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1. Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): study protocol for a randomized controlled trial

Author

Cavalcanti, AB, Berwanger, O, Suzumura, ÉA, Amato, MB, Tallo, FS, Rezende, AC, Telles, MM, Romano, E, Guimarães, HP, Regenga, MM, Takahashi, LN, Oliveira, RP, Carvalho, VO, Díaz Quijano, FA, Carvalho, CR, Kodama, AA, Ribeiro, GF, Abreu, MO, Oliveira, IM, Guyatt, G, Ferguson, N, Walter, S, Vasconcelos, MO, Segundo, VJ, Ferraz, ÍL, Silva, RS, de Oliveira Filho, W, Silva, NB, Heirel, C, Takatani, RR, Neto, JA, Neto, JC, Almeida, SD, Chamy, G, Neto, GJ, Dias, AP, Silva, RR, Tavares, RC, Souza, ML, Decio, JC, Lima, CM, Neto, FF, Oliveira, KR, Dias, PP, Brandão, AL, Ramos, JE Jr, Vasconcelos, PT, Flôres, DG, Filho, GR, Andrade, IG, Martinez, A, França, GG, Monteiro, LL, Correia, EI, Ribeiro, W, Pereira, AJ, Andrade, W, Leite, PA, Feto, JE, Holanda, MA, Amorim, FF, Margalho, SB, Domingues, SM Jr, Ferreira, CS, Ferreira, CM, Rabelo, LA, Duarte, JN, Lima, FB, Kawaguchi, IA, Maia, MO, Correa, FG, Ribeiro, RA, Caser, E, Moreira, CL, Marcilino, A, Falcão, JG, Jesus, KR, Tcherniakovisk, L, Dutra, VG, Thompson, MM, Piras, C, Giuberti, J. Jr, Silva, AS, Santos, JR, Potratz, JL, Paula, LN, Bozi, GG, Gomes, BC, Vassallo, PF, Rocha, E, Lima, MH, Ferreira, A. F, Gonçalves, F, Pereira, SA, Nobrega, MS, Caixeta, CR, Moraes, AP, Carvalho, AG, Alves, JD, Carvalho, FB, Moreira, FB, Starling, CM, Couto, WA, Bitencourt, WS, Silva, SG, Felizardo, LR, Nascimento, FJ, Santos, D, Zanta, CC, Martins, MF, Naves, SA, Silva, FD, Laube, G. Jr, Galvão, EL, Sousa, MF, Souza, MM, Carvalho, FL, Bergo, RR, Rezende, CM, Tamazato, EY, Sarat, SC Jr, Almeida, PS, Gorski, AG, Matsui, M, Neto, EE, Nomoto, SH, Lima, ZB, Inagaki, AS, Gil, FS, Araújo, MF, Oliveira, AE, Correa, TA, Mendonça, A, Reis, H, Carneiro, SR, Rego, LR, Cunha, AF, Barra, WF, Carneiro, M, Batista, RA, Zoghbi, KK, Machado, NJ, Ferreira, R, Apoena, P, Leão, RM, Martins, ER, Oliveira, ME, Odir, I, Kleber, W, Tavares, D, Araújo, ME, Brilhante, YN, Tavares, DC, Carvalho, WL, Winveler, GF, Filho, AC, Cavalcanti, RA, Grion, CM, Reis, AT, Festti, J, Gimenez, FM, Larangeira, AS, Cardoso, LT, Mezzaroba, TS, Kauss, IA, Duarte, PA, Tozo, TC, Peliser, P, Germano, A, Gurgel, SJ, Silva, SR, Kuroda, CM, Herek, A, Yamada, SS, Schiavetto, PM, Wysocki, N, Matsubara, RR, Sales, JA