Improvement of osteoblast adhesion, viability, and mineralization by restoring the cell cytoskeleton after bisphosphonate discontinuation in vitro.

OBJECTIVE: Bisphosphonates are prescribed to treat excessive bone resorption in patients with osteoporosis. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients prior to dentoalveolar surgery. Furthermore, bisphosphonate discontinuation has been studied in vivo, in humans, and in animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro. METHODOLOGY: Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated. RESULTS: Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 µM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression. CONCLUSION: Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.

Unveiling the role of hydroxyapatite and hydroxyapatite/silver composite in osteoblast-like cell mineralization: An exploration through their viscoelastic properties.

Mechanical properties are becoming fundamental for advancing the comprehension of cellular processes. This study addresses the relationship between viscoelastic properties and the cellular mineralization process. Osteoblast-like cells treated with an osteogenic medium were employed for this purpose. Additionally, the study explores the impact of hydroxyapatite (HA) and hydroxyapatite/silver (HA/Ag) composite on this process. AFM relaxation experiments were conducted to extract viscoelastic parameters using the Fractional Zener (FZ) and Fractional Kelvin (FK) models. Our findings revealed that the main phases of mineralization are associated with alterations in the viscoelastic properties of osteoblast-like cells. Furthermore, HA and HA/Ag treatments significantly influenced changes in the viscoelastic properties of these cells. In particular, the HA/Ag treatment demonstrated a marked enhancement in cell fluidity, suggesting a possible role of silver in accelerating the mineralization process. Moreover, the study underscores the independence observed between fluidity and stiffness, indicating that modifications in one parameter may not necessarily correspond to changes in the other. These findings shed light on the factors involved in the cellular mineralization process and emphasize the importance of using viscoelastic properties to discern the impact of treatments on cells.

High Doses of Phytase Alleviate the Negative Effects of Calcium and Phosphorus Imbalance on Growth Performance and Bone Mineralization in Broiler Chickens

This study investigated the effect of calcium (Ca) and phytase interaction on growth performance and bone quality in 1-42-day-old broiler chickens. A total of 624 female one-day-old Ross 308 broilers were allotted to 13 treatments with four replicates and 12 birds per replicate. A 2 × 6 factorial experiment was designed to test the combinations of 0.50% and 1.00% Ca with 0, 500, 1,000, 2,500, 5,000, and 10,000 FTU/kg phytase in the basal diet (0.25% non-phytate phosphorus, NPP). The control diet contained adequate Ca and phosphorus (P). Dietary Ca, phytase, and their interaction affected growth performance and bone mineralization of broilers at 1-42 days of age (p<0.05). The broilers fed with 1.00% Ca had lower body weight gain (BWG) and feed intake (FI) compared with the birds fed with 0.50% Ca (p<0.05). The BWG, FI, leg bone weight, and ash weight of the broilers fed with 0.25% NPP were lower than those of birds fed with the control diet (p<0.05). The addition of 500-10,000 FTU/kg phytase improved growth rate and leg bone quality, especially at 1.00% Ca (p<0.05). No differences were observed in growth performance and bone quality of 42-day-old broilers fed with 1.00% Ca + 2,500-10,000 FTU/kg phytase and the control diet (p>0.05). These data indicated that high doses of phytase (2,500-10,000 FTU/kg) alleviate the negative effects of Ca and P imbalance (Ca-to-NPP ratio = 4.0) on growth performance and bone mineralization of broiler chickens.(AU)

Telmisartan impairs the in vitro osteogenic differentiation of mesenchymal stromal cells from spontaneously hypertensive male rats.

