed. In the 3rd group, osteoblasts seemed to secrete bone matrix and accelerate osteoinductive effect with increased osteopregenitor activity and positive osteopontin and osteonectin expressions. Conclusion: Resveratrol treatment was thought to be an alternative and supportive drug for implant application by inducing new bone formation in the calvaral defect region as a result of short-term treatment.

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.

Effect of risperidone on proliferation and apoptosis of MC3T3-E1 cells

This aim of this study was to assess the molecular mechanism of osteoporosis in schizophrenia patients with risperidone use. Here, we investigated the effects of risperidone on cellular proliferation and apoptosis of a preosteoblast cell line, MC3T3-E1. Cell viability and apoptotic rate of MC3T3-E1 were detected by cell counting kit-8 and flow cytometry at a serial dose of risperidone and at different time points, respectively. Bone transformation relevant gene serum osteocalcin (BGP), collagen 1, tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels were determined by real-time PCR (qPCR). Their protein expression patterns were evaluated using western blot. The results revealed that risperidone dramatically inhibited MC3T3-E1 cell proliferation in a dose-dependent manner. It also significantly induced MC3T3-E1 cell apoptosis. TNF-α gene and protein levels were greatly enhanced after risperidone treatment. In contrast, BGP, collagen 1, OPG, and RANKL gene and protein levels were markedly downregulated. Our study indicated that risperidone suppressed MC3T3-E1 cell proliferation and induced apoptosis. It also regulated BGP gene and protein expression.

Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition

Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI−, while Z-IETD-FMK caused a shift in the cell population from AV+PI− to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α.

Addition of zirconium oxide to Biodentine increases radiopacity and does not alter its physicochemical and biological properties

Abstract Objectives: To evaluate the radiopacity of Biodentine (BD) and BD associated with 15% calcium tungstate (BDCaWO4) or zirconium oxide (BDZrO2), by using conventional and digital radiography systems, and their physicochemical and biological properties. Materials and Methods: Radiopacity was evaluated by taking radiographs of cement specimens (n=8) using occlusal film, photostimulable phosphor plates or digital sensors. Solubility, setting time, pH, cytocompatibility and osteogenic potential were also evaluated. Data were analyzed using one-way ANOVA and Tukey post-test or two-way ANOVA and Bonferroni post-test (α=0.05). Results: BD radiopacity was lower than 3 mm Al, while BD ZrO2 and BD CaWO4 radiopacity was higher than 3 mm Al in all radiography systems. The cements showed low solubility, except for BDCaWO4. All cements showed alkaline pH and setting time lower than 34 minutes. MTT and NR assays revealed that cements had greater or similar cytocompatibility in comparison with control. The ALP activity in all groups was similar or greater than the control. All cements induced greater production of mineralized nodules than control. Conclusions: Addition of 15% ZrO2 or CaWO4 was sufficient to increase the radiopacity of BD to values higher than 3 mm Al. BD associated with radiopacifiers showed suitable properties of setting time, pH and solubility, except for BDCaWO4, which showed the highest solubility. All cements had cytocompatibility and potential to induce mineralization in Saos-2 cells. The results showed that adding 15% ZrO2 increases the radiopacity of BD, allowing its radiography detection without altering its physicochemical and biological properties.

Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Systemic supplement with resveratrol increased bone formation in rats' alveolar socket / Aumento de la formación ósea en el hueso alveolar de rata con suplemento sistémico con resveratrol

Resveratrol in cell culture media increases osteoblastic markers. Also results from previous studies provide evidence for resveratrol positive effects on bone healing and bone production. In this preclinical study we investigated bone healing in rats by resveratrol systemic application. 30 Wistar male rats were divided into two groups (study group and control group). At first, maxillary second molars of rats were extracted. The rats were kept in laboratory for next 28 days. Study group received resveratrol 20 mg/kg by abdominal injection every day. The control group received placebo in the same manner that study group. Rats were sacrificed after 28 days and bone samples were collected from center of maxillary second molar socket. Samples were evaluated histologically for new bone formation, inflammation, necrosis, fibrosis and foreign body reaction. The mean difference of new bone formation in control group (28.30 %) and study group (45 %) were statistically significant (P=0.014). There were no significant differences in inflammation, fibrosis, necrosis and foreign body reaction (P>0.05). Resveratrol has positive effects on bone healing but more evidence needed from more clinical and animal studies.