Jr, Laprovita, MP, Pena, FM, Sá, A, Vianna, A, Verdeal, JC, Martins, GA, Salgado, DR, Coelho, AM, Coelho, M, Morong, AS, Poquiriqui, RM, Ferreira, AP, Lucena, DN, Marino, NF, Moreira, MA, Uratani, CC, Severino, MA, Silva, PN, Medeiros, LG, Filho, FG, Guimarães, DM, Rezende, VM, Carbonell, RC, Trindade, RS, Pellegrini, JA, Boniatti, MM, Santos, MC, Boldo, R, Oliveira, VM, Corrêa, VM, Nedel, W, Teixeira, C, Schaich, F, Tagliari, L, Savi, A, Schulz, LF, Maccari, JG, Seeger, GM, Foernges, RB, Rieder, MM, Becker, DA, Broilo, FP, Schwarz, P, Alencastro, A, Berto, P, Backes, F, Dias, FS, Blattner, C, Martins, ET, Scaglia, NC, Vieira, SR, Prado, KF, Fialkow, L, Franke, C, Vieira, DF, Moraes, RB, Marques, LS, Hopf, JL, Wawrzeniak, IC, Rech, TH, Albuquerque, RB, Guerreiro, MO, Teixeira, LO, Macedo, PL, Bainy, MP, Ferreira, EV, Martins, MA, Andrade, LA, Machado, FO, Burigo, AC, Pincelli, M, Kretzer, L, Maia, IS, Cordeiro, RB, Westphal, G, Cramer, AS, Dadam, MM, Barbosa, PO, Caldeira, M, Brilenger, CO, Horner, MB, Oliveira, GL, Germiniani, BC, Duarte, R, Assef, MG, Rosso, D, Bigolin, R, Vanzuita, R, Prado, LF, Oliveira, V, Reis, DL, Morais, MO, Bastos, RS, Santana, HS, Silva, AO, Cacau, LA, Almeida, MS, Canavessi, HS, Nogueira, EE, Pavia, CL, Araujo, JF, Lira, JA, Nienstedt, EC, Smith, TC, Romano, M, Barros D, Costa, AF, Takahashi, L, Werneck, V, Farran, J, Henriques, LA, Miura, C, Lopes, RD, Vendrame, LS, Sandri, P, Galassi, MS, Amato, P, Toufen, C. Jr, Santiago, RR, Hirota, AS, Park, M, Azevedo, LC, Malbouison, LM, Costa, MC, Taniguchi, L, Pompílio, CE, Baruzzi, C, Andrade, AH, Taira, EE, Taino, B, Oliveira, CS, Silva, AC, Ísola, A, Rezende, E, Rodrigues, RG, Rangel, VP, Luzzi, S, Giacomassi, IW, Nassar, AP Jr, Souza, AR, Rahal, L, Nunes, AL, Giannini, F, Menescal, B, Morais, JE, Toledo, D, Morsch, RD, Merluzzi, T, Amorim, DS, Bastos, AC, Santos, PL, Silva, SF, Gallego, RC, Santos, GD, Tucci, M, Costa, RT, Santos, LS, Demarzo, SE, Schettino, GP, Suzuki, VC, Patrocinio, AC, Martins, ML, Passos, DB, Cappi, SB, Gonçalves, I. Jr, Borges, MC, Lovato, W, Tavares, MV, Morales, D, Machado, LA, Torres, FC, Gomes, TM, Cerantola, RB, Góis, A, Marraccini, T, Margarida, K, Cavalcante, E, Machado, FR, Mazza, BF, Santana, HB, Mendez, VM, Xavier, PA, Rabelo, MV, Schievano, FR, Pinto, WA, Francisco, RS, Ferreira, EM, Silva, DC, Arduini, RG, Aldrighi, JR, Amaro, AF, Conde, KA, Pereira, CA, Tarkieltaub, E, Oliver, WR, Guadalupe, EG, Acerbi, PS, Tomizuka, CI, Oliveira, TA, Geha, NN, Mecatti, GC, Piovesan, MZ, Salomão, MC, Moreno, MS, Orsatti, VN, Miranda, W, Ray, A, Guerra, A, Filho, ML, Ferreira, FH Jr, Filho, EV, Canzi, RA, Giuberti, AF, Garcez, MC, Sala, AD, Suguitani, EO, Kazue, P, Oliveira, LR, Infantini, RM, Carvalho, FR, Andrade, LC, Santos, TM, Carmona, CV, Figueiredo, LC, Falcão, A, Dragosavak, D, Filho, WN, Lunardi, MC, Lago, R, Gatti, C, Chiasso, TM, Santos, GO, Araujo, AC, Ornellas, IB, Vieira, VM, Hajjar, LA, Figueiredo, AC, Damasceno, B, Hinestrosa, A, Diaz