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Universidade Estadual De Campinas Faculdade De Ciências Médicas Carlos Vinícius Buarque De Gusmão Efeito Do Ultrassom, Ondas

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Universidade Estadual De Campinas Faculdade De Ciências Médicas Carlos Vinícius Buarque De Gusmão Efeito Do Ultrassom, Ondas UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE CIÊNCIAS MÉDICAS CARLOS VINÍCIUS BUARQUE DE GUSMÃO EFEITO DO ULTRASSOM, ONDAS DE CHOQUE E DE PRESSÃO RADIAL NO REPARO DE DEFEITOS ÓSSEOS NAS TÍBIAS DE RATOS AVALIADO PELA EXPRESSÃO E ATIVIDADE DE AKT, BMP-2, ERK-2, FAK E TGF-β1 EFFECT OF ULTRASOUND, EXTRACORPOREAL SHOCKWAVES AND RADIAL PRESSURE WAVES ON AKT, BMP-2, ERK-2, FAK AND TGF-β1 DURING BONE HEALING IN RAT TIBIAL DEFECTS CAMPINAS 2018 CARLOS VINÍCIUS BUARQUE DE GUSMÃO EFEITO DO ULTRASSOM, ONDAS DE CHOQUE E DE PRESSÃO RADIAL NO REPARO DE DEFEITOS ÓSSEOS NAS TÍBIAS DE RATOS AVALIADO PELA EXPRESSÃO E ATIVIDADE DE AKT, BMP-2, ERK-2, FAK E TGF-β1 EFFECT OF ULTRASOUND, EXTRACORPOREAL SHOCKWAVES AND RADIAL PRESSURE WAVES ON AKT, BMP-2, ERK-2, FAK AND TGF-β1 DURING BONE HEALING IN RAT TIBIAL DEFECTS Tese apresentada à Faculdade de Ciências Médicas da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutor em Ciências. Thesis presented to the Faculty of Medical Sciences of the State University of Campinas as part of the requirements to obtain the title of Doctor in Sciences. ORIENTADOR: PROF. DR. WILLIAM DIAS BELANGERO ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELO ALUNO CARLOS VINÍCIUS BUARQUE DE GUSMÃO, E ORIENTADO PELO PROF. DR. WILLIAM DIAS BELANGERO. CAMPINAS 2018 Agência(s) de fomento e nº(s) de processo(s): CAPES, 02-P-3369/2017; FAPESP, 2014/26729-0 Ficha catalográfica Universidade Estadual de Campinas Biblioteca da Faculdade de Ciências Médicas Maristella Soares dos Santos - CRB 8/8402 Gusmão, Carlos Vinícius Buarque de, 1986- G972e Efeito do ultrassom, ondas de choque e de pressão radial no reparo de defeitos ósseos nas tíbias de ratos avaliado pela expressão e atividade de Akt, BMP-2, ERK-2, FAK e TGF-b1 / Carlos Vinícius Buarque de Gusmão. – Campinas, SP : [s.n.], 2018. Orientador: William Dias Belangero. Tese (doutorado) – Universidade Estadual de Campinas, Faculdade de Ciências Médicas. 1. Osteogênese. 2. Ondas ultrassônicas. 3. Fraturas ósseas. 4. Mecanotransdução celular. 5. Ondas de choque. I. Belangero, William Dias, 1952-. II. Universidade Estadual de Campinas. Faculdade de Ciências Médicas. III. Título. Informações para Biblioteca Digital Título em outro idioma: Effect of ultrasound, extracorporeal shockwaves and radial pressure waves on Akt, BMP-2, ERK-2, FAK and TGF-b1 during bone healing in rat tibial defects Palavras-chave em inglês: Osteogenesis Ultrasonic waves Mechanotransduction, Cellular Mechanotransduction Shock waves Área de concentração: Fisiopatologia Cirúrgica Titulação: Doutor em Ciências Banca examinadora: William Dias Belangero [Orientador] Fernando Baldy dos Reis José Marcos Alves Ana Amélia Rodrigues José Barreto Campello Carvalheira Data de defesa: 27-02-2018 Programa de Pós-Graduação: Ciências da Cirurgia BANCA EXAMINADORA DA DEFESA DE DOUTORADO CARLOS VINÍCIUS BUARQUE DE GUSMÃO ORIENTADOR: PROF. DR. WILLIAM DIAS BELANGERO MEMBROS: 1. PROF. DR. WILLIAM DIAS