The Effects of Milk Supplementation on Bone Health Indices in Adults: A Meta-Analysis of Randomized Controlled Trials.

Milk contains a number of bone-beneficial nutrients. However, milk, due to the D-galactose content, might have unfavorable effects on bone health. A meta-analysis of randomized controlled trials (RCTs) was performed to clarify the effects of milk supplementation on bone mineral density (BMD), bone turnover markers [N-terminal telopeptide of type I collagen (NTx), C-terminal telopeptide of type 1 collagen (CTx), osteocalcin, bone alkaline phosphatase (BALP), and procollagen type 1 N-propeptide (P1NP)], and hormonal indices related to bone metabolism [parathyroid hormone (PTH), 25-hydroxyvitamin D [25(OH)D], and insulin-like growth factor 1 (IGF-1)] in adults. The PubMed and Web of Science databases were searched. A random-effects model was used to estimate the pooled effect sizes. A total of 20 RCTs were included. The trial duration ranged from 1 mo to 36 mo. Milk supplementation resulted in a small but significant increase in BMD at the hip (+0.004 g/cm2; n = 9 RCTs) and lumbar spine (+0.025 g/cm2; n = 7), but did not significantly affect whole-body BMD (n = 3) and femoral neck BMD (n = 7). Milk supplementation reduced the concentrations of P1NP (-5.20 ng/mL; n = 9), CTx (-0.16 ng/mL; n = 9), and NTx (-8.66 nmol bone collagen equivalents/mmol creatinine; n = 3). The concentrations of osteocalcin (n = 9) and BALP (n = 3) were not affected by milk supplementation. Reduced parathyroid hormone PTH (-1.01 pg/mL; n = 13) concentrations and increased IGF-1 (+1.79 nmol/l; n = 4) concentrations were observed with milk supplementation. 25(OH)D (+3.73 ng/mL; n = 11) concentrations were increased with vitamin-D fortified milk supplementation. The addition of milk to the diet may potentially increase the likelihood of preventing bone loss by restoring bone homeostasis through the modulation of the calcium-vitamin D-PTH axis, bone remodeling rate, and growth hormone/IGF-1 axis.

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

BACKGROUND: 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.

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.

Combination therapy of curcumin and alendronate modulates bone turnover markers and enhances bone mineral density in postmenopausal women with osteoporosis.

OBJECTIVE: This study evaluated the effects of combination therapy of curcumin and alendronate on BMD and bone turnover markers in postmenopausal women with osteoporosis. SUBJECTS AND METHODS: In a randomized, double-blind trial study, 60 postmenopausal women were divided into three groups: control, alendronate, and alendronate + curcumin. Each group included 20 patients. Total body, total hip, lumbar spine and femoral neck BMDs were measured by dual-energy X-ray absorptiometry (DXA) at baseline and after 12 months of therapy. Bone turnover markers such as bone-specific alkaline phosphatase (BALP), osteocalcin and C-terminal cross-linking telopeptide of type I collagen (CTx) were measured at the outset and 6 months later. RESULTS: Patients in the control group suffered a significant decrease in BMD and increased bone turnover markers at the end of study. The group treated with only alendronate showed significantly decreased levels of BALP and CTx and increased levels of osteocalcin compared to the control group. The alendronate group also showed significant increases in the total body, total hip, lumbar spine and femoral neck BMDs at the end of study compared to the control group. In the curcumin + alendronate group, BALP and CTx levels decreased and osteocalcin levels increased significantly at the end of study compared to the control and alendronate groups. BMD indexes also increased in four areas significantly at the end of study compared to the control and alendronate groups. CONCLUSION: The combination of curcumin and alendronate has beneficial effects on BMD and bone turnover markers among postmenopausal women with osteoporosis. Arch Endocrinol Metab. 2018;62(4):438-45.

Combination therapy of curcumin and alendronate modulates bone turnover markers and enhances bone mineral density in postmenopausal women with osteoporosis