Quijano, FA, CORTEGIANI, Andrea, RAINERI, Santi Maurizio, Cavalcanti, AB, Berwanger, O, Suzumura, ÉA, Amato, MB, Tallo, FS, Rezende, AC, Telles, MM, Romano, E, Guimarães, HP, Regenga, MM, Takahashi, LN, Oliveira, RP, Carvalho, VO, Díaz-Quijano, FA, Carvalho, CR, Kodama, AA, Ribeiro, GF, Abreu, MO, Oliveira, IM, Guyatt, G, Ferguson, N, Walter, S, Vasconcelos, MO, Segundo, VJ, Ferraz, ÍL, Silva, RS, de Oliveira Filho, W, Silva, NB, Heirel, C, Takatani, RR, Neto, JA, Neto, JC, Almeida, SD, Chamy, G, Neto, GJ, Dias, AP, Silva, RR, Tavares, RC, Souza, ML, Decio, JC, Lima, CM, Neto, FF, Oliveira, KR, Dias, PP, Brandão, AL, Ramos, JE Jr, Vasconcelos, PT, Flôres, DG, Filho, GR, Andrade, IG, Martinez, A, França, GG, Monteiro, LL, Correia, EI, Ribeiro, W, Pereira, AJ, Andrade, W, Leite, PA, Feto, JE, Holanda, MA, Amorim, FF, Margalho, SB, Domingues, SM Jr, Ferreira, CS, Ferreira, CM, Rabelo, LA, Duarte, JN, Lima, FB, Kawaguchi, IA, Maia, MO, Correa, FG, Ribeiro, RA, Caser, E, Moreira, CL, Marcilino, A, Falcão, JG, Jesus, KR, Tcherniakovisk, L, Dutra, VG, Thompson, MM, Piras, C, Giuberti, J Jr, Silva, AS, Santos, JR, Potratz, JL, Paula, LN, Bozi, GG, Gomes, BC, Vassallo, PF, Rocha, E, Lima, MH, Ferreira, A F, Gonçalves, F, Pereira, SA, Nobrega, MS, Caixeta, CR, Moraes, AP, Carvalho, AG, Alves, JD, Carvalho, FB, Moreira, FB, Starling, CM, Couto, WA, Bitencourt, WS, Silva, SG, Felizardo, LR, Nascimento, FJ, Santos, D, Zanta, CC, Martins, MF, Naves, SA, Silva, FD, Laube, G Jr, Galvão, EL, Sousa, MF, Souza, MM, Carvalho, FL, Bergo, RR, Rezende, CM, Tamazato, EY, Sarat, SC Jr, Almeida, PS, Gorski, AG, Matsui, M, Neto, EE, Nomoto, SH, Lima, ZB, Inagaki, AS, Gil, FS, Araújo, MF, Oliveira, AE, Correa, TA, Mendonça, A, Reis, H, Carneiro, SR, Rego, LR, Cunha, AF, Barra, WF, Carneiro, M, Batista, RA, Zoghbi, KK, Machado, NJ, Ferreira, R, Apoena, P, Leão, RM, Martins, ER, Oliveira, ME, Odir, I, Kleber, W, Tavares, D, Araújo, ME, Brilhante, YN, Tavares, DC, Carvalho, WL, Winveler, GF, Filho, AC, Cavalcanti, RA, Grion, CM, Reis, AT, Festti, J, Gimenez, FM, Larangeira, AS, Cardoso, LT, Mezzaroba, TS, Kauss, IA, Duarte, PA, Tozo, TC, Peliser, P, Germano, A, Gurgel, SJ, Silva, SR, Kuroda, CM, Herek, A, Yamada, SS, Schiavetto, PM, Wysocki, N, Matsubara, RR, Sales, JA