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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 runtrelated 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.

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.

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.

SEMEGHINI, Mayara Sgarbi. Effect of carotenoid lycopene on bone metabolism and functional activity of cells from the osteogenic lineage of ovariectomized rats. Ribeirão Preto, 2019. 72 p. Thesis (Doctorate in Oral Biology). Faculty of Dentistry of Ribeirão Preto, University of São Paulo. Osteoporosis is a silence disease characterized by a decrease in bone mass leading to morbidity and high risk of fractures. Its incidence is higher in women after menopause because of hormonal changes, as well as genetic propension and life styles such as sedentarism, alcoholism, tabagism and poor food intake. Besides calcium rich foods that favour the maintenance of bone mass and its density, there are several other nutrients that may influence in the prevention of diseases associated to bone metabolism, such as carotenoids. Among carotenoids, lycopene has been associated to prevention of bone diseases because of its antioxidant properties. The purpose of this investigation was to perform in vitro and in vivo studies about the effect of lycopene in bone metabolism of ovariectomized 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 into control (sham), ovariectomized (Ovx) and ovariectomized + 1 μmol/L lycopene (Ovx/Lic) groups. After the experimental periods, there were evaluated cell proliferation, alkaline phosphatase in situ detection, mineralization and quantitative expression of genes associated to bone metabolism. Besides, in vivo studies were carried out to evaluate femur remodeling by tomographic, histomorphometric and estereological evaluation after daily intake of 10 mg/kg of lycopene for 60 days after ovariectomy. All quantitative data were analyzed by means of one-way ANOVA statistical test with significance of p ≤ 0.05. The in vitro results showed that the presence of lycopene favored cell proliferation and in situ detection of ALP in late culture stages, as well as promoted a significative increase in the deposition of mineralized nodules. Lycopene significatively increased the expression of genes Alp, Runx2, Bglap and Bmp4, besides decreasing the Rankl/Opg ratio when compared to Ovx group. In vivo experiments showed that oral administration of lycopene promoted a significant maintenance of tissue and bone volume in femoral epiphysis, as well as of bone trabeculae and number of osteocytes. Results suggest that lycopene can benefit bone metabolism with the onset of osteoporosis.

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expressão gênica não só das células diferenciadas da calvária, mas também aquelas ainda presentes na medula óssea, influenciando, assim, a formação adequada de tecido ósseo em uma situação de osteoporose.Palavras-chave: Osteoporose, Microarray, miRNA, Calvária, Medula Óssea.ABSTRACT SEMEGHINI, MS. Large scale gene expression evaluation of osteoblastic cells from calvaria and bone marrow of ovariectomized rats. Ribeirão Preto, 2014. 128f. Dissertação AbstractBone remodeling is a physiological process which maintains skeleton integrity replacing old bone by a young bone matrix. In some diseases such as osteoporosis bone formation is impaired due to the decrease in the number of osteoblasts and reduction of their activities leading to a loss of bone tissue microarchitecture, making it fragile and susceptible to fracture. The objective of this study was to evaluate behavioral differences in osteoblast-like cells from calvaria and bone marrow of rats induced to osteoporosis by ovariectomy through large-scale gene expression analysis. Eighteen Wistar rats were used and divided into control and ovariectomized groups. After 150 days of surgery, the rats of both groups were sacrificed for collection of femurs and calvaria fragments. The cells collected from bone marrow and calvaria were grown in 24-well plates (n = 5) for cell proliferation and viability analysis, as well as grown in culture bottles for total RNA extraction of these cells.The RNA was quantified and its integrity assessed by gel electrophoresis in a microfluidic system. Identification of gene modulation as well as microRNAs involved in gene inhibition was performed by cDNA microarray. Bone marrow cells from the control group (MC) showed a significant decrease in proliferation compared with cells from the calvaria control group (CC) in all periods (p <0.05). On the other hand, bone marrow cells of ovariectomized rats (MO) revealed a significant increase in the proliferation rate after 7 and 10 days (p<0.01) compared to calvaria cells of ovariectomized rats (CO). Cell viability was higher in all periods of CC and CO groups compared to MC and MO groups (p <0.05). Microarray data were normalized using quantile and after statistical analysis by unpaired t-test with p-value ≤ 0.005 and subsequent fold change ≥ 2.0, 5.447 differentially expressed mRNAs were detected in calvaria cells and 4.399 mRNAs in bone marrow cells. The miRNAs data were also normalized using the quantile and after statistical analysis by moderate ttest with p-value ≤ 0.05 and subsequent fold change ≥ 1.5, 84 miRNAs differentially expressed in calvaria cells were detected and 55 miRNAs in bone marrow cells. In CC group, there may be highlighted repressed genes such as Notch1, Dlx5, Fgf1, Fgfr2 and Il6 as regulators of cell proliferation and caspases 6 and 12 in response to organic substances. The same group showed induced genes such as Gli1 and Bmp7 genes as regulators of cell proliferation, Gli1, Bmp3 and Mmp2 involved in the biological process of bone development and Lrp1, Mmp2 and Tgfβ1...

Osteoporosis is one of the most important diseases associated with aging. The aim of this study was to determine if there is a distinct expression of genes associated with osteoblast differentiation in bone marrow mesenchymal stem cells (MSCs) and calvaria osteoblastic cells (COs) of rats induced to osteoporosis through ovariectomy. Cells from bone marrow and calvaria of control and osteoporotic rats were cultured in osteogenic medium and divided into four different groups: calvaria/control (CC); calvaria/osteoporosis (CO); bone marrow/control (MC) and bone marrow/osteoporosis (MO). After 3, 7 and 10 days, real‐time RT‐PCR for key bone markers was carried out in quintuplicate (n=5) and data were compared by Kruskal‐Wallis and Mann‐Whitney tests (p<0.05). Alkaline phosphatase and runt‐related transcription factor 2 expressions were significantly increased in MO and decreased in CO in all periods. Osteocalcin expression was significantly increased in MO at 7 and 10 days, and decreased in CO in all periods. The ratio of receptor activator of nuclear factor kappa‐B ligand /osteoprotegerin expression was significantly decreased in MO at 7 and 10 days and in CO in all periods. Our results show a distinct gene expression pattern for MSCs and COs after osteoporosis induction and suggest that MSCs undergo a metabolic adjustment in order to compensate the effects of estrogen decrease in this osteoporosis model. Grant Funding Source: Supported by FAPESP