ABSTRACT Objective: This study evaluated the effects of combination therapy of curcumin and alendronate on BMD and bone turnover markers in postmenopausal women with osteoporosis. Subjects and methods: In a randomized, double-blind trial study, 60 postmenopausal women were divided into three groups: control, alendronate, and alendronate + curcumin. Each group included 20 patients. Total body, total hip, lumbar spine and femoral neck BMDs were measured by dual-energy X-ray absorptiometry (DXA) at baseline and after 12 months of therapy. Bone turnover markers such as bone-specific alkaline phosphatase (BALP), osteocalcin and C-terminal cross-linking telopeptide of type I collagen (CTx) were measured at the outset and 6 months later. Results: Patients in the control group suffered a significant decrease in BMD and increased bone turnover markers at the end of study. The group treated with only alendronate showed significantly decreased levels of BALP and CTx and increased levels of osteocalcin compared to the control group. The alendronate group also showed significant increases in the total body, total hip, lumbar spine and femoral neck BMDs at the end of study compared to the control group. In the curcumin + alendronate group, BALP and CTx levels decreased and osteocalcin levels increased significantly at the end of study compared to the control and alendronate groups. BMD indexes also increased in four areas significantly at the end of study compared to the control and alendronate groups. Conclusion: The combination of curcumin and alendronate has beneficial effects on BMD and bone turnover markers among postmenopausal women with osteoporosis. Arch Endocrinol Metab. 2018;62(4):438-45

Effects of Syzygium aromaticum, Cinnamomum zeylanicum, and Salvia triloba extracts on proliferation and differentiation of dental pulp stem cells.

INTRODUCTION: Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. OBJECTIVES: We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs) proliferation, differentiation, and immune responses. MATERIAL AND METHODS: Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 µg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. RESULTS: The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor alpha (TNF-α), S. triloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. triloba as a potent anti-inflammatory agent, which could be used safely in biocomposite or scaffold fabrications for dentistry. CONCLUSIONS: Because calcium granule formation and cell viability play a critical role in hard tissue formation, S. aromaticum in dentistry should be strictly controlled, and the mechanism leading to reduced calcium granule formation should be identified.

Effects of Syzygium aromaticum, Cinnamomum zeylanicum, and Salvia triloba extracts on proliferation and differentiation of dental pulp stem cells

Abstract Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. Objectives: We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs) proliferation, differentiation, and immune responses. Material and Methods: Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 μg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. Results: The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor alpha (TNF-α), S. triloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. triloba as a potent anti-inflammatory agent, which could be used safely in biocomposite or scaffold fabrications for dentistry. Conclusions: Because calcium granule formation and cell viability play a critical role in hard tissue formation, S. aromaticum in dentistry should be strictly controlled, and the mechanism leading to reduced calcium granule formation should be identified.

Low-level laser therapy (780 nm) combined with collagen sponge scaffold promotes repair of rat cranial critical-size defects and increases TGF-ß, FGF-2, OPG/RANK and osteocalcin expression.

The aim of this study was to evaluate the effect of collagen sponge scaffold (CSS) implantation associated with low-level laser therapy (LLLT) on repairing bone defects. A single 5-mm cranial defect was surgically created in forty Wistar rats, which then received one of the following four interventions (n = 10 per group): no treatment (G0); bone defect implanted with collagen sponge scaffold (CSS) alone (G1); defect treated with low-level laser therapy (LLLT) (wavelength 780 nm; total energy density 120 J/cm2 ; power 50 mW) alone (G2); and CSS associated with LLLT treatment (G3). After surgery, animals in each group were euthanized at 21 days and 30 days (n = 5 per euthanasia time group). Bone formation was monitored by X-ray imaging analysis. Biopsies were collected and processed for histological analysis and immunohistochemical evaluation of transforming growth factor-beta (TGF-ß), fibroblast growth factor-2 (FGF-2), osteoprotegerin (OPG) and receptor activator of nuclear factor ƙ (RANK). Osteocalcin (OCN) was detected by immunofluorescence analysis. Compared to the G0 group, defects in the 30-day G3 group exhibited increased bone formation, both by increase in radiopaque areas (P < 0.01) and by histomorphometric analysis (P < 0.001). The histopathological analysis showed a decreased number of inflammatory cells (P < 0.001). The combined CCS + LLLT (G3) treatment also resulted in the most intense immunostaining for OPG, RANK, FGF-2 and TGF-ß, and the most intense and diffuse OCN immunofluorescent labelling at 30 days postsurgery (G3 vs. G0 group, P < 0.05). Therefore, the use of CCS associated with LLLT could offer a synergistic advantage in improving the healing of bone fractures.

Metformin Decreases Reactive Oxygen Species, Enhances Osteogenic Properties of Adipose-Derived Multipotent Mesenchymal Stem Cells In Vitro, and Increases Bone Density In Vivo.

