miR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells.

BACKGROUND: The autoimmune regulator (Aire) gene is critical for the appropriate establishment of central immune tolerance. As one of the main controllers of promiscuous gene expression in the thymus, Aire promotes the expression of thousands of downstream tissue-restricted antigen (TRA) genes, cell adhesion genes and transcription factor genes in medullary thymic epithelial cells (mTECs). Despite the increasing knowledge about the role of Aire as an upstream transcriptional controller, little is known about the mechanisms by which this gene could be regulated. RESULTS: Here, we assessed the posttranscriptional control of Aire by miRNAs. The in silico miRNA-mRNA interaction analysis predicted thermodynamically stable hybridization between the 3'UTR of Aire mRNA and miR-155, which was confirmed to occur within the cellular milieu through a luciferase reporter assay. This finding enabled us to hypothesize that miR-155 might play a role as an intracellular posttranscriptional regulator of Aire mRNA. To test this hypothesis, we transfected a murine mTEC cell line with a miR-155 mimic in vitro, which reduced the mRNA and protein levels of Aire. Moreover, large-scale transcriptome analysis showed the modulation of 311 downstream mRNAs, which included 58 TRA mRNAs. Moreover, miR-155 mimic-transfected cells exhibited a decrease in their chemotaxis property compared with control thymocytes. CONCLUSION: Overall, the results indicate that miR-155 may posttranscriptionally control Aire mRNA, reducing the respective Aire protein levels; consequently, the levels of mRNAs encode tissue-restricted antigens were affected. In addition, miR-155 regulated a crucial process by which mTECs allow thymocytes' migration through chemotaxis.

Lycopene prevents bone loss in ovariectomized rats and increases the number of osteocytes and osteoblasts.

Osteoporosis is a prevalent disease with a high incidence in women at the onset of menopause mainly because of hormonal changes, genetics, and lifestyle, leading to decreased bone mass and risk of fractures. Maintaining bone mass is a challenge for postmenopausal women, with calcium-rich food intake being essential for bone health. Nevertheless, other nutrients such as carotenoids may influence bone metabolism because of their high antioxidant properties. This study aimed to evaluate the effect of the carotenoid lycopene on bone cells and in the microarchitecture of ovariectomized rats employing in vitro and in vivo assays. After 8 weeks of ovariectomy, femurs were removed to isolate bone marrow mesenchymal cells to be cultured in osteogenic medium (sham and ovariectomized/OVX) or with 1 µmol/L lycopene (OVX/Lyc). There were performed assays for alkaline phosphatase activity and its in situ detection, mineralization nodules, and quantitative expression of genes associated with osteogenesis. Daily ingestion of 10 mg/kg of lycopene by oral gavage for 8 weeks after ovariectomy was conducted for stereological evaluation of the number and volume of osteoblasts, osteoclasts, and osteocytes of femur distal epiphysis and for microtomographic evaluation of the bone microarchitecture of the femoral proximal epiphysis. Data were normalized and analyzed by comparison among the groups using one-way ANOVA followed by post hoc tests with the significance level set out at 5%. Results showed that lycopene promoted an increase in ALP in situ detection as well as a significant increase in mineralized nodules deposition and expression of genes Runx2 and Bglap when compared with the OVX group. The administration by oral gavage of lycopene increased the total number of osteoblasts and osteocytes when compared to sham and ovariectomized groups. Additionally, it decreased the volume and number of osteoclasts and also reduced the volume of osteocytes compared to the sham group. These results suggest that lycopene improves bone cell metabolism and bone remodeling with the onset of osteoporosis. Future studies with different concentrations and periods of administration should be carried out to shed further light on it.

Texturized P(VDF-TrFE)/BT membrane enhances bone neoformation in calvaria defects regardless of the association with photobiomodulation therapy in ovariectomized rats.

