UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
CILENE CRISTINA CERRI AHMAD
CARACTERÍSTICAS MECÂNICAS DO TECIDO ÓSSEO DO FÊMUR
DE RATOS WISTAR JOVENS E ADULTOS
MECHANICAL CHARACTERISTICS OF FEMUR BONE FROM
YOUNG AND ADULT WISTAR RATS
Piracicaba 2019
CILENE CRISTINA CERRI AHMAD
CARACTERÍSTICAS MECÂNICAS DO TECIDO ÓSSEO DO
FÊMUR DE RATOS WISTAR JOVENS E ADULTOS
MECHANICAL CHARACTERISTICS OF FEMUR BONE FROM
YOUNG AND ADULT WISTAR RATS
Dissertação apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Mestra em Biologia Buco-Dental, na Área de Anatomia.
Dissertation presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Master in Oral Biology, in Anatomy area.
Orientador: Prof. Dr. Felippe Bevilacqua Prado
ESTE EXEMPLAR CORRESPONDE A VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA CILENE CRISTINA CERRI AHMAD ORIENTADA PELO PROF. DR. FELIPPE BEVILACQUA PRADO.
Piracicaba 2019
DEDICATÓRIA
Ao montar qualquer trabalho de pesquisa e tentar compor, devemos agradecer primeiramente às oportunidades, e estimular outros colegas a cada dia reportar experimentos e torná-los conhecidos.
Acredito que todos que se interessam pela ciência, estudo e conhecimento não se decepcionarão.
Este tema tratado é só um início. Sempre as informações aqui contidas são, serão, ou foram úteis a alguém, com objetivo de evolução e superação de técnicas e dificuldades.
Agradeço para tal feito a Deus e minha família, que são meu esteio e incentivo para tudo.
AGRADECIMENTOS
À Universidade Estadual de Campinas, na pessoa do Magnifico Reitor Prof. Dr. Marcelo Knobel.
À Faculdade de Odontologia de Piracicaba, na pessoa do Senhor Diretor, Prof. Dr. Francisco Haiter Neto.
A Coordenadoria de Pós Graduação, na figura da Senhora Coordenadora Prof. Dr.ª Karina Gonzales Silvério Ruiz.
Ao programa de pós graduação em Biologia Buco-dental, na figura da coordenadora Prof ª. Dr.ª Ana Paula de Souza.
Ao meu orientador, Prof. Dr. Felippe Bevilacqua Prado, por ter acreditado. A professora Prof ª. Dr.ª Ana Claudia Rossi, por colaborar no delineamento do projeto e fornecer os modelos animais.
Ao professor Dr. Alexandre Rodrigues Freire, pela enorme colaboração com o desenvolvimento do trabalho e simulações computacionais.
Aos servidores da biblioteca da FOP-UNICAMP.
A aqueles que mesmo de forma direta e indireta colaboraram com este estudo.
RESUMO
Comparar os parâmetros da microarquitetura óssea do fêmur de ratos em diferentes fases da vida ("jovem" e "adulto") utilizando o cálculo computacional. Serão utilizados seis ratos Wistar, separados em dois grupos: Grupo Jovem com 3 ratos (60 dias de vida) e Grupo Adulto com 3 ratos (210 dias de vida), pois atingem a maturidade do músculo esquelético. Os ratos "jovens" foram eutanasiados e os fêmures direitos em cada rato foram retirados com 60 dias de vida e os ratos adultos foram eutanasiados e os fêmures direitos em cada rato foram removidos com eutanásia de 210 dias e o fêmur direito foi removido. O software CTAnalyzer foi utilizado para realizar os cálculos. A estrutura óssea do fêmur de ratos wistar "normais" foi avaliada pelo volume ósseo (VB), porcentagem de volume ósseo (Bv / Tv), espessura trabecular (Tb.Th), número trabecular (Tb.N), porosidade total ( Po (tot)), porcentagem de porosidade (Po) e volume total de espaço poroso (Po.V (tot)), uma vez que esses parâmetros estão diretamente relacionados à densidade mineral do tecido ósseo. No presente estudo, os resultados das propriedades mecânicas do tecido ósseo do fêmur de ratos jovens e adultos mostraram que a variável idade não está associada ao volume ósseo (VB), Volume ósseo percentual (Bv / Tv), Espessura trabecular. Th) e Número trabecular (Tb.N), Porosidade total (por cento) (Po (tot)) e Volume total do espaço poroso (Po.V (tot)). As informações obtidas podem servir de base para a caracterização do tecido ósseo e mais importante para futuros experimentos informatizados, especialmente com a Análise de Elementos Finitos (AEF).