Telmisartan (TELM) is an angiotensin II (Ang II) type 1 receptor (Agtr1) antagonist, with partial agonism for Pparg, and has been shown to affect bone metabolism. Therefore, the aim of this study was to investigate the effects of TELM in the in vitro osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSC) from spontaneously hypertensive rats (SHRs). BMSC were obtained from male SHR, and the osteogenic medium (OM) was added to the cells concomitantly with TELM (0.005, 0.05, and 0.5 µM). Undifferentiated BMSC, in control medium (CM), showed an increased viability, while the addition of OM reduced this parameter, and TELM did not show cytotoxicity in the concentrations used. BMSC in OM had an alkaline phosphatase (ALP) activity peak at d10, which decreased at d14 and d21, and TELM reduced ALP at d10 in a dose-dependent manner. Mineralization was observed in the OM at d14, which intensified at d21, but was inhibited by TELM. Agtr1b was increased in the OM, and TELM inhibited its expression. TELM reduced Opn, Ocn, and Bsp and increased Pparg expression, and at the higher concentration TELM also increased the expression of adipogenic markers, Fabp4 and Adipoq. In addition, TELM 0.5 µM increased Irs1 and Glut4, insulin and glucose metabolism markers, known to be regulated by Pparg and to be related to adipogenic phenotype. Our data shows that TELM inhibited the osteogenic differentiation and mineralization of SHR BMSC, by favoring an adipogenic prone phenotype due to Pparg upregulation.

Low concentrations of caffeic acid phenethyl ester stimulate osteogenesis in vitro.

OBJECTIVE: The aim of this study was to evaluate the effects of caffeic acid phenethyl ester (CAPE) on osteoblast-like cell cultures (SAOS-2). METHODS: SAOS-2 were exposed to CAPE at 1 nM, 10 nM, 100 nM, 1 µM, and 10 µM. Non-exposed cultures were used as control. The following parameters were assayed: 1) cell viability at 1, 3, and 7 days; 2) alkaline phosphatase (ALP) activity at 5 and 10 days; 3) matrix mineralization at 14 days; and 4) Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (SPP1), and osteocalcin (BGLAP) gene expression at 5 and 10 days. The data were analyzed by ANOVA two-way or Kruskal-Wallis (α = 5%). RESULTS: At day 1, cell viability was similar among all groups (p > 0.05). At days 3 and 7, cultures exposed to CAPE at 10 µM exhibited a significant reduction in cell viability compared with the others groups (p < 0.05). At day 5, ALP activity was similar among all experimental groups; at day 10, however, the stain intensity was higher in cultures exposed to CAPE at 100 nM and 10 nM in comparison with the other groups (p < 0.05). At days 5 and 10, RUNX2, ALP, SPP1, and BGLAP gene expression was greater in cultures exposed to CAPE in comparison with the control (p < 0.05). At day 14, matrix mineralization was similar in cultures exposed to CAPE at 1 nM and 10 nM (p > 0.05), but superior to those ones observed in the other experimental groups (p < 0.05). CONCLUSION: CAPE at low concentrations can positively module the osteogenesis in vitro.

Mineralization of Phosphorylated Fish Skin Collagen/Mangosteen Scaffolds as Potential Materials for Bone Tissue Regeneration.

In this study, a potential hard tissue substitute was mimicked using collagen/mangosteen porous scaffolds. Collagen was extracted from Tilapia fish skin and mangosteen from the waste peel of the respective fruit. Sodium trimetaphosphate was used for the phosphorylation of these scaffolds to improve the nucleation sites for the mineralization process. Phosphate groups were incorporated in the collagen structure as confirmed by their attenuated total reflection Fourier transform infrared (ATR-FTIR) bands. The phosphorylation and mangosteen addition increased the thermal stability of the collagen triple helix structure, as demonstrated by differential scanning calorimetry (DSC) and thermogravimetry (TGA) characterizations. Mineralization was successfully achieved, and the presence of calcium phosphate was visualized by scanning electron microscopy (SEM). Nevertheless, the porous structure was maintained, which is an essential characteristic for the desired application. The deposited mineral was amorphous calcium phosphate, as confirmed by energy dispersive X-ray spectroscopy (EDX) results.

DNMT1 Inhibitor Restores RUNX2 Expression and Mineralization in Periodontal Ligament Cells.

Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.

Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation.