OBJECTIVES: The purpose of this investigation was to evaluate in vivo the response of bone tissue to photobiomodulation when associated with texturized P(VDF-TrFE)/BT in calvaria defects of ovariectomized rats. MATERIALS AND METHODS: Wistar Hannover rats were submitted to ovariectomy/control surgery. Calvaria bone defects of 5-mm diameter were performed after 90 days of ovariectomy. The animals were divided into OVX (without laser (L) and membrane), OVX + P(VDF-TrFE)/BT, OVX + P(VDF-TrFE)/BT + L, and OVX + PTFE + L. It was utilized a low-intensity gallium-aluminum-arsenide laser (GaAlAs) with 780-nm wavelength and 30-J/cm2 energy density in 12 sessions (120 s). Thirty days after the bone defect the animals were euthanized for histological, microtomographic, and molecular evaluation. Quantitative analysis was analyzed by statistical software for p < 0.05. RESULTS: Histological parameters showed bone tissue formation at the borders of all group defects. The association of photobiomodulation and texturized P(VDF-TrFE)/BT was not synergistic and did not show significant changes in morphometric analysis and biomarkers gene expression. Nevertheless, texturized P(VDF-TrFE)/BT membrane enhanced bone repair regardless of the association with photobiomodulation therapy, with an increase of connectivity density when compared to the OVX + PTFE + L group. The association of photobiomodulation therapy and PTFE was synergistic, increasing the expression of Runx2, Alp, Bsp, Bglap, Sp7, and Rankl, even though not enough to reflect significance in the morphometric parameters. CONCLUSIONS: The utilization of texturized P (VDF-TrFE)/BT, regardless of the association with photobiomodulation therapy, enhanced bone repair in an experimental model of osteoporosis.

Preadministration of yerba mate (Ilex paraguariensis) helps functional activity and morphology maintenance of MC3T3-E1 osteoblastic cells after in vitro exposition to hydrogen peroxide.

Natural substances with antioxidant effects may benefit prevention and treatment of people with or prone to bone diseases after menopause, such as osteoporosis. This study aimed to evaluate the in vitro effect of preadministration of yerba mate extract (YM) in the metabolism of MC3T3-E1 osteoblasts exposed to hydrogen peroxide (H2O2). The cells (MC3T3-E1) were cultured in 24-well plates with the concentration of 1 µg/mL yerba mate extract dissolved in culture medium throughout the culture period. Four hours before each experiment, 400 µmol/L H2O2 was added per well to simulate oxidative stress. There were evaluated cell adhesion and proliferation, in situ detection of alkaline phosphatase (ALP), mineralized nodules, and immunolocalization of osteocalcin (OCN), bone sialoprotein (BSP) and alkaline phosphatase (ALP) proteins. The results showed that YM preadministration to H2O2 exposition significatively increased cell adhesion after 3 days as well as proliferation and in situ ALP detection after 10 and 7 days respectively, when compared to H2O2 group. Besides, staining of OCN and BSP proteins was less intense and scattered in poor spread cells with cytoskeletal changes in H2O2 group when compared to control and YM H2O2 group. ALP staining was restrained to intracellular regions and similar in all experimental groups. Our results suggest that preadministration of yerba mate extract may prevent deleterious effects in the morphology and functional activity of osteoblasts exposed to H2O2, which could enable the maintenance of extracellular matrix in the presence of oxidative stress.

Photobiomodulation therapy does not depend on the differentiation of dental pulp cells to enhance functional activity associated with angiogenesis and mineralization.

The purpose of this study is to analyze the influence of InGaAlP diode laser (660 nm) with or without an odontogenic medium (OM) in the functional activity of OD-21 cells. Undifferentiated OD-21 pulp cells were cultivated with or without OM and divided into four groups (n = 5): nonirradiated control (C -), nonirradiated + OM (C +), irradiated (L -), and irradiated + OM (L +). Laser application was performed in two sessions of a 24-h interval with an irradiance of 11.3 mW/cm2, energy density of 1 J/cm2, and total cumulative energy/well of 4.6 J. Cell proliferation, VEGF-164 expression, mineralization, and expression of Alp, Runx2, and Dmp1 genes, as well as immunolocalization of RUNX2 and MEPE proteins, were evaluated. Data were analyzed by statistical tests (α = 0.05). All studied groups showed a similar increase in cell proliferation with or without OM. After 7 and 10 days, a significatively higher concentration of VEGF-164 in L - group when compared to C - group was observed. A significant increase in mineralized nodules in the L + was noted when compared to C + in the same conditions. Photobiomodulation upregulated significantly Runx2 and Dmp1 expression after 10 days in L - and after 7 days in L + , with downregulation of Dmp1 after 10 days in L + group. Immunolocalization of RUNX2 and MEPE was expressive after 7 days of culture in the cytoplasm adjacent to the nucleus with a decrease after 10 days, regardless of the presence of OM. Photobiomodulation enhances metabolism associated with angiogenesis, gene expression, and mineralization regardless of the odontogenic medium in OD-21 cells.