BELANGERO 2. PROF. DR. FERNANDO BALDY DOS REIS 3. PROF. DR. JOSÉ MARCOS ALVES 4. PROFª. DRª. ANA AMÉLIA RODRIGUES 5. PROF. DR. JOSÉ BARRETO CAMPELLO CARVALHEIRA Programa de Pós-Graduação em Ciências da Cirurgia da Faculdade de Ciências Médicas da Universidade Estadual de Campinas. A ata de defesa com as respectivas assinaturas dos membros da banca examinadora encontra-se no processo de vida acadêmica do aluno. Data: 27/02/2018 DEDICATÓRIA Àqueles que buscam o conhecimento para aliviar as chagas do próximo; Àqueles que trabalham e estudam para beneficiar a humanidade; Àqueles que se felicitam pelo bem produzido; E aos humildes de coração. AGRADECIMENTOS Deus, princípio de tudo e Quem nos permite existir e gozar da existência; Carlos Alberto Buarque de Gusmão e Maria Angelica Barcellos de Gusmão, pai e mãe, genitores, educadores, trabalhadores, exemplos, motivadores...; Prof. Dr. William Dias Belangero, meu orientador desde o 1º ano da graduação, que me ensinou os caminhos da ciência, e ensina ainda hoje; Nilza Alzira Batista, amiga, conselheira, companheira, cuja presença foi fundamental para todo o desenrolar da tese; Valéria Trombini Vidotto Lemes e Wilson Leite Maia Neto, amigos que auxiliaram valiosamente para a produção dos resultados; Prof. Dr. José Marcos Alves, que auxiliou com equipamentos e literatura de precioso valor. Prof. Dr Orlando Petrucci Junior, que cedeu gentilmente as dependências de seu laboratório para execução de parte dos experimentos. Antonio Ramos Calixto, Cristina Tanikawa, Daniela Diogenes de Carvalho, Isabella Bonilha de Oliveira e José Ricardo Lenzi Mariolani sempre dispostos para auxiliar quanto às dúvidas e atividades experimentais. Amigos muitos, nem sempre visíveis e próximos fisicamente, que me auxiliam desde há muito tempo. A FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), processo n° 2014/26729-0 pelo financiamento desta pesquisa como parte do Auxílio Regular intitulado “Estudo Biomecânico e Biomolecular de Métodos Terapêuticos de Regeneração Óssea”. A CAPES (Coordenação de Aperfeiçoamento Pessoal de Nível Superior) pela concessão de bolsa de doutorado (processo 02-P-3369/2017). RESUMO Ondas acústicas, como ultrassom pulsado de baixa intensidade, ondas de choque e ondas de pressão radial interferem na osteogênese por meio da modulação da expressão e do perfil de atividade de Akt, BMP-2 (bone morphogenetic protein-2), ERK-2 (extracellular signal-regulated kinase-2), FAK (focal adhesion kinase) e TGF- β1 (transforming growth factor-β1). Apesar do uso habitual na prática médica, não está claro se as terapias acústicas aceleram o reparo de fraturas agudas. Ademais, nenhum estudo comparou o efeito do ultrassom, ondas de choque e de pressão radial na modulação da expressão e do perfil de atividade de Akt, BMP-2, ERK-2, FAK e TGF-β1 durante o reparo ósseo. A presente tese foi desenvolvida para determinar se o ultrassom, as ondas de choque e as de pressão radial modulam a expressão e o perfil de atividade de Akt, BMP-2, ERK-2, FAK e TGF-β1 durante o reparo de defeitos ósseos produzidos em tíbias de ratos; e, caso afirmativo, se a modulação proteica difere entre as terapias acústicas. Para tanto, defeitos ósseos nas tíbias de ratos foram expostos a uma sessão de 500 pulsos e 0.12mJ/mm2 de ondas de choque, uma sessão de 500 