ABSTRACT
To compare the bone micro arquitecture parameters of the femur of rats at different stages of life ("young" and "adult") using the computational calculation. Six Wistar rats will be used, separated into two groups: Young Group with 3 rats (60 days of life) and Adults Group with 3 rats (210 days of life) were used, because they reach skeletal muscle maturity. The "young" rats were euthanized and the right femurs in each rat were removed with 60 days of life and the adult rats were euthanized and the right femurs in each rat were removed with 210-day-old euthanasia and the right femur were removed. The CTAnalyzer software were used to perform the calculations. Student's t-test were used to compare the information obtained in the two groups. the bone structure of the femur of "normal" wistar rats was evaluated by bone volume (BV), percentage of bone volume (Bv / Tv), trabecular thickness (Tb.Th), trabecular number (Tb.N ), total porosity (Po (tot)), percentage of porosity (Po) and total volume of pore space (Po.V (tot)), since these parameters are directly related to mineral density of bone tissue. In the present study, the results of the mechanical properties of the bone tissue of the femur of young and adult rats showed that the age variable is not associated with Bone volume (BV), Percent bone volume (Bv / Tv), Trabecular thickness .Th) and Trabecular number (Tb.N), Total porosity (percent) (Po (tot)) and Total volume of pore space (Po.V (tot)). The information obtained may serve as a basis for characterizing the bone tissue and more important for future computerized experiments, especially with Finite Element Analysis (AEF).
SUMÁRIO
1 INTRODUÇÃO...10 2 Artigo: CARACTERÍSTICAS MECÂNICAS DO TECIDO ÓSSEO DO FÊMUR DE RATOS WISTAR JOVENS E ADULTOS...12 3 Conclusão...23 Referências...24 Anexos
Anexo 1 - COMPROVANTE DE SUBMISSÃO DO ARTIGO NA REVISTA...25 Anexo 2 - COMPROVANTE DO COMITÊ DE ÉTICA - CEUA/UNICAMP...26 Anexo 3 - COMPROVANTE DO SOFTWARE ANTI-PLÁGIO...27
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1 INTRODUÇÃO
Os estudos com Modelos de Elementos Finitos e as simulações computacionais dão condições para a avaliação de situações que de outra maneira não poderiam ser alcançadas por estudos envolvendo modelos em vivo. Esses ensaios in sílico biomecânicos computadorizados utilizam de parâmetros que dependem da características dos materiais biológicos para que possamos melhor compreender a ação de forças e suas interações com tais estruturas (Prado et al., 2016).
A quantificação por meio de critérios da estrutura arquitetônica óssea esponjosa e cortical pode ser executada por metodologias por assim dizer que destroem os próprios tecidos, ou seja, destrutivas, como o caso da histomorfometria convencional, ou metodologias chamada de não destrutiva, nesse caso as técnicas de ressonância magnética e a micro tomografia computadorizada tridimensional (MicroTC 3D) (Karatas e toy, 2014).