Green tea extract rich in epigallocatechin gallate impairs alveolar bone loss in ovariectomized rats with experimental periodontal disease.

Periodontal disease and osteoporosis are characterized by bone resorption, and researchers have shown an association between these two diseases through increasing loss of systemic bone mass and triggering alveolar bone loss. Green tea is a common and easily accessible beverage, and evidences show that flavonoid epigallocatechin gallate (EGCG) could decrease bone loss in pathologies such as osteoporosis and periodontal disease. In order to verify its possible effects and apply them in the treatment and prevention of these diseases, this investigation aimed to evaluate the influence of green tea extract (GTE) on bone metabolism of ovariectomized rats after experimental periodontal disease (EPD) by histological, morphological and microtomographic parameters. Wistar female rats were divided into Sham, Sham + EPD, Sham + EPD + GTE, OVX, OVX + EPD and OVX + EPD + GTE groups. Immediately after surgery, gavage administration of 50 mg/kg of green tea extract (GTE) was performed for 60 days, with subsequent induction of periodontal disease by ligature 15 days before euthanasia. Mandible and femur samples were collected for histological, morphometric and microtomographic analysis. The results were analysed by means of statistical software with significance set at 5%. Histological and morphometric analysis showed a significant decrease in alveolar and femoral trabecular bone loss in groups that received GTE. Microtomographic results showed that trabecular thickness and bone surface density values in alveolar bone interradicular septum of the OVX + EPD + GTE groups were similar to the Sham group. The results obtained suggest that green tea extract may improve bone metabolism in osteoporotic rats with periodontal disease.

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.

Menopause transition promotes distinct modulation of mRNAs and miRNAs expression in calvaria and bone marrow osteoblastic cells.

Investigation on functional genome research may contribute to the knowledge of functional roles of different mRNAs and miRNAs in bone cells of osteoporotic animals. Currently, few studies indicate the changes in gene modulation that osteoporosis causes in osteoblastic cells from different sites. Thus, the purpose of this investigation was to evaluate cell viability, alkaline phosphatase activity and modulation of mRNAs/miRNAs in osteoblastic cells from calvaria and bone marrow by means of microarray technology. Wistar female rats were divided in sham operated and ovariectomized groups. After 150 days of ovariectomy, cells were isolated from both sites to perform cell culture. Results showed that calvaria cells from ovariectomized rats had a decrease in viability when compared to control groups and to bone marrow cells from osteoporotic rats after 3 days. Alkaline phosphatase activity decreased in calvaria cells from ovariectomized rats whereas it was increased in bone marrow osteoblastic cells in the same group. Microarray data analysis showed 5447 differentially expressed mRNAs and 82 differentially expressed miRNAs in calvaria cells. The same way, 4399 mRNAs and 54 miRNAs were expressed in bone marrow cells. mRNAs associated with bone metabolism such as Anxa5, Sp7, Spp1, Notch1 were distinctively modulated in both sites, as well as miRNAs such as miR-350, miR-542-3p, miR-204-5p, and miR-30e-3p. The RNA species identified in this study could be further used as targets for treatment or prevention of osteoporosis.

Lycopene enhances bone neoformation in calvaria bone defects of ovariectomized rats.