Due to its pleiotropic effects, the commonly used drug metformin has gained renewed interest among medical researchers. While metformin is mainly used for the treatment of diabetes, recent studies suggest that it may have further application in anticancer and antiaging therapies. In this study, we investigated the proliferative potential, accumulation of oxidative stress factors, and osteogenic and adipogenic differentiation potential of mouse adipose-derived stem cells (MuASCs) isolated from mice treated with metformin for 8 weeks. Moreover, we investigated the influence of metformin supplementation on mice bone density and bone element composition. The ASCs isolated from mice who were treated with metformin for 8 weeks showed highest proliferative potential, generated a robust net of cytoskeletal projections, had reduced expression of markers associated with cellular senescence, and decreased amount of reactive oxygen species in comparison to control group. Furthermore, we demonstrated that these cells possessed greatest osteogenic differentiation potential, while their adipogenic differentiation ability was reduced. We also demonstrated that metformin supplementation increases bone density in vivo. Our result stands as a valuable source of data regarding the in vivo influence of metformin on ASCs and bone density and supports a role for metformin in regenerative medicine.

Incorporation of Fucoidan in ß-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage.

In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology.

Clinical and biochemical comparison of guided tissue regeneration versus guided tissue regeneration plus low-level laser therapy in the treatment of class II furcation defects: A clinical study.

OBJECTIVE: The present study was aimed to compare the clinical and biochemical effectiveness of guided tissue regeneration (GTR) alone and combined with low-level laser therapy (LLLT) application in the treatment of furcation II periodontal defects, over a period of 6 months. MATERIAL AND METHODS: Thirty-three furcation defects were included in the study. Seventeen of these defects were treated with GTR plus LLLT, and sixteen of them were treated with GTR alone. Probing pocket depth (PPD), clinical attachment level (CAL), horizontal probing depth (HPD), and alkaline phosphatase (ALP) and osteocalcin (OC) levels in the gingival crevicular fluid (GCF) were recorded at baseline and at postoperative 3rd and 6th months. RESULTS: Healing was uneventful in all cases. At the 3rd and 6th months, both treatment modalities-GTR and GTR plus LLLT--showed improved PPD, CAL, and HPD values compared to their baseline values. ALP and OC levels in GCF increased after the treatment in both groups (p < 0.05). When compared the two groups, at the 6th month, PPD, CAL, HPD, and ALP values showed significantly more improvement in laser group than non-laser group (p < 0.05). CONCLUSIONS: The results of this study showed that both treatments led to significantly favorable clinical improvements in furcation periodontal defects. LLLT plus GTR may be a more effective treatment modality compared to GTR alone.

Preparation of Quenchbodies by protein transamination reaction.

Quenchbody (Q-body) is an antibody fragment labeled with fluorescent dye(s), which functions as a biosensor via the antigen-dependent removal of the quenching effect on fluorophores. It is based on the principle that the fluorescence of the dye(s) attached to the antibody N-terminal region is quenched primarily by the tryptophan residues present in the variable regions, and this quenching is released when the antigen binds to the antibody, resulting in increased fluorescence intensity. Hence Q-body is utilized in various immunoassays for the rapid and sensitive detection of analytes. So far, Q-bodies have been prepared by using a cell-free translation system or by combining Escherichia coli expression and post-labeling steps. However, the above methods need antibody gene cloning, and are time-consuming. In this study, we report a novel approach to prepare Q-bodies by protein N-terminal transamination. We used the antigen-binding fragment (Fab) of an antibody against the bone-Gla-protein (BGP), a biomarker for bone diseases, which was expressed in E. coli. The purified Fab was treated with Rapoport's salt to convert the amino group at the N-terminus to a ketone group, which in turn was allowed to react with fluorescent probes that have aminooxy or hydrazide groups, to prepare a Q-body. The Q-body prepared by this method could detect the BGP-C7 antigen at concentrations as low as 10 nM. Since the approach can label the protein N-terminus directly, it could be applied for preparing Q-bodies from natural antibodies and for the rapid screening of high-performance Q-bodies.

Exogenous and endogenous determinants of vitamin K status in cystic fibrosis.