Para a avaliação quantitativa ou caracterização da estrutura histomorfometrica óssea esponjosa ou trabecular são empregados cinco básicos parâmetros: a razão entre o volume do tecido ósseo e o volume total da amostra (BV/TV - %), o número de trabéculas ósseas por milímetro de tecido, significando além disso um número que noticia a densidade trabecular (TbN - 1/mm), a razão entre a grau da área superficial do osso e o seu volume (BS/BV - %), a espessura trabecular (TbTh - mm) e a separação trabecular (TbSp - mm). A obtenção desses parâmetros do tecido ósseo de ratos wistar jovens e adultos por si parecer ser uma contribuição para o desenvolvimento de futuras simulações computacionais e para o entendimento de diversas situações clínica da área médica e odontológica, como, por exemplo, fraturas e traumas em ossos longos e da face, instalação de implantes ósseo integrados, reparo tecidual ósseo, entre outras (Daegling e Hylander, 2000; Prado et al., 2016; Pryor et al., 2016).
Dessa maneira, o conhecimento da interna arquitetura do osso trabecular e esponjoso faz-se necessária melhorar a compreensão do comportamento do tecido ósseo normal em jovens e adultos e o fornecimento de dados sobre as características do tecido biológico para as futuras pesquisas onde são realizados ensaios computadorizados como por exemplo a análise de elementos finitos.
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Diante do exposto o objeto deste estudo foi determinar as propriedades mecânicas do tecido ósseo do fêmur de ratos jovens e adultos, uma vez que esse modelo animal e um dos mais utilizados e vem ganhando força como um método in silico para criação de hipótese para os procedimentos clínicos odontológicos e cirúrgicos da maxilo mandibulares.
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2 ARTIGO: MECHANICAL CHARACTERISTICS OF FEMUR BONE FROM YOUNG AND ADULT WISTAR RATS
Artigo submetido ao periódico Journal of Applied of Oral Science (Anexo 1).
Authors: Cilene Cristina Cerri Ahmad1, Alexandre Rodrigues Freire1, Ana Cláudia Rossi1, Felippe Bevilacqua Prado1
1 Department of Biociences, Anatomy Division, Piracicaba Dental School, University of Campinas, Piracicaba, São Paulo, Brazil.
Corresponding author’s email address: Prof. Dr. Felippe Bevilacqua Prado
Avenida Limeira, n / 901 – Areião. Piracicaba, São Paulo – Brazil. Tel: +55 19 21065721 Postal Code: 13414-903
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ABSTRACT
To compare the bone micro arquitecture parameters of the femur of rats at different stages of life ("young" and "adult") using the computational calculation. Six Wistar rats will be used, separated into two groups: Young Group with 3 rats (60 days of life) and Adults Group with 3 rats (210 days of life) were used, because they reach skeletal muscle maturity. The "young" rats were euthanized and the right femurs in each rat were removed with 60 days of life and the adult rats were euthanized and the right femurs in each rat were removed with 210-day-old euthanasia and the right femur were removed. The CTAnalyzer software were used to perform the calculations. Student's t-test were used to compare the information obtained in the two groups. the bone structure of the femur of "normal" wistar rats was evaluated by bone volume (BV), percentage of bone volume (Bv / Tv), trabecular thickness (Tb.Th), trabecular number (Tb.N ), total porosity (Po (tot)), percentage of porosity (Po) and total volume of pore space (Po.V (tot)), since these parameters are directly related to mineral density of bone tissue. In the present study, the results of the mechanical properties of the bone tissue of the femur of young and adult rats showed that the age variable is not associated with Bone volume (BV), Percent bone volume (Bv / Tv), Trabecular thickness .Th) and Trabecular number (Tb.N), Total porosity (percent) (Po (tot)) and Total volume of pore space (Po.V (tot)). The information obtained may serve as a basis for characterizing the bone tissue and more important for future computerized experiments, especially with Finite Element Analysis (AEF).
Keywords: Femur; Rats; Computed tomography.
INTRODUCTION
Computed tomography (micro-CT) has become a gold standard and important tool for the 3D bone structure analysis [1-3], even though there are still "good" correlations between histomorphometric and microtomographic analysis [4,5].