Osteoporosis can affect a significant part of the population and fractures are the most common complications associated with this disease, leading to high public health costs. Thus, the prevention of fractures is relevant to individuals with signs and symptoms as well as to the health system. Postmenopausal osteoporosis has been associated with oxidative stress, emphasizing the importance of an efficient defense system to maintain bone health. Lycopene is a carotenoid with antioxidant properties that may stimulate osteoblastogenesis and inhibit osteoclastogenesis. The purpose of this investigation was to analyze the influence of lycopene in the bone neoformation of calvaria defects in ovariectomized rats utilizing the concentration of 45 mg/kg. Wistar Hannover female rats were divided into ovariectomized and sham groups. The ovariectomized animals received 45 mg/kg lycopene (OvxL) or water (Ovx) by daily gavage the day after ovariectomy/sham surgery for 16 weeks. Twelve weeks after ovariectomy, there were performed 5-mm calvaria defects followed by euthanasia after 4 weeks. Samples of bone tissue were collected to perform morphological and morphometrical analysis of the neoformed bone area, and percentage with Software Image J. Morphological evaluation showed mature bone with more osteocytes in the group OVxL when compared to the other groups. The morphometrical analysis demonstrated a significant increase of bone neoformation in the group OvxL (p<0.05). The data obtained suggest that lycopene benefits bone repair in the absence of estrogenic hormones.

Low concentrations of grape seed extract maintain osteoblast morphology, cell adhesion, and mineralization.

The increase in life expectancy has led to a higher incidence of osteoporosis, characterized by an imbalance in bone remodeling. Several drugs are used for its treatment, but most promote undesirable side effects. The present investigation evaluated the effects of two low concentrations of grape seed extract (GSE) rich in proanthocyanidins on MC3T3-E1 osteoblastic cells. The cells were cultured in an osteogenic medium and divided into control (C), 0.1 µg/mL GSE (GSE0.1), and 1.0 µg/mL GSE (GSE1.0) groups to evaluate cell morphology, adhesion, and proliferation, in situ alkaline phosphatase (ALP) detection, mineralization and immunolocalization of osteopontin (OPN). The data obtained were analyzed by statistical tests for a significance of 5%. Cell morphology was maintained with both GSE concentrations, whereas cell adhesion significantly increased within three days in all groups. Cell proliferation increased significantly at seven days of culture, followed by a significant decrease in all experimental periods, with no statistical difference among them. In situ detection of ALP and mineralization increased with time, but within each period, no statistical differences among groups were observed. The expression of osteopontin was distributed regularly with more intensity after 24 hours in the GSE0.1 group. After three days, OPN expression was more intense in the control group, followed by GSE0.1 and GSE1.0 groups. Data obtained suggest that low concentrations of GSE do not affect the morphology and may stimulate the functional activity of osteoblastic cells.

Osteointegration of autogenous bone graft associated with osteoblastic cells under treatment with caffeine.

PURPOSE: The present study investigated osteointegration of autogenous bone (AB) from calvaria graft associated with osteoblastic cells (OC) in bone defects in rats subjected to daily administration of caffeine. MATERIALS AND METHODS: Male rats received daily intraperitoneal injection of 1.5% caffeine (0.2 mL/100 g body weight) or saline solution for 30 days. Then they were anesthetized, submitted to the extraction of the upper right incisor, and implanted with AB only and AB + OC. The animals were killed on 7th, 21st, and 42nd days after surgery, and their maxilla were processed for obtaining semiserial sections (5 µm) stained with hematoxylin and eosin. Through image analysis system, the bone volume and the quality of graft in adjacent areas were estimated. RESULTS: The results showed that in caffeine treatment, the AB + OC graft showed no foreign body and acute inflammatory reactions inside the defect when compared to AB. The histometric results revealed that the association AB + OC produced significant increase (10%-15%) in bone volume in later experimental period (42 days) when compared with saline solution group (P ≤ 0.01). CONCLUSIONS: It was concluded that the association of AB from calvaria + OC demonstrated progressive osteointegration and accelerated the repair of bone defects in animals treated with daily caffeine.

Osteoporosis Affects Functional Activity and Gene Expression of Osteoblastic Cells Derived from Rat Alveolar Bone.