Histatin-1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin-1 has been shown to promote re-epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad-spectrum activity of Histatin-1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation. Specifically, Histatin-1 promoted cell adhesion, spreading, and migration of SAOS-2 cells and MC3T3-E1 preosteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin-1 induced the expression of osteogenic genes, including osteocalcin, osteopontin, and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin-1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS-2 and MC3T3-E1 cells. Mechanistically, although Histatin-1 failed to activate ERK1/2, FAK, and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear relocalization of ß-catenin. Strikingly, the effects of Histatin-1 were recapitulated in cells that are nonosteogenically committed, since it promoted surface adhesion, migration, and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin-1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration.

Bone marrow coagulated and low-level laser therapy accelerate bone healing by enhancing angiogenesis, cell proliferation, osteoblast differentiation, and mineralization.

The present study evaluated bone marrow aspirate (BMA) and low-level laser therapy (LLLT) on bone healing. It was created critical-size defects (CSD) of 5 mm diameter in rat calvaria of 64 rats. Animals were randomly divided into four groups: Control (blood clot), BMA (coagulated BMA), LLLT (laser irradiation and blood clot), and BMA/LLLT (laser irradiation and coagulated BMA). Euthanasia was performed at 15 or 30 days postoperative. Immunohistochemical reactions were performed to identify vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN). The markers were quantified, and data were statistically analyzed. Groups BMA/LLLT and LLLT presented significantly higher VEGF expression than group control. Group BMA/LLLT presented a significantly higher expression of PCNA than all experimental groups. Groups BMA and BMA/LLLT presented significantly higher expression of BMP-2 than all experimental groups. Groups LLLT and BMA/LLLT presented significantly higher expression of OPN than groups control and BMA. Groups LLLT, BMA, and BMA/LLLT presented a significantly higher expression of OCN than group control. It can be concluded that the association of BMA and LLLT enhanced bone healing by improving expression of VEGF, PCNA, Runx2, BMP-2, OPN, and OCN.

Bone morphogenetic proteins in biomineralization of two endodontic restorative cements.

To assess the effect of biodentine (BD) and MTA-angelus (MTA) on biocompatibility, BMP2, BMP4, and osteocalcin (OC) expression. Subcutaneously implanted tubes of four groups (MTA, BD, Control, and Sham) were kept over 15, 30, and 60 days; histological analyses were performed using H&E and Von Kossa; ELISA quantified IL-1ß and IL-8 expression; and qRT-PCR verified gene expression of BMPs and OC. Sham showed slight changes in profile/intensity of inflammatory infiltrate in all periods. Control had an inflammatory score significantly higher than Sham at 15 days (p < .05). BD revealed a similar inflammatory response to Sham, without significant changes over periods. MTA group exhibited an increase in chronic inflammatory profile at 30 days, with significant reduction at 60 days, when compared to Sham (p < .05). At 30/60 days, experimental groups presented birefringent areas. At 30/60 days, BD and MTA significantly increase IL-1ß compared to Control, whereas an increase in IL-8 was observed only in BD. At 30/60 days, BD produces an expression of BMP2 whereas MTA influenced BMP4 and OC. Materials tested are biocompatible and they have osteoinductive activity; the materials influenced the expression of the tested mediators differently, suggesting different affinities with the substrate and the dental substrates.

In vivo evaluation of Vivere bovine pericardium valvular bioprosthesis with a new anti-calcifying treatment.

The objective of this study was to evaluate the effect of chemical treatment with glutamic acid to avoid calcification of biological cardiac valves. The bovine pericardium (BP) tissues were fixed with 0.5% glutaraldehyde (BP/GA), followed by treatment with glutamic acid (BP/GA + Glu) for neutralization of the free aldehyde groups. Microscopic analysis showed that the wavy structure of collagen fibrils was preserved, but changes in elastin's integrity occurred. However, the treatment did not promote undesirable changes in the thermal and mechanical properties of the modified BPs. These samples were systematically studied in rat subcutaneous tissue: control (BP/GA) and anticalcificant (BP/GA + Glu). After 60 days, both groups induced similar inflammatory reactions. In terms of calcification, BP/GA + Glu remained more stable with a lower index (3.1 ± 0.2 µg Ca2+ /mg dry tissue), whereas for BP/GA it was 5.7 ± 1.3 µg Ca2+ /mg dry tissue. Bioprostheses made from BP/GA + Glu were implanted in the pulmonary position in sheep, and in vivo echocardiographic analyses revealed maintenance of valvar function after 180 days, with low gradients and minimal valve insufficiency. The explanted tissues of the BP/GA + Glu group had a lower average calcium content 3.8 ± 3.0 µg Ca2+ /mg dry tissue. The results indicated high anticalcification efficiency of BP/GA + Glu in both subcutaneous implant in rats and in the experimental sheep model, which is an advantage that should encourage the industrial application of these materials for the manufacture of bioprostheses.