Currently the micro-CT has been widely used in science to evaluate the characteristics of the trabecular bone microstructure in different age groups [6-7] and in diverse anatomical structure and locations [8-9]. In addition to studies targeting the age variable, the micro-CT has been used to assess the micro-architectural changes related to different mechanical tests [10-11].
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Few micro-CT few studies mention the effect of age and sex on the microstructure of the trabecular bone structure [12,13] and the scientific literature did not allow us to find any combination of age and sex in Wister rats femur.
It is necessary to improve the understanding of the behavior of normal bone tissue in young and adult rats and provide specific data about the characteristics of biological tissue for future research where computerized tests such as finite element analysis are performed.
The aim of this study was to determine the mechanical properties of the bone tissue of the femur of young and adult rats.
MATERIAL AND METHODS
ANIMAL MODEL
All procedures performed on the animals were approved (protocol CEUA 5206-1 / 205206-19) by the Animal Research Ethics Committee of the State University of Campinas (Anexo 2).
For this project the right femurs of the rats of the 2 groups, young and adult at different stages of life ("young" and "adult") were used using to calculate the mechanical properties of bone by three-dimensional computerized micro tomography (Microtc-3D).
Six Wistar rats were separated into two groups: Young, with three "young" rats (60 days of life) and three adult rats (210 days of life) were used, because they reached skeletal muscle maturity (14). Young rats were euthanized and the right and left femurs were removed 60 days old and the adult rats were euthanized and the right and left femurs were removed at 210 days of age. During the whole procedure, the animals were kept in the FOP / UNICAMP School of Dentistry of Piracicaba. Euthanasia was performed at the School of Dentistry of Piracicaba. Euthanasia was performed through the over-dosing of ketamine (150mg / kg, intravenous). Ketamine (50mg / kg) were administered intravenously, with triple the inducing dose and Xylazine: 2% - 10 to 13Mg / kg of intraperitoneal (I.P) weight. Xilasin will be twice the dose. The femurs were removed with the aid of appropriate instruments after euthanasia of the rats. Femurs were stored in sterile hydrated gauze in saline and stored at 20 ° C. After storage, the femurs were brought to the Department of Morphology of the FOP / UNICAMP, where they were scanned in a computerized micro-computerized tomograph SkyScan 1174 (SkyScan, Belgium) with 50kV and 800mA, and a voxel size of 30 micrometers, bone
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density, shape and volume of bone. The femurs were scanned in the micrograph of the conic beam SkyScan 1176 (SkyScan, Kontich, Belgium).
The images projected on the computerized microtomography were reconstructed using the Skyscan software. The trabecular bone compartment in the right femoral diaphysis region was segmented into 100 slices in a region of approximately 0.5mm. Starting below the condyle crest of the femur in the most central portion of the shaft in each animal. The same procedure was performed in the femoral neck region now using 20 slices. The images were binarized (90-255 threshold).
The following mechanical parameters were analyzed by three-dimensional computerized microtomography (Microtc-3D) for the femoral neck and diaphysis of "young and adult" rats, respectively:
Femur Neck: Bone volume (BV), Percent bone volume (Bv/Tv), Trabecular thickness (Tb.Th) and Trabecular number (Tb.N).
Diaphysis: Bone volume (BV), Total porosity (percent) (Po(tot)) and Total volume of pore space (Po.V(tot)).
STATISCTICAL ANALYSIS
The mechanical characteristics data were tabulated and analyzed by the GraphPAD Prism Software using the Unpaired Student T test with p value: P <0.05.
RESULTS
Femur Neck:
Figure 1 shows the results of the comparison of the micrographs parameters Bone Volume (BV), Percent Bone Volume (Bv / Tv), Trabecular Thickness (Tb.Th) and Trabecular Number (Tb.N) in the neck region of the femur of "Young" and "Adult" rats showed no significant differences (p: 0.5083) between the groups.
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Figure 1 Graph of trabecular microarchitecture variables for the femoral neck region. Data extracted from the right femur of Wistar rats. Results of non-parametric comparison between "Young" and "Adults". P <0.001.