Recent studies suggest that osteoporosis, in addition to the damage caused in long bones, may cause deterioration in the jaws, especially in alveolar bone sites, with effects in the progress of periodontal disease as well as in bone healing. The aim of this study was to evaluate the effect of osteoporosis in the metabolism of rat alveolar bone osteoblasts. There were used 10 female rats divided in two experimental groups (Sham and OVX), which were ovariectomized and after 8 weeks euthanized to collect mandibular bone samples in order to isolate osteoblastic cells. The cells were cultured in 24-well plates to perform the in vitro experiments. After 7, 10 and 14 days, there were evaluated cell proliferation by MTT assay, in situ detection of alkaline phosphatase (ALP) as well as mineralized nodules and expression of genes associated to bone remodeling. Results showed that at 7, 10 and 14 days cell proliferation was lower for OVX group. In situ detection of ALP was higher at 7 days and lower at 10 and 14 days in OVX group. At 17 and 21 days, OVX group had a significative decrease of mineralization nodules. There was a downregulation in the expression of Alp, Bglap and Runx2 genes and an upregulation of Opg in OVX group, whereas Opn and Rankl modulation was similar between the evaluated groups. Our results suggest that osteoporosis has a deleterious effect on alveolar bone cells from ovariectomized rats, which might affect the treatment of diseases associated to the jaw bones.

Impact of calcium aluminate cement with additives on dental pulp-derived cells.

Calcium aluminate cement (CAC) has been highlighted as a promising alternative for endodontic use aiming at periapical tissue repair. However, its effects on dental pulp cells have been poorly explored. OBJECTIVE: This study assessed the impact of calcium chloride (CaCl2) and bismuth oxide (Bi2O3) or zinc oxide (ZnO) additives on odontoblast cell response to CAC. METHODOLOGY: MDPC-23 cells were exposed for up to 14 d: 1) CAC with 2.8% CaCl2 and 25% ZnO (CACz); 2) CAC with 2.8% CaCl2 and 25% Bi2O3 (CACb); 3) CAC with 10% CaCl2 and 25% Bi2O3 (CACb+); or 4) mineral trioxide aggregate (MTA), placed on inserts. Non-exposed cultures served as control. Cell morphology, cell viability, gene expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), and dentin matrix protein 1 (DMP-1), ALP activity, and extracellular matrix mineralization were evaluated. Data were compared using ANOVA (α=5%). RESULTS: Lower cell density was detected only for MTA and CACb+ compared with Control, with areas showing reduced cell spreading. Cell viability was similar among groups at days one and three (p>0.05). CACb+ and MTA showed the lowest cell viability values at day seven (p>0.05). CACb and CACb+ promoted higher ALP and BSP expression compared with CACz (p<0.05); despite that, all cements supported ALP activity. Matrix mineralization were enhanced in CACb+ and MTA. CONCLUSION: In conclusion, CAC with Bi2O3, but not with ZnO, supported the expression of odontoblastic phenotype, but only the composition with 10% CaCl2 promoted mineralized matrix formation, rendering it suitable for dentin-pulp complex repair.

Osteogenic potential of autogenous bone associated with bone marrow osteoblastic cells in bony defects: a histomorphometric study.

PURPOSE: Because of limitations of autogenous grafts, allografts, xenografts, alloplasts, and hydroxyapatite as graft materials, researchers have been using bone tissue engineering as a strategy for bone regeneration. The aim of this work was to study the effect of bone tissue engineering, associating bone marrow osteoblastic cells, and autogenous bone in defects created by dental extraction in rats. MATERIALS AND METHODS: Eighty male rats from 250 to 300 g were anesthetized, submitted to the extraction of the superior incisor, and divided in control group (C), implanted with osteoblastic cells (OC), autogenous bone (AB), and osteoblastic cells + autogenous bone (OC + AB). The animals were killed on 10th and 20th days after surgery and their maxilla were processed for obtaining fine semiserial sections (5 mum), and then stained with hematoxylin-eosin. Through image analysis system, bone volume in areas adjacent to the implants was estimated. RESULTS: The histometric results revealed that the association OC + AB produced significant increase (10%-15%) of bone in both experimental periods when compared with the control group (P < or = 0.01). CONCLUSIONS: Osteoblastic cells associated with autogenous bone accelerated the repair of bone defect, and the action of the osteoblastic cells was more effective until the 10th day and of the autogenous bone after this period.