Jr, Laprovita, MP, Pena, FM, Sá, A, Vianna, A, Verdeal, JC, Martins, GA, Salgado, DR, Coelho, AM, Coelho, M, Morong, AS, Poquiriqui, RM, Ferreira, AP, Lucena, DN, Marino, NF, Moreira, MA, Uratani, CC, Severino, MA, Silva, PN, Medeiros, LG, Filho, FG, Guimarães, DM, Rezende, VM, Carbonell, RC, Trindade, RS, Pellegrini, JA, Boniatti, MM, Santos, MC, Boldo, R, Oliveira, VM, Corrêa, VM, Nedel, W, Teixeira, C, Schaich, F, Tagliari, L, Savi, A, Schulz, LF, Maccari, JG, Seeger, GM, Foernges, RB, Rieder, MM, Becker, DA, Broilo, FP, Schwarz, P, Alencastro, A, Berto, P, Backes, F, Dias, FS, Blattner, C, Martins, ET, Scaglia, NC, Vieira, SR, Prado, KF, Fialkow, L, Franke, C, Vieira, DF, Moraes, RB, Marques, LS, Hopf, JL, Wawrzeniak, IC, Rech, TH, Albuquerque, RB, Guerreiro, MO, Teixeira, LO, Macedo, PL, Bainy, MP, Ferreira, EV, Martins, MA, Andrade, LA, Machado, FO, Burigo, AC, Pincelli, M, Kretzer, L, Maia, IS, Cordeiro, RB, Westphal, G, Cramer, AS, Dadam, MM, Barbosa, PO, Caldeira, M, Brilenger, CO, Horner, MB, Oliveira, GL, Germiniani, BC, Duarte, R, Assef, MG, Rosso, D, Bigolin, R, Vanzuita, R, Prado, LF, Oliveira, V, Reis, DL, Morais, MO, Bastos, RS, Santana, HS, Silva, AO, Cacau, LA, Almeida, MS, Canavessi, HS, Nogueira, EE, Pavia, CL, Araujo, JF, Lira, JA, Nienstedt, EC, Smith, TC, Romano, M, Barros D, Costa, AF, Takahashi, L, Werneck, V, Farran, J, Henriques, LA, Miura, C, Lopes, RD, Vendrame, LS, Sandri, P, Galassi, MS, Amato, P, Toufen, C Jr, Santiago, RR, Hirota, AS, Park, M, Azevedo, LC, Malbouison, LM, Costa, MC, Taniguchi, L, Pompílio, CE, Baruzzi, C, Andrade, AH, Taira, EE, Taino, B, Oliveira, CS, Silva, AC, Ísola, A, Rezende, E, Rodrigues, RG, Rangel, VP, Luzzi, S, Giacomassi, IW, Nassar, AP Jr, Souza, AR, Rahal, L, Nunes, AL, Giannini, F, Menescal, B, Morais, JE, Toledo, D, Morsch, RD, Merluzzi, T, Amorim, DS, Bastos, AC, Santos, PL, Silva, SF, Gallego, RC, Santos, GD, Tucci, M, Costa, RT, Santos, LS, Demarzo, SE, Schettino, GP, Suzuki, VC, Patrocinio, AC, Martins, ML, Passos, DB, Cappi, SB, Gonçalves, I Jr, Borges, MC, Lovato, W, Tavares, MV, Morales, D, Machado, LA, Torres, FC, Gomes, TM, Cerantola, RB, Góis, A, Marraccini, T, Margarida, K, Cavalcante, E, Machado, FR, Mazza, BF, Santana, HB, Mendez, VM, Xavier, PA, Rabelo, MV, Schievano, FR, Pinto, WA, Francisco, RS, Ferreira, EM, Silva, DC, Arduini, RG, Aldrighi, JR, Amaro, AF, Conde, KA, Pereira, CA, Tarkieltaub, E, Oliver, WR, Guadalupe, EG, Acerbi, PS, Tomizuka, CI, Oliveira, TA, Geha, NN, Mecatti, GC, Piovesan, MZ, Salomão, MC, Moreno, MS, Orsatti, VN, Miranda, W, Ray, A, Guerra, A, Filho, ML, Ferreira, FH Jr, Filho, EV, Canzi, RA, Giuberti, AF, Garcez, MC, Sala, AD, Suguitani, EO, Kazue, P, Oliveira, LR, Infantini, RM, Carvalho, FR, Andrade, LC, Santos, TM, Carmona, CV, Figueiredo, LC, Falcão, A, Dragosavak, D, Filho, WN, Lunardi, MC, Lago, R, Gatti, C, Chiasso, TM, Santos, GO, Araujo, AC, Ornellas, IB, Vieira, VM, Hajjar, LA, Figueiredo, AC, Damasceno, B, Hinestrosa, A, Diaz-Quijano, FA, Raineri, SM, and Cortegiani, A

