F.S. Oliveira

Nanotopography directs mesenchymal stem cells to osteoblast lineage through regulation of microRNA-SMAD-BMP-2 circuit.

The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time‐point, miR‐sequencing analysis revealed that 20 miRs were upregulated (>twofold) while 20 miRs were downregulated (>threefold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR‐4448, ‐4708, and ‐4773, which were significantly downregulated (>fivefold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP‐2) osteogenic signal, which were upregulated by Ti with nanotopography. Overexpression of miR‐4448, ‐4708, and 4773 in MC3T3‐E1 pre‐osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP‐2 displayed a higher osteogenic effect on MC3T3‐E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP‐2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR‐SMAD‐BMP‐2 circuit governs the osteoblast differentiation induced by Ti with nanotopography. J. Cell. Physiol. 229: 1690–1696, 2014.

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non‐osteogenic medium with or without purmorphamine (2 µM) for periods of up to 14 days. Purmorphamine up‐regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204–208, 2012.

Poly(vinylidene-trifluoroethylene)/barium titanate composite for in vivo support of bone formation

In this study, we evaluated the effect of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane on in vivo bone formation. Rat calvarial bone defects were implanted with P(VDF-TrFE)/BT and polytetrafluoroethylene (PTFE) membranes, and at 4 and 8 weeks, histomorphometric and gene expression analyses were performed. A higher amount of bone formation was noticed on P(VDF-TrFE)/BT compared with PTFE. The gene expression of RUNX2, bone sialoprotein, osteocalcin, receptor activator of nuclear factor-kappa B ligand, and osteoprotegerin indicates that P(VDF-TrFE)/BT favored the osteoblast differentiation compared with PTFE. These results evidenced the benefits of using P(VDF-TrFE)/BT to promote new bone formation, which may represent a promising alternative to be employed in guided bone regeneration.

Diet carotenoid lutein modulates the expression of genes related to oxygen transporters and decreases DNA damage and oxidative stress in mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.

Participation of TNF-α in Inhibitory Effects of Adipocytes on Osteoblast Differentiation.

Mesenchymal stem cells from bone marrow (BM‐MSCs) and adipose tissue (AT‐MSCs) are attractive tools for cell‐based therapies to repair bone tissue. In this study, we investigated the osteogenic and adipogenic potential of BM‐MSCs and AT‐MSCs as well as the effect of crosstalk between osteoblasts and adipocytes on cell phenotype expression. Rat BM‐MSCs and AT‐MSCs were cultured either in growth, osteogenic, or adipogenic medium to evaluate osteoblast and adipocyte differentiation. Additionally, osteoblasts and adipocytes were indirectly co‐cultured to investigate the effect of adipocytes on osteoblast differentiation and vice versa. BM‐MSCs and AT‐MSCs exhibit osteogenic and adipogenic potential under non‐differentiation‐inducing conditions. When exposed to osteogenic medium, BM‐MSCs exhibited higher expression of bone markers compared with AT‐MSCs. Conversely, under adipogenic conditions, AT‐MSCs displayed higher expression of adipose tissue markers compared with BM‐MSCs. The presence of adipocytes as indirect co‐culture repressed the expression of the osteoblast phenotype, whereas osteoblasts did not exert remarkable effect on adipocytes. The inhibitory effect of adipocytes on osteoblasts was due to the release of tumor necrosis factor alpha (TNF‐α) in culture medium by adipocytes. Indeed, the addition of exogenous TNF‐α in culture medium repressed the differentiation of BM‐MSCs into osteoblasts mimicking the indirect co‐culture effect. In conclusion, our study showed that BM‐MSCs are more osteogenic while AT‐MSCs are more adipogenic. Additionally, we demonstrated the key role of TNF‐α secreted by adipocytes on the inhibition of osteoblast differentiation. Thus, we postulate that the higher osteogenic potential of BM‐MSCs makes them the first choice for inducing bone repair in cell‐based therapies. J. Cell. Physiol. 230: 204–214, 2016.

Progression of osteogenic cell cultures grown on microtopographic titanium coated with calcium phosphate and functionalized with a type I collagen-derived peptide.