Effect of grape seed extract (GSE) on functional activity and mineralization of OD-21 and MDPC-23 cell lines.

Recent studies on functional tissue regeneration have focused on substances that favor cell proliferation and differentiation, including the bioactive phenolic compounds present in grape seed extract (GSE). The aim of this investigation was to evaluate the stimulatory potential of GSE in the functional activity of undifferentiated pulp cells and odontoblast-like cells. OD-21 and MDPC-23 cell lines were cultivated in odontogenic medium until subconfluence, seeded in 24-well culture plates in a concentration of 2x104/well and divided into: 1) OD-21 without GSE; 2) OD-21+10 µg/mL of GSE; 3) MDPC-23 without GSE; 4) MDPC-23+10 µg/mL of GSE. Cell proliferation, in situ detection of alkaline phosphatase (ALP) and total protein content were assessed after 3, 7 and 10 days, and mineralization was evaluated after 14 days. The data were analyzed by ANOVA statistical tests set at a 5% level of significance. Results revealed that cell proliferation increased after 10 days, and protein content, after 7 days of culture in MDPC-23 cells. In situ ALP staining intensity was higher in undifferentiated pulp cells and odontoblast-like cells after 7 and 10 days, respectively. A discrete increase in MDPC-23 mineralization after GSE treatment was observed despite OD-21 cells presenting a decrease in mineralized nodule deposits. Data suggest that GSE favors functional activity of differentiated cells more broadly than undifferentiated cells (OD-21). More studies with different concentrations of GSE must be conducted to confirm its benefits to cells regarding dentin regeneration.

Low-level laser therapy enhances the number of osteocytes in calvaria bone defects of ovariectomized rats.

BACKGROUND: Osteoporosis can make bone repair difficult. Low-level laser therapy (LLLT) has been shown to be a promising tool for bone neoformation. This study aimed to analyze the effect of LLLT on calvaria bone defects of ovariectomized rats using stereology. METHODS: Fifty-four Wistar rats were subjected to bilateral ovariectomy, and bone defects were created in calvaria after 150 days. The animals were divided into nine groups (n =  6 per group), and 24 hours after the bone defects were created they received 3, 6 or 12 sessions of LLLT at 0, 20 or 30 J/cm2, using a 780-nm low-intensity GaAlAs laser. One-way ANOVA followed by Tukey's post hoc test was used for data processing. A difference of P < 0.05 was considered statistically significant. The parameters evaluated were osteocyte density (Nv ost), total osteocyte number (Nto ost), trabecular surface density (Sv t), and trabecular surface area (Sa t). RESULTS: Data obtained showed that Nto ost, Sv t, and Sa t in group G2 rats were significantly different from G1 (0 J/cm2) (P < 0.05). Compared to group G4, G5 presented higher values for the parameters Sv t and Sa t, and G6 presented significantly higher values for almost all the analyzed parameters (Nv ost, Nto ost, Sv t, and Sa t) (P < 0.05). Compared to group G7, G8 showed a higher value only for the parameter Sa t, and G9 showed significantly higher values for parameters Nv ost, Nto ost, Sv t, and Sa t. CONCLUSION: We conclude that LLLT stimulated bone neoformation and contributed to an increase in the total number of osteocytes, especially with a laser energy density of 30 J/cm2 given for 6 and 12 sessions.

Cell therapy stimulates bone neoformation in calvaria defects in rats subjected to local irradiation.

BACKGROUND: The purpose of the study was to analyze the effect of cell therapy on the repair process in calvaria defects in rats subjected to irradiation. METHODS: Bone marrow mesenchymal cells were characterized for osteoblastic phenotype. Calvariae of male Wistar rats were irradiated (20 Gy) and, after 4 weeks, osteoblastic cells were placed in surgically created defects in irradiated (IRC) and control animals (CC), paired with untreated irradiated (IR) and control (C) animals. After 30 days, histological and microtomographic evaluation was performed to establish significant (P < 0.05) differences among the groups. RESULTS: Higher alkaline phosphatase detection and activity, along with an increase in mineralized nodules, in the IRC, C and CC groups compared to the IR group, confirmed an osteoblastic phenotype. Histology showed impaired bone neoformation following irradiation, affecting bone marrow composition. Cell therapy in the IRC group improved bone neoformation compared to the IR group. Microtomography revealed increased bone volume, bone surface and trabecular number in IRC group compared to the IR group. CONCLUSION: Cell therapy may improve bone neoformation in defects created after irradiation.

