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.

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.

Resumo Estudos recentes sugerem que a osteoporose, além dos danos provocados em ossos longos, pode causar deterioração dos ossos maxilares, especialmente na região do osso alveolar, com efeitos na progressão da doença periodontal assim como no reparo ósseo. O objetivo deste estudo foi avaliar o efeito da osteoporose no metabolismo de osteoblastos do osso alveolar mandibular de ratos. Foram utilizadas 10 ratas fêmeas divididas em dois grupos experimentais (Sham e OVX), que foram ovariectomizadas e após 8 semanas, eutanasiadas para coletar amostras do osso mandibular e isolar as células osteoblásticas. As células foram cultivadas em placas de cultura de 24 poços para serem realizados os experimentos in vitro. Após 7, 10 e 14 dias foram avaliados a proliferação celular pelo ensaio de MTT, detecção in situ de fosfatase alcalina (ALP) assim como de nódulos mineralizados e expressão quantitativa de genes associados à remodelação óssea. Os resultados mostraram que aos 7, 10 e 14 dias a proliferação celular foi menor para o grupo OVX. A detecção in situ de ALP foi maior aos 7 dias e menor aos 10 e 14 dias no grupo OVX. Aos 17 e 21 dias o grupo OVX apresentou uma diminuição dos nódulos mineralizados. Houve uma repressão na expressão dos genes Alp, Bglap e Runx2 e uma indução do gene Opg no grupo OVX, enquanto que a modulação dos genes Opn e Rankl foi similar entre os grupos experimentais. Nossos resultados sugerem que a osteoporose tem um efeito deletério no metabolismo de células do osso alveolar em ratas ovariectomizadas, o que pode afetar o tratamento de doenças associadas aos ossos maxilares

Abstract 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.

Abstract 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.

SUMMARY: Impairing osteoporosis progression is a challenge, and recently the role of antioxidants has been associated to bone metabolism. Green tea extract is rich in catechins, especially epigallocatechin gallate (EGCG), which may help control osteoporosis damage in bone tissue. This investigation evaluated the efficacy of green tea ingestion containing different concentrations of EGCG in calvaria bone repair of ovariectomized rats. Wistar rats (n=15) were ovariectomized and divided into 3 groups: ovariectomized (OVX), ovariectomized + GTE 15 % EGCG (OVX/GTE15), and ovariectomized + GTE 94 % EGCG (OVX/GTE94). Green tea extract was administered by gavage in the concentration of 50 mg/kg and sham group (n=5) received water. Bone defects were performed in the calvaria 60 days after ovariectomy followed by 4 weeks until euthanasia. Bone samples were collected to perform qualitative and quantitative histological analysis of bone formation. Data obtained were submitted to normality and ANOVA statistical test for p<0.05. The mean values of neoformed bone for Sham, OVX, OVX/GTE15 and OVX/GTE94 were respectively: 21.11 ± 3.91; 19.92 ± 2.20; 33.05 ± 1.26 e 34.75 ± 0.54 (p<0.05). Results show that continuous ingestion of green tea extract immediately after ovariectomy shows positive effects in the prevention of bone loss in osteoporosis, even with low concentrations of EGCG.

RESUMEN: La disminución en la progresión de la osteoporosis es un desafío, y recientemente el papel de los antioxidantes se ha asociado al metabolismo óseo. El extracto de té verde es rico en catequinas, especialmente el galato de epigalocatequina (EGCG), lo que puede ayudar a controlar el daño de la osteoporosis en el tejido óseo. Esta investigación evaluó la eficacia de la ingesta de té verde con diferentes concentraciones de EGCG en la reparación ósea de calvaria de ratas ovariectomizadas. Las ratas Wistar (n = 15) fueron ovariectomizadas y divididas en 3 grupos: ovariectomizadas (OVX), ovariectomizadas + GTE 15 % EGCG (OVX / GTE15), y ovariectomizadas + GTE 94 % EGCG (OVX / GTE94). El extracto de té verde se administró por sonda en una concentración de 50 mg/kg y el grupo simulado (n = 5) recibió agua. Los defectos óseos se realizaron en la calvaria 60 días después de la ovariectomía, seguido de 4 semanas hasta la eutanasia. Se obtuvieron muestras de hueso para realizar un análisis histológico cualitativo y cuantitativo de la formación ósea. Los datos obtenidos se sometieron a normalidad y prueba estadística ANOVA (p<0,05). Los valores medios de hueso neoformado para Sham, OVX, OVX / GTE15 y OVX / GTE94 fueron: 21,11 ± 3,91; 19,92 ± 2,20; 33,05 ± 1,26 y 34,75 ± 0,54 (p <0,05), respectivamente. Los resultados muestran que la ingesta continua de extracto de té verde, inmediatamente después de la ovariectomía, muestra efectos positivos en la prevención de la pérdida ósea ocurrida en la osteoporosis, incluso con concentraciones bajas de EGCG.

Abstract 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.

O objetivo foi avaliar o potencial odontogênico de células indiferenciadas da polpa (OD-21) por meio de indução química in vitro. As células foram divididas em grupos: controle (OD-21), induzido (OD-21 em meio suplementado/OD-21 + OM), e células odontoblastóides (MDPC-23). Após 3, 7, 10 e 14 dias, avaliou-se proliferação e viabilidade celular, proteína total e fosfatase alcalina (ALP), mineralização, imunolocalização da proteína da matriz dentinária 1 (DMP1), ALP e osteopontina (OPN), assim como a expressão dos genes ALP, OSTERIX (Osx), DMP1 e fator de transcrição RUNX2 por PCR em tempo real. Os dados foram avaliados pelo teste de Kruskal-Wallis seguido pelo teste de Mann-Whitney U (p<0.05). Houve diminuição na proliferação celular em OD-21 + OM, com viabilidade celular similar em todos os grupos, exceto aos sete dias. O conteúdo de proteína total foi maior no grupo OD-21 + OM em todos os períodos; o mesmo ocorreu com a atividade de ALP quando comparada com o grupo OD-21, além de apresentar resultados similares ao grupo MDPC-23. A mineralização foi maior em OD-21 + OM quando comparada com o controle negativo. A imunolocalização demonstrou expressão de DMP1 e ALP em MDPC-23 e OD-21 + OM, enquanto que todos os grupos foram positivos para OPN. A expressão gênica de DMP1 e ALP foi maior nas culturas de MDPC-23, enquanto que a de RUNX2 foi menor para estas células e maior no controle negativo. A expressão de OSTERIX foi menor em OD-21 + OM quando comparada aos outros grupos. Sugere-se que as células indiferenciadas da polpa da linhagem OD-21 apresentam potencial odontogênico e poderiam ser usadas para a engenharia tecidual

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.