BACKGROUND The functionalization of metallic surfaces aims at promoting the cellular response at the biomaterial-tissue interface. This study investigates the effects of the functionalization of titanium (Ti) microtopography with a calcium phosphate (CaP) coating with and without peptide 15 (P-15), a synthetic peptide analog of the cell-binding domain of collagen I, on the in vitro progression of osteogenic cells. METHODS Sandblasting and acid etching (SBAE; control) Ti microtopography was coated with CaP, enabling the loading of two concentrations of P-15: 20 or 200 μg/mL. A machined Ti was also examined. Rat calvarial osteogenic cells were cultured on Ti disks with the surfaces mentioned above for periods up to 21 days (n = 180 per group). RESULTS CaP coating exhibited a submicron-scale needle-shaped structure. Although all surfaces were hydrophobic at time zero, functionalization increased hydrophilicity at equilibrium. Microtopographies exhibited a lower proportion of well-spread cells at 4 hours of culture and cells with long cytoplasmic extensions at day 3; modified SBAE supported higher cell viability and larger extracellular osteopontin (OPN) accumulation. For SBAE and modified SBAE, real-time polymerase chain reaction showed the following results: 1) lower levels for runt-related transcription factor 2 at 7 days and for bone sialoprotein at days 7 and 10 as well as higher OPN levels at days 7 and 10 compared to machined Ti; and 2) higher alkaline phosphatase levels at day 10 compared to day 7. At 14 and 21 days, modified SBAE supported higher proportions of red-dye-stained areas (calcium content). CONCLUSION Addition of a CaP coating to SBAE Ti by itself may affect key events of in vitro osteogenesis, ultimately resulting in enhanced matrix mineralization; additional P-15 functionalization has only limited synergistic effects.

Study comparing midazolam and nitrous oxide in dental anxiety control.

Abstract The purpose of this study is to comparatively assess the effect of midazolam and nitrous oxide associated with oxygen, in lower third molar extractions, on the change in the anxiety level of patients by salivary cortisol dosage. Twenty-eight male patients underwent lower third molar extraction under sedation with midazolam and nitrous oxide. Objective (salivary cortisol dosage) and subjective (Corah Dental Anxiety Scale) data have been obtained. By salivary cortisol, 40 minutes after midazolam administration, there has been a statistically significant difference compared with the mean baseline value. Midazolam was the most effective sedation method for reducing salivary cortisol level.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

Treatment of Intrabony Defects with Anorganic Bone Matrix/P-15 or Guided Tissue Regeneration in Patients with Aggressive Periodontitis

Intrabony periodontal defects present a particular treatment problem, especially in patients with generalized aggressive periodontitis (G-AgP). Regenerative procedures have been indicated for this clinical situation. The aim of this study was to compare treatment outcomes of intrabony periodontal defects with either anorganic bone matrix/cell binding peptide (ABM/P-15) or guided tissue regeneration (GTR) in patients with G-AgP. Fifteen patients, with two intrabony defects ≥3 mm deep, were selected. Patients were randomly allocated to be treated with ABM/P-15 or GTR. At baseline and at 3 and 6 months after surgery, clinical and radiographic parameters and IL-1β and IL-6 gingival fluid concentrations were recorded. There was a significant probing pocket depth reduction (p<0.001) for both groups (2.27 ± 0.96 mm for ABM/P-15 group and 2.57 ± 1.06 mm for GTR group). Clinical attachment level gain (1.87 ± 0.94 mm for ABM/P-15 group and 2.09 ± 0.88 mm for GTR group) was also observed. There were no statistically significant differences in clinical parameters between the groups. The radiographic bone fill was more expressive in ABM/P-15 group (2.49 mm) than in GTR group (0.73 mm). In subtraction radiographs, the areas representing gain in density were 93.16% of the baseline defect for ABM/P-15 group versus 62.03% in GRT group. There were no statistically significant differences in inter-group and intra-group comparisons with regards to IL-1β and IL-6 quantification. Treatment of intrabony periodontal defects in patients with G-AgP with ABM/P-15 and GTR improved significantly the clinical outcomes. The use of ABM/P-15 promoted a better radiographic bone fill.

Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model

Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.