Tooth pigmentation caused by bilirubin: a case report and histological evaluation.

Systemic disorders in pediatric patients, such as congenital biliary atresia, acute liver failure, and biliary hypoplasia, may be the indications for a need of liver transplantation. One of the manifestations of these disorders is the elevated serum levels of bilirubin (hyperbiliru-binemia), a product of hemoglobin degradation, which is deposited in different tissues, including mineralized and soft tissues. When hyperbilirubinemia occurs during the period of dental development, these teeth can develop a green coloration, which remains permanently, because, after maturation, these tissues loose their metabolic activity. This case report describes a 9-year-old girl who required a liver transplant due to biliary atresia when she was three years old. Some of her pigmented teeth needed extraction and afterwards were submitted for histological analysis and compared with sound teeth.

Low concentrations of grape seed extract maintain osteoblast morphology, cell adhesion, and mineralization

Abstract The increase in life expectancy has led to a higher incidence of osteoporosis, characterized by an imbalance in bone remodeling. Several drugs are used for its treatment, but most promote undesirable side effects. The present investigation evaluated the effects of two low concentrations of grape seed extract (GSE) rich in proanthocyanidins on MC3T3-E1 osteoblastic cells. The cells were cultured in an osteogenic medium and divided into control (C), 0.1 µg/mL GSE (GSE0.1), and 1.0 µg/mL GSE (GSE1.0) groups to evaluate cell morphology, adhesion, and proliferation, in situ alkaline phosphatase (ALP) detection, mineralization and immunolocalization of osteopontin (OPN). The data obtained were analyzed by statistical tests for a significance of 5%. Cell morphology was maintained with both GSE concentrations, whereas cell adhesion significantly increased within three days in all groups. Cell proliferation increased significantly at seven days of culture, followed by a significant decrease in all experimental periods, with no statistical difference among them. In situ detection of ALP and mineralization increased with time, but within each period, no statistical differences among groups were observed. The expression of osteopontin was distributed regularly with more intensity after 24 hours in the GSE0.1 group. After three days, OPN expression was more intense in the control group, followed by GSE0.1 and GSE1.0 groups. Data obtained suggest that low concentrations of GSE do not affect the morphology and may stimulate the functional activity of osteoblastic cells.

Resumo O aumento da expectativa de vida tem levado a uma maior incidência de osteoporose, caracterizada por um desequilíbrio na remodelação óssea. Vários medicamentos são utilizados para o seu tratamento, contudo, a maioria promove efeitos colaterais indesejáveis. A presente investigação avaliou os efeitos de duas baixas concentrações de extrato de semente de uva (GSE) rico em proantocianidinas em células osteoblásticas MC3T3-E1. As células foram cultivadas em meio osteogênico e divididas em grupos controle (C), 0,1 µg/mL de GSE (GSE0.1) e 1,0 µg/mL de GSE (GSE1.0) para avaliar morfologia, adesão e proliferação celular, detecção in situ de fosfatase alcalina (ALP), mineralização e imunolocalização da proteína osteopontina (OPN). Os dados obtidos foram analisados por testes estatísticos para um nível de significância de 5%. A proliferação celular aumentou significativamente aos sete dias de cultura, seguido de uma diminuição significativa em todos os períodos experimentais, sem diferença estatística entre eles. A detecção in situ de ALP e mineralização aumentou com o tempo, mas dentro de cada período não foram observadas diferenças estatísticas entre os grupos. A morfologia celular foi mantida com ambas as concentrações de GSE, enquanto a adesão celular aumentou significativamente aos três dias em todos os grupos. A expressão de osteopontina distribuiu-se regularmente com maior intensidade após 24 horas no grupo GSE0.1. Após três dias, a expressão de OPN foi mais intensa no grupo controle, seguida pelos grupos GSE0.1 e GSE1.0. Os dados obtidos sugerem que baixas concentrações de GSE não afetam a morfologia e podem estimular a atividade funcional das células osteoblásticas.