El resveratrol en los medios de cultivo celular aumenta los marcadores osteoblásticos. Los resultados de estudios anteriores proporcionan evidencia de efectos positivos del resveratrol sobre la curación ósea y la producción ósea. En este estudio preclínico, investigamos la curación ósea en ratas mediante la aplicación sistémica de resveratrol. Se dividieron 30 ratas macho Wistar en dos grupos (estudio y control). Inicialmente se extrajeron los segundos molares maxilares de las ratas y los animales se mantuvieron en el laboratorio durante los siguientes 28 días. El grupo de estudio recibió todos los días resveratrol 20 mg/kg por inyección abdominal . El grupo control recibió placebo de la misma manera que el grupo estudio. Las ratas fueron sacrificadas después de 28 días y se recogieron muestras de hueso del centro del segundo molar maxilar. Las muestras se evaluaron histológicamente para la formación de hueso nuevo, inflamación, necrosis, fibrosis y reacción de cuerpo extraño. La media de formación de hueso nuevo en el grupo control (28,30 %) y en el grupo estudio (45 %) fueron estadísticamente significativas (P=0,014). No hubo diferencias significativas en la inflamación, fibrosis, necrosis y reacción al cuerpo extraño (P>0,05). El resveratrol tiene efectos positivos sobre la curación de los huesos, pero aún es necesario realizar más pruebas de estudios clínicos, como también en animales.

Statins and bonehealth: a mini review / Estatinas y salud ósea: una minirevisión

Statins are a widely prescribed class of medications that inhibit similar pathways as the anti-resorptive bisphosphonate drugs. Statins target the mevalonate pathway by blocking HMG-CoA reductase. Several recent meta-analyses concluded statins are osteoprotective in the general population. Here we present current literature exploring the mechanisms underlying the putative osteoprotective effects of statins. We also review recent clinical studies, ranging from observational cohort studies to randomized clinical trials, testing the effect of statins on bone health in various populations. (AU)

Las estatinas son un grupo de drogas prescriptas en forma habitual, con la capacidad de bloquear vías de señalización similares a las inhibidas por los amino-bisfosfonatos. Las estatinas inhiben la vía del mevalonato, a través del bloqueo de diferentes enzimas. Varios metaanálisis recientes llevaron a la conclusión de que las estatinas tienen capacidad osteoprotectora en la población general. En esta revisión presentamos la literatura actual describiendo los mecanismos que subyacen en el potencial efecto osteoprotector de las estatinas, como así también estudios observacionales y clínicos aleatorizados sobre el efecto de estatinas en la salud ósea en diversas poblaciones. (AU)

Avaliação do efeito antiproliferativo da apocinina e diapocinina em células de osteossarcoma humano / Evaluation of the antiproliferative effect of apocynin and diapocynin on human osteosarcoma cells