Femur Diaphysis:
Figure 2 shows the results of the comparison of the micro-tomographic parameters, volume (BV), Total porosity (Po (tot)) and Total volume of pore space (Po.V (tot)) in the femoral diaphysis region of "Young" and "Adult" rats showed no significant differences (p: 0.5083) between the groups.
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Figure 2: Graph of the variables of the trabecular microarquitetura for the femoral diaphysis region. Data extracted from the right femur of Wistar rats. Results of non-parametric comparison between "Young" and "Adults". P <0.001.
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DISCUSSION
The structure of a long bone consists of many parts; proximal and distal epiphysis, the spongy bone and the diaphysis constituted by the medullary cavity, endosteum, periosteum and nutrient foramen. Thus the anatomical structure of the long bone is divided into two main parts, the epiphysis and the diaphysis, the first is the widest section at each end of the bone and the second, makes up the greater part of the length of the bone. This is the main difference between the epiphysis and the diaphysis. The regions of the diaphysis and the epiphysis of the femur present distinct anatomical conformation as well as their cortical and spongy bone formation, which can lead to information differences as to their mechanical characteristics. Within this context, those on the characteristics or properties of these structures are fundamental and to a certain extent primordial for the accomplishment of future computerized biomechanical in silico studies that increasingly require parameters and data of the mechanical properties of the biological structures in order to understand aa relationship between forces and biological structure.
The acquisition of data and mechanical parameters of the bone tissue of young and adult wistar rats is not always easy and the literature is still very scarce when it comes to studies of the mechanical properties of the bone of the rats, mainly young, and "Adults" without systemic changes or influences (15,16).
In this context, the present study may contribute to the increase of computational simulations through Finite Element Analysis with the incorporation of new data of the mechanical characteristics of the bone tissue of young and adult rats. Furthermore, understanding different medical or dental clinical circumstances, such as fractures and / or trauma to long bones or face, implanting of bone implants or prostheses, bone tissue repair, among other modalities, may be facilitated with introduction of new data on the mechanical characteristics of rat biological tissue, since this is the animal model currently used by many scientists.
Our results present new findings on the mechanical properties of the bone tissue of the femur of "young and adult" wistar rats "normal", that is, without systemic alterations. We chose to evaluate the mechanical properties of the bone tissue of wistar rats of different ages and without systemic alterations because there is a need to start the bone model in order to understand the characteristics of the bone tissue differently than most of studies and Finite Element Analysis (17).
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In the present study, the bone structure of the femur of "normal" wistar rats was evaluated by bone volume (BV), percentage of bone volume (Bv / Tv), trabecular thickness (Tb.Th), trabecular number (Tb.N ), total porosity (Po (tot)), percentage of porosity (Po) and total volume of pore space (Po.V (tot)), since these parameters are directly related to mineral density of bone tissue. In the present study, the results of the mechanical properties of the bone tissue of the femur of young and adult rats showed that the age variable is not associated with Bone volume (BV), Percent bone volume (Bv / Tv), Trabecular thickness .Th) and Trabecular number (Tb.N), Total porosity (percent) (Po (tot)) and Total volume of pore space (Po.V (tot)). Some authors, however, show that both mechanical loads as well as the age variable are factors that influence the characteristics of the bone structural properties of the rats (18, 19).
It is necessary to consider that in the present study, young Wistar rats, 2 months old and 6 months old, were used. The results of the mechanical properties of the femoral bone tissue of these groups in the present study when compared showed that advancing age is not associated with Bone volume (BV), Percent bone volume (Bv / Tv), Trabecular thickness (Tb. Th) and Trabecular number (Tb.N), Total porosity (percent) (Po (tot)) and Total volume of pore space (Po.V (tot)). The results presented by Dominguez et al. 2009 are both interesting and different from the present study. These authors noted that aging is associated with low values for the trabecular bone volume (BV / TV,%) = percentage bone volume (Bv / Tv) parameter for the trabecular bone volume in the distal femoral region, While the number of trabecular bone trabeculae (Trabecular number Tb.N) decreased in the elderly animals (17).