Cystic fibrosis (CF) patients are at high risk for vitamin K deficiency. The effects of vitamin K supplementation are very ambiguous. Therefore, we aimed to define the determinants of vitamin K deficiency in a large cohort of supplemented - 146 (86.9%) and non-supplemented - 22 (13.1%) CF patients. Vitamin K status was assessed using prothrombin inducted by vitamin K absence (PIVKA-II) and undercarboxylated osteocalcin (u-OC). The pathological PIVKA-II concentration (≥ 2 ng/ml) and abnormal percentage of osteocalcin (≥ 20%) were found in 72 (42.8%) and 60 (35.7%) subjects, respectively. We found that liver involvement, diabetes, and glucocorticoid therapy were potential risk factors for vitamin K deficiency. Pathological concentrations of PIVKA-II occurred more frequently in patients with pancreatic insufficiency and those who have two severe mutations in both alleles of the CFTR gene. Pathological percentage of u-OC was found more frequently in adult CF patients and those not receiving vitamin K. However, it seems that there are no good predictive factors of vitamin K deficiency in CF patients in everyday clinical care. Early vitamin K supplementation in CF patients seems to be warranted. It is impossible to clearly determine the supplementation dose. Therefore, constant monitoring of vitamin K status seems to be justified.

Periodontal Health in Women With Early-Stage Postmenopausal Breast Cancer Newly on Aromatase Inhibitors: A Pilot Study.

BACKGROUND: Aromatase inhibitor (AI) use results in low estrogen levels, which in turn affect bone mineral density (BMD). Periodontitis, alveolar bone loss, and tooth loss are associated with low BMD. The goal of this study is to assess the prevalence of periodontitis and perceived oral health and evaluate salivary biomarkers in postmenopausal women who are survivors of early-stage (I to IIIA) breast cancer (BCa) and receive adjuvant AI therapy. METHODS: Participants included 58 postmenopausal women: 29 with BCa on AIs and 29 controls without BCa diagnoses. Baseline periodontal status was assessed with: 1) periodontal probing depth (PD); 2) bleeding on probing (BOP); and 3) attachment loss (AL). Demographic and dental utilization information was gathered by questionnaire. Linear regression modeling was used to analyze the outcomes. RESULTS: No differences were found in mean PD or number of teeth. The AI group had significantly more sites with BOP (27.8 versus 16.7; P = 0.02), higher worst-site AL (5.2 versus 4.0 mm; P <0.01), and more sites with dental calculus (18.2 versus 6.4; P <0.001) than controls. Linear regression adjusted for income, tobacco use, dental insurance, and previous radiation and chemotherapy exposure demonstrated that AI use increased AL by >2 mm (95% confidence interval, 0.46 to 3.92). Median salivary osteocalcin and tumor necrosis factor-α levels were significantly higher in the AI group than the control group. CONCLUSION: This first investigation of the periodontal status of women initiating adjuvant AI therapy identifies this population as having an increased risk for periodontitis.

Modulatory effects of l-arginine and soy enriched diet on bone homeostasis abnormalities in streptozotocin-induced diabetic rats.

Diabetes mellitus is a complex syndrome which is responsible for numerous complications affecting the whole body. Osteoporosis is regarded as one of the chronic complications of diabetes mellitus that results from reduced bone formation and increased resorption. In this context, we searched for dietary supplements that preserve diabetic bone loss. Parathyroid hormone (PTH) has been suggested as a possible mechanism affecting bone homeostasis in streptozotocin (STZ)-induced diabetic rats. The osteoprotective effects of l-arginine and soy enriched diet were also investigated. Male Wistar rats were allocated into four groups; normal control, untreated STZ-diabetic rats and STZ-diabetic rats treated with either l-arginine (10mg/kg/day) or fed soy enriched diet (200 g/kg diet) for 12 weeks. l-Arginine and soy enriched diet normalized serum PTH level and increased serum osteocalcin level; bone osteocalcin, osteoprotegerin and runt-related transcription factor2 mRNA levels compared to diabetic rats. A decrease in serum pyridinoline, C-terminal telopeptides of type I collagen, cathepsin k levels and bone cathepsin k mRNA level was observed in both treated groups. Both treatments increased serum insulin and insulin like growth factor-1 levels and decreased urinary calcium excretion. In conclusion, l-arginine and soy enriched diet are effective in prevention of osteoporosis associated with diabetes mellitus.

Effect of avocado/soybean unsaponifiables on osseointegration: a proof-of-principle preclinical in vivo study.