O osteossarcoma (OS) é o tumor maligno primário mais comum do tecido ósseo, caracterizado pela formação de osteócitos anormais. Apesar do avanço nas terapias convencionais (quimioterapia e retirada do tumor), essas não conseguem eliminar totalmente as células tumorais e impedir a progressão da doença. Recentemente, agentes derivados de fontes naturais ganharam considerável atenção por causa de sua segurança, eficácia e disponibilidade imediata. Nesse sentido, a apocinina, inibidor do complexo NADPH-oxidase, vem sendo estudada como agente antitumoral em alguns tipos de câncer como: pâncreas, próstata, pulmão e mama. Apocinina é um pró-fármaco e sua ação parece estar relacionada à sua conversão produzindo a diapocinina, a qual se mostrou mais efetiva do que a apocinina. Portanto, o objetivo desse estudo é avaliar, in vitro, o potencial antitumoral da apocinina e diapocinina em células de osteossarcoma humano. Para isso, foram utilizados osteoblastos humanos normais (HOb) e osteossarcoma humano imortalizadas (SaOS-2) tratados ou não com apocinina e diapocinina em diversas concentrações. Foram realizados os ensaios de viabilidade celular, alterações morfológicas, apoptose celular, produção de espécies reativas de oxigênio (EROs), formação de colônias, migração, invasão e expressão do fator indutor de hipóxia-1alfa (HIF-1). Também foram conduzidos ensaios para verificar a atividade de metaloproteinase de matriz (MMP) 2 e 9. Os resultados em SaOS-2 mostraram que o tratamento com apocinina nas concentrações de 1,5 e 3 mM; e diapocinina nas concentrações de 0,75 e 1,5 mM reduziram a viabilidade; aumentaram o número de células em apoptose e diminuíram a produção de EROs; sem causar danos às células HOb. Além disso, essas mesmas concentrações inibiram a migração e invasão celular; diminuíram a expressão de HIF-1; e reduziram a atividade de MMP-2 em SaOS-2. Considerando os resultados obtidos, concluímos que a apocinina e diapocinina podem atuar como possíveis moduladores de células tumorais, sendo que a diapocinina mostrou ser mais efetiva nos parâmetros testados.(AU)

Osteosarcoma (OS) is the most common primary malignant tumor of bone tissue, characterized by the formation of abnormal osteocytes. Despite advances in conventional therapies (chemotherapy and surgery) they cannot completely eliminate tumor cells and prevent the progression of the disease. Recently, agents derived from natural sources have achieved considerable attention because of their safety, efficacy and immediate availability of therapies. In this way, apocynin, an inhibitor of the NADPH-oxidase complex, has been studied as an antitumor agent in some types of cancer, such as pancreas, prostate, lung and breast. Apocynin is a prodrug and its action indicate to be related to its conversion to diapocynin, which has been shown to be more efficient than apocynin itself. Thus, the aim of this study is to evaluate, in vitro, the antitumor potential of apocynin and diapocynin in human osteosarcoma cells. For this, normal human osteoblasts (HOb) and immortalized human osteosarcoma cells (SaOS-2) were treated or no-treated with apocynin and diapocynin in various concentrations. Cell viability assay, morphological alterations, cellular apoptosis, reactive oxygen species (ROS) production, colony formation, migration, invasion and expression of hypoxia-inducible factor-1 alpha (HIF-1) were performed. We also performed assays to verify the activity of matrix metalloproteinase (MMP) 2 and 9. The results in SaOS-2 showed that treatment with apocynin at concentrations of 1,5 e 3 mM; and diapocynin at concentrations of 0,75 e 1,5 mM reduced cell viability; increased the number of cells in apoptosis and decreased the production of ROS; without damaging HOb cells. Moreover, these same concentrations inhibited cell migration and invasion; decreased HIF-1 expression; and reduced MMP 2 activity in SaOS-2. Considering the results, we suggest that apocynin and diapocynin may act as possible modulators of tumor cells, and diapocynin has been shown to be more effective.(AU)

A SERM increasing the expression of the osteoblastogenesis and mineralization-related proteins and improving quality of bone tissue in an experimental model of osteoporosis

Abstract Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.

Short term sodium alendronate administration improves the peri-implant bone quality in osteoporotic animals