Lycopene enhances bone neoformation in calvaria bone defects of ovariectomized rats

Abstract Osteoporosis can affect a significant part of the population and fractures are the most common complications associated with this disease, leading to high public health costs. Thus, the prevention of fractures is relevant to individuals with signs and symptoms as well as to the health system. Postmenopausal osteoporosis has been associated with oxidative stress, emphasizing the importance of an efficient defense system to maintain bone health. Lycopene is a carotenoid with antioxidant properties that may stimulate osteoblastogenesis and inhibit osteoclastogenesis. The purpose of this investigation was to analyze the influence of lycopene in the bone neoformation of calvaria defects in ovariectomized rats utilizing the concentration of 45 mg/kg. Wistar Hannover female rats were divided into ovariectomized and sham groups. The ovariectomized animals received 45 mg/kg lycopene (OvxL) or water (Ovx) by daily gavage the day after ovariectomy/sham surgery for 16 weeks. Twelve weeks after ovariectomy, there were performed 5-mm calvaria defects followed by euthanasia after 4 weeks. Samples of bone tissue were collected to perform morphological and morphometrical analysis of the neoformed bone area, and percentage with Software Image J. Morphological evaluation showed mature bone with more osteocytes in the group OVxL when compared to the other groups. The morphometrical analysis demonstrated a significant increase of bone neoformation in the group OvxL (p<0.05). The data obtained suggest that lycopene benefits bone repair in the absence of estrogenic hormones.

Resumo A osteoporose afeta grande parte da população e as fraturas são as complicações mais importantes relacionadas a essa doença, gerando altos gastos para o poder público. Dessa forma, a prevenção de fraturas decorrentes da osteoporose torna-se relevante tendo em vista que gera benefícios tanto para o indivíduo acometido pela doença quanto para o sistema de saúde. A osteoporose pós menoupasa tem sido associada ao estresse oxidativo, portanto, um eficiente sistema de defesa antioxidante é primordial para a manutenção da saúde óssea. O licopeno é um carotenoide antioxidante que aparentemente estimula a osteoblastogênese e inibe a osteoclastogênese. O objetivo deste estudo foi analisar a influência do licopeno na neoformação óssea em defeitos de calvária em ratas ovariectomizadas utilizando a concentração de 45 mg/kg. Foram utilizados 15 ratas Wistar Hannover pesando aproximadamente 200g, sendo que 10 animais foram submetidos à ovariectomia bilateral e 5 (Grupo Sham) foram submetidos à simulação da cirurgia de ovariectomia bilateral. Os animais ovariectomizados foram divididos aleatoriamente em 2 grupos: Ovariectomizado (Ovx) e Ovariectomizado Licopeno (OvxL) que receberam água e licopeno respectivamente, por sonda gástrica, diariamente. As administrações iniciaram-se no dia seguinte à cirurgia de ovariectomia e/ou da exposição dos ovários e foram mantidas por 120 dias, data de realização da eutanásia. O grupo Sham recebeu água diariamente. Noventa dias após a ovariectomia bilateral foram confecionados defeitos ósseos nas calvárias de todos os animais e após trinta dias as ratas foram eutanasiadas. As amostras de tecido ósseo foram coletadas e foi realizado o processamento para a obtenção das lâminas histológicas. Foram realizadas as análises morfológicas e morfométrica, onde foi estimada a área (mm2) e porcentagem (%) relativa de osso neoformado utilizando o Software Image J. A avaliação morfológica evidenciou a ação benéfica do licopeno pois os animais que receberam esse antioxidante apresentaram um tecido ósseo mais maduro, com maior presença de osteócitos quando comparados aos demais grupos. Por meio das análises morfométricas verificou-se maior neoformação óssea para os animais que receberam o licopeno (p<0,05). Diante dos resultados obtidos, concluiu-se que o licopeno na concentração de 45 mg/Kg teve efeito benéfico no processo de reparação, promovendo significante formação óssea frente à ausência de hormônios estrogênicos.