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

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

Osteogenic potential of sol-gel bioactive glasses containing manganese.

Bioactive glasses (BGs) are widely used for bone regeneration, and allow the incorporation of different ions with therapeutic properties into the glass network. Amongst the different ions with therapeutic benefits, manganese (Mn) has been shown to influence bone metabolism and activate human osteoblasts integrins, improving cell adhesion, proliferation and spreading. Mn has also been incorporated into bioceramics as a therapeutic ion for improved osteogenesis. Here, up to 4.4 mol% MnO was substituted for CaO in the 58S composition (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5) and its effects on the glass properties and capability to influence the osteogenic differentiation were evaluated. Mn-containing BGs with amorphous structure, high specific surface area and nanoporosity were obtained. The presence of Mn2+ species was confirmed by X-ray photoelectron spectroscopy (XPS). Mn-containing BGs presented no cytotoxic effect on human mesenchymal stem cells (hMSCs) and enabled sustained ion release in culture medium. hMSCs osteogenic differentiation stimulation and influence on the mineralisation process was also confirmed through the alkaline phosphatase (ALP) activity, and expression of osteogenic differentiation markers, such as collagen type I, osteopontin and osteocalcin, which presented higher expression in the presence of Mn-containing samples compared to control. Results show that the release of manganese ions from bioactive glass provoked human mesenchymal stem cell (hMSC) differentiation down a bone pathway, whereas hMSCs exposed to the Mn-free glass did not differentiate. Mn incorporation offers great promise for obtaining glasses with superior properties for bone tissue regeneration.

Treatment with an inhibitor of fatty acid synthase attenuates bone loss in ovariectomized mice.

Bone and fat cells have an antagonistic relationship. Adipocytes exert a toxic effect on bone cells in vitro through the secretion of fatty acids, which are synthesized by fatty acid synthase (FAS). Inhibition of FAS in vitro rescues osteoblasts from fat-induced toxicity and cell death. In this study, we hypothesized that FAS inhibition would mitigate the loss of bone mass in ovariectomized (OVX) mice. We treated OVX C57BL/6 mice with cerulenin (a known inhibitor of FAS) for 6 weeks and compared their bone phenotype with vehicle-treated controls. Cerulenin-treated mice exhibited a significant decrease in body weight, triglycerides, leptin, and marrow and subcutaneous fat without changes in serum glucose or calciotropic hormones. These effects were associated with attenuation of bone loss and normalization of the bone phenotype in the cerulenin-treated OVX group compared to the vehicle-treated OVX group. Our results demonstrate that inhibition of FAS enhances bone formation, induces uncoupling between osteoblasts and osteoclasts, and favors mineralization, thus providing evidence that inhibition of FAS could constitute a new anabolic therapy for osteoporosis.

Lycopene influences osteoblast functional activity and prevents femur bone loss in female rats submitted to an experimental model of osteoporosis.