Abstract Sodium alendronate is a bisphosphonate drug that exerts antiresorptive action and is used to treat osteoporosis. Objective The aim of this study was to evaluate the bone repair process at the bone/implant interface of osteoporotic rats treated with sodium alendronate through the analysis of microtomography, real time polymerase chain reactions and immunohistochemistry (RUNX2 protein, bone sialoprotein (BSP), alkaline phosphatase, osteopontin and osteocalcin). Material and Methods A total of 42 rats were used and divided in to the following experimental groups: CTL: control group (rats submitted to fictitious surgery and fed with a balanced diet), OST: osteoporosis group (rats submitted to a bilateral ovariectomy and fed with a low calcium diet) and ALE: alendronate group (rats submitted to a bilateral ovariectomy, fed with a low calcium diet and treated with sodium alendronate). A surface treated implant was installed in both tibial metaphyses of each rat. Euthanasia of the animals was conducted at 14 (immunhostochemistry) and 42 days (immunohistochemistry, micro CT and PCR). Data were subjected to statistical analysis with a 5% significance level. Results Bone volume (BV) and total pore volume were higher for ALE group (P<0.05). Molecular data for RUNX2 and BSP proteins were significantly expressed in the ALE group (P<0.05), in comparison with the other groups. ALP expression was higher in the CTL group (P<0.05). The immunostaining for RUNX2 and osteopontin was positive in the osteoblastic lineage cells of neoformed bone for the CTL and ALE groups in both periods (14 and 42 days). Alkaline phosphatase presented a lower staining area in the OST group compared to the CTL in both periods and the ALE at 42 days. Conclusion There was a decrease of osteocalcin precipitation at 42 days for the ALE and OST groups. Therefore, treatment with short-term sodium alendronate improved bone repair around the implants installed in the tibia of osteoporotic rats.

Mecanismos intracelulares induzidos por leucotrienos durante a diferenciação osteogênica / Leukotriene-induced intracellular mechanisms during osteogenic differentiation

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico.(AU)

Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNFα in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.(AU)

Tridax procumbens flavonoids: a prospective bioactive compound increased osteoblast differentiation and trabecular bone formation

BACKGROUND: The Tridax procumbens extracts (TPE) are known for their ethno-medicinal properties to increase osteogenic functioning in mesenchymal stem cells. Recently, we found that the T. procumbens flavonoids (TPF) significantly suppressed the RANKL-induced osteoclasts differentiation and bone resorption. The TPF also promoted osteoblasts differentiation and bone formation demonstrated by increasing bone formation markers in cultured mouse primary osteoblasts. However, the effects of the TPF on in vivo bone formation remain unclear. In this study, we investigated the effects of the TPF on in vivo bone formation, injected the TPF (20 mg/kg) twice a day in the low calcium diet mice and killed them after 21 day. Radiographic and histomorphometric analyses were performed on the dissected bones to determine the anabolic effects of the TPF. RESULTS: Bone mineral density and bone mineral content of the TPF-treated mice were significantly increased compared to the control mice. Bone formation-related indices like osteoblast number, osteoblast surface, bone volume, mineralizing surface, mineral apposition rate and bone formation rate were significantly increased in the TPF-treated mice compared to the control mice. CONCLUSION: Our findings point towards the stimulation of bone formation by TPF, suggested that the TPF could be a potential natural anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

BiodentineTM is cytocompatible with human primary osteoblasts

Abstract Calcium silicate-based materials have been widely studied due to their resemblance to, and similar applicability of, mineral trioxide aggregate (MTA). Among these, Biodentine™ (BD) was specifically designed as a "dentin replacement" material for applications such as root perforations, apexification, treatment of resorptive lesions, and as a retrograde filling material. The present study aimed to assess the in vitro response of human primary osteoblasts to BD using MTA AngelusTM as a reference material, by simultaneously analyzing three different cell viability parameters, namely mitochondrial activity, membrane integrity, and cell density. BD and MTA extracts were prepared by incubation on culture media for 24 h or 42 days after mixing. Primary human osteoblasts were exposed to extracts for 24 h, at 37oC with 5% CO2, and cell viability was evaluated by the XTT, NRU, and CVDE assays. Both materials induced cell viability levels higher than 70% when extracted for 24 h. However, when cells were exposed to extracts with increased conditioning times, MTA presented significant cytotoxic effects (p < 0.05) in comparison to the control and MTA at 24 h. After 42 days, the XTT assay identified a significant reduction in cell viability by BD when compared to the control (p<0.05), despite the fact that levels above the 70% viability cutoff were attained for biocompatible materials. It can be concluded that BD is cytocompatible with human primary osteoblasts, indicating its adequacy in direct contact with bone tissues.