Subjects
Research design, ARDS, medicine.medical_specialty, Time Factors, Ventilator-Induced Lung Injury, Alveolar recruitment, Treatment outcome, Randomized, Medicine (miscellaneous), Settore MED/41 - Anestesiologia, Hospital mortality, law.invention, Positive-Pressure Respiration, Study Protocol, Mechanical ventilation, Clinical trials, Randomized controlled trial, Clinical Protocols, law, Medicine, Humans, Pharmacology (medical), Hospital Mortality, PEEP, Protocol (science), Respiratory Distress Syndrome, Acute respiratory distress syndrome, business.industry, respiratory system, Length of Stay, medicine.disease, Clinical trial, Pulmonary Alveoli, Intensive Care Units, Treatment Outcome, Multicenter study, Barotrauma, Research Design, Physical therapy, business, Brazil
Abstract

Background Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH2O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure ≤30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method. Trial registration ClinicalTrials.gov Identifier: NCT01374022

Published
2012

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

Author

Ricardo V, Sousa LG, Regalo IH, Pitol DL, Bombonato-Prado KF, Regalo SCH, and Siessere S

Subjects
Rats, Female, Animals, Humans, Lycopene pharmacology, Rats, Wistar, Skull, Carotenoids pharmacology, Ovariectomy adverse effects, Bone Density, Osteoporosis etiology
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.

Published
2023
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3. Low concentrations of grape seed extract maintain osteoblast morphology, cell adhesion, and mineralization.

Author

Coelho MC, Vetucci VR, Fernandes RR, Sanchez PKV, Siessere S, and Bombonato-Prado KF

Subjects
Osteopontin metabolism, Cell Adhesion, Osteogenesis, Cell Proliferation, Osteoblasts, Cell Differentiation, Alkaline Phosphatase metabolism, Grape Seed Extract pharmacology
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.

Published
2023
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4. Lycopene prevents bone loss in ovariectomized rats and increases the number of osteocytes and osteoblasts.

Author

Semeghini MS, Scalize PH, Coelho MC, Fernandes RR, Pitol DL, Tavares MS, de Sousa LG, Coppi AA, Siessere S, and Bombonato-Prado KF

Subjects
Animals, Bone Density, Epiphyses, Female, Humans, Lycopene metabolism, Lycopene pharmacology, Osteoblasts, Osteocytes, Osteogenesis, Ovariectomy, Rats, Bone Diseases, Metabolic metabolism, Osteoporosis prevention & control
Abstract

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., (© 2022 Anatomical Society.)

Published
2022
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5. miR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells.

Author

Tanaka PP, Oliveira EH, Vieira-Machado MC, Duarte MJ, Assis AF, Bombonato-Prado KF, and Passos GA

Subjects
Animals, Epithelial Cells metabolism, Mice, RNA, Messenger genetics, RNA, Messenger metabolism, AIRE Protein, MicroRNAs metabolism, Transcription Factors metabolism
Abstract

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., (© 2022. The Author(s).)

Published
2022
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6. Green tea extract rich in epigallocatechin gallate impairs alveolar bone loss in ovariectomized rats with experimental periodontal disease.