Antioxidant properties of several nutrients may influence bone metabolism, affording protection against damaging effects caused by oxidative stress. Thus, we hypothesized that lycopene may benefit bone tissue metabolism and functional activity of osteoblastic cells from bone marrow of osteoporotic female rats. Wistar rats were ovariectomized and paired with sham animals. In vitro evaluations were performed after 60 days of surgery, when cells were cultured in osteogenic medium and divided in control (C), ovariectomized (OVX) and ovariectomized + 1 µmol/L lycopene (OVXL) groups. Besides, in vivo studies were carried out to evaluate femur bone remodeling by histological and histomorphometric analyses after daily intake of 10 mg/kg of lycopene for 30 and 60 days after ovariectomy. Cell proliferation was significantly higher in OVX and OVXL groups after 10 days of culture. Alkaline phosphatase activity (ALP) was higher in OVXL group in later periods of cell culture, whereas its in situ detection was higher for this group in all experimental periods; nevertheless, mineralization did not show significant differences among the groups. There was a significant upregulation of genes Sp7, Runx2 and Bsp after 3 days and genes Runx2 and Bglap after 10 days from OVXL when compared to OVX. In vivo results demonstrated that daily intake of 10 mg/kg of lycopene for 60 days decreased bone loss in femur epiphysis in ovariectomized rats by maintaining trabecular bone similar to controls. Data obtained suggest that lycopene might benefit the functional activity of osteoblastic cells from ovariectomized rats, as well as avoid further bone resorption.

Impact of homocysteine on vasculogenic factors and bone formation in chicken embryos.

Developmental endochondral ossification requires constant blood supply, which is provided by the embryonic vascular network. High levels of homocysteine (Hcy) have vasculotoxic properties, but it remains unclear how Hcy disrupts blood vessel formation in endochondral ossification. Thus, we investigated the toxicity of Hcy on contents of vasculogenic factors (VEGF, VCAM-1, NOS3) and osteocalcin, using developing limbs as model. Chicken embryos were submitted to treatment with 20 µmol D-L Hcy at 12H&H and the analyses occur at 29H&H and 36H&H. We did not identify differences in the area of limb ossification in Hcy-treated (7.5 × 105 µm2 ± 3.9 × 104) and untreated embryos (7.6 × 105 µm2 ± 3.3 × 104) at 36H&H. In Hcy-treated embryos, we observed a significantly decrease of 46.8% at 29H&H and 26.0% at 36H&H in the number of VEGF-reactive cells. Also, treated embryos showed decrease of 98.7% in VCAM-1-reactive cells at 29H&H and 34.6% at 36H&H. The number of NOS3-reactive cells was reduced 54.0% at 29H&H and 91.5% at 36H&H, in the limbs of Hcy-treated embryos. Finally, in Hcy-treated embryos at 36H&H, we observed a reduction of 58.86% in the number of osteocalcin-reactive cells. Here, we demonstrated for the first time that the toxicity of Hcy is associated with a reduction in the contents of proteins involved in blood vessel formation and bone mineralization, which interferes with endochondral ossification of the limb during embryonic development. Graphical abstract.

Synthesis of Sr-morin complex and its in vitro response: decrease in osteoclast differentiation while sustaining osteoblast mineralization ability.

Strontium ranelate (SrR) has been used as the ultimate choice for osteoporosis treatment. However, the development of more tolerable and bioactive Sr2+ carriers is still a need. The design of Sr2+-based platforms has moved towards the obtention of anion carriers that can also exhibit a positive effect on bone metabolism. In this sense, we used morin, a natural flavonoid, as a new arrangement for Sr2+ carriage in the synthesis of an Sr2+ complex. It has been claimed that phenolic compounds promote bone health. Therefore, we hypothesized that the association of Sr2+ with morin could improve its anabolic effects. Complexes with the general formula [(C15H9O7)Sr(H2O)2]Cl·3H2O were synthesized and characterized by elemental analysis, thermogravimetry, UV-Vis and infrared absorption spectroscopies and 1H-nuclear magnetic resonance. We showed that the complexation between morin and Sr2+ occurred among the 3-OH and 4C[double bond, length as m-dash]O groups of morin. Preosteoclasts cultures with the Sr-morin complex exhibited a reduced osteoclast differentiation rate and sustained osteoblast mineralization ability. The response of Sr-morin was higher than that observed for SrR at the same concentration range. Considering the above-mentioned observations, the Sr-morin complex could be an interesting approach to be further exploited not only as an alternative treatment for osteoporosis but also in the design of materials for faster osteointegration.

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.

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.

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

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.