PURPOSE: To evaluate the influence of administration of avocado/soybean unsaponifiables (ASU) on implant osseointegration in rat tibiae. MATERIALS AND METHODS: Thirty rats were randomly assigned into one of three equal-sized groups: (1) ASU1: administration of ASU starting 7 days prior to implant placement; (2) ASU2: administration of ASU starting on the day of implant placement, and (3) CTL: administration of saline solution. In all animals, one titanium implant was placed in each tibia. All animals received ASU or saline solution by gavage daily until sacrifice 60 days postoperatively. Implant osseointegration and bone maturation were assessed by biomechanical and radiographic bone density analysis; descriptive histology; immunohistochemistry for bone morphogenetic protein 2 (BMP-2), transforming growth factor beta 1 (TGF-ß1), and osteocalcin; and histomorphometric evaluation of bone-to-implant contact (BIC) and mineralized bone area fraction within the threads of the implant. RESULTS: ASU1 and ASU2 showed three times higher expression of BMP-2 and nine times higher expression of TGF-ß1 compared with CTL (P < .05). Histomorphometric analysis, however, showed that both ASU1 and ASU2 groups presented significantly higher BIC values only in the cortical bone compartment when compared to CTL (P < .05). CONCLUSION: ASU consumption seems to exert only a subtle effect on implant osseointegration.

Periodontal fibroblasts modulate proliferation and osteogenic differentiation of embryonic stem cells through production of fibroblast growth factors.

BACKGROUND: Periodontal ligament fibroblasts (PLFs) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. It is hypothesized that PLFs induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GFs) and skin fibroblasts (SFs). METHODS: PLFs and GFs were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SFs were purchased commercially. mES cells were incubated with culture supernatants of these fibroblasts or cocultured directly with the cells. Proliferation and mineralization in mES cells were determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured. RESULTS: In cocultures, PLFs stimulated proliferation of mES cells more effectively than GFs or SFs. Similarly, the addition of culture supernatant of PLFs induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor (FGF)4 or the c-Jun N-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of mES cells in the order PLFs > GFs > SFs. These activities of PLFs were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pretreatment of mES cells with the extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin. CONCLUSION: PLFs regulate the proliferation and osteogenic differentiation of mES cells more strongly than GFs and SFs via the secretion of FGF through a mechanism that involves mitogen-activated protein kinase-mediated signaling.

Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors.

Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm; 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3 J/cm(2). The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

Effect of 940 nm low-level laser therapy on osteogenesis in vitro.

Bone regeneration is essential in medical treatment, such as in surgical bone healing and orthodontics. The aim of this study is to examine the effect of different powers of 940 nm diode low-level laser treatment (LLLT) on osteoblast cells during their proliferation and differentiation stages. A human fetal osteoblast cell line was cultured and treated with LLLT. The cells were divided into experimental groups according to the power delivered and periods of exposure per day for each laser power. The (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide) (MTT) assay was used to determine cell proliferation. Both alkaline phosphatase and osteocalcin activity assays were assessed for cell differentiation. All treatment groups showed a significant increase in cell proliferation and differentiation compared to the control group. Regarding the exposure time, the subgroups treated with the LLLT for 6 min showed higher proliferation and differentiation rates for the powers delivered, the 300-mW LLLT group significantly increased the amount of cell proliferation. By contrast, the 100 and 200 mW groups showed significantly greater amounts of cell differentiation. These results suggest that the use of LLLT may play an important role in stimulating osteoblast cells for improved bone formation.

Assessment of different decalcifying protocols on Osteopontin and Osteocalcin immunostaining in whole bone specimens of arthritis rat model by confocal immunofluorescence.

Confocal immunofluorescence is a valuable technique for the detection of relevant molecules in the pathogenesis of arthritis in rat models; however, it requires efficient processing of tissues including bone decalcification. The decalcification process must ensure the complete removal of calcium and also a proper preservation of cellular structures and, specially, the antigenicity of the tissue to allow the immunodetection of the molecules of interest. In the present study, we evaluated the effect of four different decalcifying solutions: the Morse´s solution, 10% EDTA (pH 7.4), 7% HCl/2% EDTA and 5% Nitric acid, as well as four different treatments of the tissues (including microwave irradiation) in the processes of decalcification for large pieces of adult rat bones (hind paw, fore paw, knee and column). We assessed the time of decalcification, the easiness of slicing, the morphological preservation and finally, the antigenicity of two different bone proteins (Osteopontin (OPN) and Osteocalcin (OC)) measured by its immunofluorescence intensity under controlled confocal microscopy conditions. Our results showed that the specimen size and the presence of skin are critical factors for the rate of decalcification, and no significant benefit was found if microwave irradiation is applied to the tissue. The comprehensive statistical analysis showed that the optimal solution for the detection of OPN and OC by confocal immunofluorescence is the 5% Nitric Acid, and followed by 10% EDTA (pH 7.4), Ana Morse solution and 7% HCl/2% EDTA.