Author

Vargas-Sanchez PK, Pitol DL, de Sousa LG, Beloti MM, Rosa AL, Rossi AC, Siéssere S, and Bombonato-Prado KF

Subjects
Animals, Bone Density drug effects, Catechin analogs & derivatives, Female, Osteoporosis pathology, Periodontal Diseases pathology, Rats, Rats, Wistar, Antioxidants pharmacology, Periodontal Diseases drug therapy, Plant Extracts pharmacology
Abstract

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., (© 2020 Company of the International Journal of Experimental Pathology (CIJEP).)

Published
2020
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7. Osteoporosis Affects Functional Activity and Gene Expression of Osteoblastic Cells Derived from Rat Alveolar Bone.

Author

Vargas-Sanchez PK, Fernandes RR, Furlaneto FAC, Sousa LG, Siéssere S, and Bombonato-Prado KF

Subjects
Alkaline Phosphatase, Animals, Bone Density, Bone and Bones, Female, Humans, Osteoblasts, Ovariectomy, Rats, Osteoporosis genetics
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.

Published
2020
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8. Impact of calcium aluminate cement with additives on dental pulp-derived cells.

Author

Messias NS, Grisote G, Martorano AS, Fernandes RR, Oliveira IR, Bombonato-Prado KF, Oliveira PT, and Castro-Raucci LMS

Subjects
Alkaline Phosphatase analysis, Alkaline Phosphatase drug effects, Animals, Bismuth chemistry, Bismuth pharmacology, Calcium Chloride chemistry, Calcium Chloride pharmacology, Cell Survival drug effects, Cells, Cultured, Drug Combinations, Gene Expression drug effects, Materials Testing, Mice, Odontoblasts drug effects, Oxides chemistry, Oxides pharmacology, Reproducibility of Results, Silicates chemistry, Silicates pharmacology, Time Factors, Zinc Oxide chemistry, Zinc Oxide pharmacology, Aluminum Compounds chemistry, Aluminum Compounds pharmacology, Calcium Compounds chemistry, Calcium Compounds pharmacology, Dental Cements chemistry, Dental Cements pharmacology, Dental Pulp cytology, Dental Pulp drug effects
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.

Published
2019
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9. Cell therapy stimulates bone neoformation in calvaria defects in rats subjected to local irradiation.

Author

Sório ALR, Vargas-Sanchez PK, Fernandes RR, Pitol DL, de Sousa LG, Bianchini ALB, de Melo GB, Siessere S, and Bombonato-Prado KF

Abstract

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., Competing Interests: None., (© 2019 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Published
2019
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10. Low-level laser therapy enhances the number of osteocytes in calvaria bone defects of ovariectomized rats.

Author

Scalize PH, de Sousa LG, Gonçalves LMN, Pitol DL, Palinkas M, Coppi AA, Righeti MA, Ricardo V, Bombonato-Prado KF, Regalo SCH, and Siessere S

Abstract

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/cm 2 , 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/cm 2 ) ( 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/cm 2 given for 6 and 12 sessions., Competing Interests: None.

Published
2019
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11. Effect of grape seed extract (GSE) on functional activity and mineralization of OD-21 and MDPC-23 cell lines.

Author

Coelho MC, Sanchez PKV, Fernandes RR, Souza FPP, Siéssere S, and Bombonato-Prado KF

Subjects
Animals, Cell Differentiation drug effects, Cell Line, Cells, Cultured, Dentin cytology, Dentin drug effects, Mice, Odontogenesis drug effects, Reference Values, Reproducibility of Results, Time Factors, Cell Proliferation drug effects, Dental Pulp cytology, Dental Pulp drug effects, Grape Seed Extract pharmacology, Odontoblasts drug effects
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.

Published
2019
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12. Aire Disruption Influences the Medullary Thymic Epithelial Cell Transcriptome and Interaction With Thymocytes.