pulsos e 2.0 bar de ondas de pressão radial, ou sessões diárias de 20 minutos de ultrassom pulsado a 30mW/cm2. Após seguimento de 1, 3 e 6 semanas, o tecido ósseo foi coletado para análise da expressão e perfil de atividade de Akt, BMP-2, ERK-2, FAK e TGF-β1. Os animais expostos ao ultrassom foram acompanhados por até 3 semanas. Identificou-se que o ultrassom pulsado de baixa intensidade modulou a expressão de BMP-2 na terceira semana; as ondas de choque modularam, na primeira semana, a expressão de BMP-2 e TGF-β1, e o perfil de atividade de Akt e FAK; e as ondas de pressão radial modularam o perfil de atividade de FAK na terceira semana. Com base nesses achados, conclui-se que, durante o reparo de defeitos ósseos produzidos em tíbias de ratos, o ultrassom pulsado de baixa intensidade, as ondas de choque e as de pressão radial modularam, de maneira distinta, a expressão e o perfil de atividade de Akt, BMP-2, FAK e TGF-β1, mas não de ERK-2. Além disso, entre as terapias avaliadas, as ondas de choque exibiram o efeito mais expressivo na modulação proteica. Palavras-chave: Akt; Fator de crescimento transformador beta; Fraturas ósseas; Mecanotransdução; Ondas de choque; Ondas ultrassônicas; Osteogênese; Proteína morfogenética óssea; Quinase de adesão focal; Quinase 2 regulada por sinal extracelular; Tecido ósseo. ABSTRACT Low-intensity pulsed ultrasound (LIPUS), extracorporeal shockwaves (ESWT) and radial pressure waves (RPWT) influence osteogenesis via Akt, BMP-2 (bone morphogenetic protein-2), ERK-2 (extracellular signal-regulated kinase-2), FAK (focal adhesion kinase) and TGF-β1 (transforming growth factor-β1) modulation. In spite of that, it is not clear whether those acoustic waves are effective to accelerate fracture healing. Additionally, to our knowledge no study compared the effect of different acoustic therapies on Akt, BMP-2, ERK-2, FAK and TGF-β1 during bone healing. An experimental study was conducted to determine whether LIPUS, ESWT and RPWT modulate Akt, BMP-2, ERK-2, FAK and TGF-β1 during bone healing in rat tibial defects; and, if so, to compare the results between the different modalities of acoustic therapies. Rat tibial defects were exposed to 500 shots of ESWT delivered at 0.12mJ/mm2, 500 impulses of RPWT operated at 2.0 bar, or daily 20-minute 30mW/cm2 sonication. Following 1, 3 and 6 weeks, bones were harvested to determine the expression and activity of Akt, BMP-2, ERK-2, FAK and TGF-β1. Animals exposed to ultrasound were followed up to 3 weeks. It was demonstrated that LIPUS modulates BMP-2 expression at three weeks; ESWT modulate Akt and FAK activity profiles, and the expression of BMP-2 and TGF- β1 at one week; and RPWT modulate FAK activity profile at 3 weeks. In conclusion, LIPUS, ESWT and RPWT exhibit distinct mechanisms to modulate Akt, BMP-2, FAK and TGF-β1, but not ERK-2 – which is not modulated –, during bone healing in rat tibial defects. Keywords: Akt; Bone; Bone morphogenetic protein; Extracellular signal-regulated kinase; Focal adhesion kinase; Fractures; LIPUS; Mechanotransduction; Osteogenesis; Shockwave; Transforming growth factor beta. LISTA DE ABREVIATURAS E SIGLAS ActR: Activin Receptor ATP: Adenosine Triphosphate BMP: Bone Morphogenetic Protein BMPR: BMP Receptor BSA:
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