Author

Speck-Hernandez CA, Assis AF, Felicio RF, Cotrim-Sousa L, Pezzi N, Lopes GS, Bombonato-Prado KF, Giuliatti S, and Passos GA

Subjects
Animals, Cell Adhesion, Cell Differentiation immunology, Epithelial Cells immunology, Female, Gene Expression Profiling, Gene Expression Regulation, Mice, Mice, Inbred BALB C, Mice, Knockout, Thymocytes immunology, Thymus Gland immunology, Transcriptional Activation, AIRE Protein, Epithelial Cells metabolism, Thymocytes metabolism, Thymus Gland cytology, Transcription Factors genetics, Transcriptome
Abstract

The function of medullary thymic epithelial cells (mTECs) is associated with thymocyte adhesion, which is crucial for the negative selection of autoreactive thymocytes in the thymus. This process represents the root of central tolerance of self-components and prevents the onset of autoimmune diseases. Since thymic epithelia correspond to an important target of donor T cells during the onset of chronic graft-vs-host-disease, mTEC-thymocyte adhesion may have implications for alloimmunity. The Aire and Fezf2 genes function as transcriptome controllers in mTECs. The central question of this study is whether there is a mutual relationship between mTEC-thymocyte adhesion and the control of the mTEC transcriptome and whether Aire is involved in this process. Here, we show that in vitro mTEC-thymocyte adhesion causes transcriptome changes in mTECs and upregulates the transcriptional expression of Aire and Fezf2 , as well as cell adhesion-related genes such as Cd80 or Tcf7 , among others. Crispr-Cas9-mediated Aire gene disruption demonstrated that this gene plays a role in the process of mTEC-thymocyte adhesion. Consistent with the nuclear localization signal (NLS) encoded by Aire exon 3, which was targeted, we demonstrate that Aire KO -/- mTECs impair AIRE protein localization in the nucleus. Consequently, the loss of function of Aire reduced the ability of these cells to adhere to thymocytes. Their transcriptomes differed from their wild-type Aire +/+ counterparts, even during thymocyte adhesion. A set of mRNA isoforms that encode proteins involved in cell adhesion were also modulated during this process. This demonstrates that both thymocyte interactions and Aire influence transcriptome profiling of mTEC cells.

Published
2018
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13. Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model.

Author

Scalize PH, Bombonato-Prado KF, de Sousa LG, Rosa AL, Beloti MM, Semprini M, Gimenes R, de Almeida AL, de Oliveira FS, Hallak Regalo SC, and Siessere S

Subjects
Animals, Biocompatible Materials chemistry, Bone Regeneration, Bone Transplantation, Bone and Bones metabolism, Cathepsin K metabolism, Disease Models, Animal, Female, Gene Expression Profiling, Gene Expression Regulation, Matrix Metalloproteinase 9 metabolism, Membranes, Artificial, Osteoblasts metabolism, Osteoclasts metabolism, Osteoporosis metabolism, RANK Ligand metabolism, Rats, Rats, Wistar, Real-Time Polymerase Chain Reaction, Receptor Activator of Nuclear Factor-kappa B metabolism, Receptors, Calcitonin metabolism, X-Ray Microtomography, Barium Compounds chemistry, Hydrocarbons, Fluorinated chemistry, Osteogenesis, Osteoporosis surgery, Titanium chemistry, Vinyl Compounds chemistry
Abstract

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.

Published
2016
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14. In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells.

Author

Semeghini MS, Fernandes RR, Chimello DT, de Oliveira FS, and Bombonato-Prado KF

Subjects
Alkaline Phosphatase analysis, Animals, Cell Count, Cell Culture Techniques, Cell Differentiation drug effects, Cell Line, Cell Proliferation drug effects, Cell Survival drug effects, Core Binding Factor Alpha 1 Subunit analysis, Culture Media, Extracellular Matrix Proteins analysis, Mice, Odontoblasts drug effects, Osteopontin analysis, Phosphoproteins analysis, Proteins analysis, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Sp7 Transcription Factor, Time Factors, Tooth Calcification drug effects, Transcription Factors analysis, Dental Pulp cytology, Odontogenesis physiology
Abstract

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.

Published
2012
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