In both the present study and the Dominguez study et al. 2009, the mineral density of trabecular bone (trabecular bone mineral density) did not show significant differences with advancing age. It is important to emphasize before comparisons, that in the study by Domingues JM et al 2009 young adult rats of the Fischer-344 line of 4 to 6 months of age and elderly rats with 24-26 months of age were used. Both the age differences and the different lineages of rats should be taken into account in the present context, since they are variables that can contribute to the differences in the findings of both studies.
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CONCLUSION
The bony tissue of the proximal epiphysis and femoral diaphysis of healthy young and adult rats show no differences in the mechanical properties bone tissue.
The data of the present study could be used for future researches and computerized tests such as finite element analysis.
REFERENCES
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2 Kim, D.G., Christopherson, G.T., Dong, X.N., et al. (2004) The effect of microcomputed tomography scanning and reconstruction voxel size on the accuracy of stereological measurements in human cancellous bone. Bone, 34, 1375-1382
3 Bouxsein, M.L., Boyd, S.K., Christiansen, B.A., et al. (2010) Guidelines for assessment of bone microstructure in Rodents using micro-computed tomography. Journal of Bone and Mineral Research, 25, 1468-1486.
4 Tamminen, I.S., Isaksson, H., Aula, A.S., et al. (2011) Reproducibility and agreement of micro-CT and histomorphometry in human trabecular bone with different metabolic status. Journal of Bone and Mineral Metabolism, 29, 442-448.
5 Müller, R., Van Campenhout, H., Van Damme, B., et al. (1998) Morphometric analysis of human bone biopsies: A quantitative structural comparison of histological sections and micro-computed tomography. Bone, 23, 59-66.
6 Djuric, M., Djonic, D., Milovanovic, P., et al. (2010) Region-specific sex-dependent pattern of age-related changes of proximal femoral cancellous bone and its implications on differential bone fragility. Calcified Tissue International, 86, 192-201.
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8 Cui, W.Q., Won, Y.Y., Baek, M.-H., et al. (2008) Age- and region-dependent changes in three-dimensional microstructural properties of proximal femoral trabeculae. Osteoporosis International, 19, 1579-1587.
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9 Chen, H., Zhou, X., Shoumura, S., Emura, S. and Bunai, Y. (2010) Age- and gender-dependent changes in three-dimensional microstructure of cortical and trabecular bone at the human femoral neck. Osteoporosis International, 21, 627-636.
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3 CONCLUSÃO
O tecido ósseo da epífise proximal e diáfise de fêmur de ratos sadios “jovens e adultos” não apresentam diferenças nas propriedades mecânicas do tecido ósseo do fêmur.
Os dados do presente estudo poderão ser utilizados para futuras pesquisas e ensaios computadorizados como a análise de elementos finitos.
Esta pesquisa melhorar a compreensão do comportamento do tecido ósseo normal em “jovens e adultos” fornecendo dados para pesquisas futuras onde serão realizados ensaios computadorizados biomecânicos como a análise de elementos finitos, e consequentemente a compreensão da ação de forças e suas interações com o tecido biológico nas situações clinicas.
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2. Karatas OH, Toy E. Three-dimensional imaging techniques: A literature review. Eur J Dent. 2014 Jan;8(1):132-40.
3. Prado FB, Freire AR, Cláudia Rossi A, Ledogar JA, Smith AL, Dechow PC, Strait DS, Voigt T, Ross CF. Review of In Vivo Bone Strain Studies and Finite Element Models of the Zygomatic Complex in Humans and Nonhuman Primates: Implications for Clinical Research and Practice. Anat Rec (Hoboken). 2016 Dec;299(12):1753-1778.
4. Pryor McIntosh L, Strait DS, Ledogar JA, Smith AL, Ross CF, Wang Q, Opperman LA, Dechow PC. Internal Bone Architecture in the Zygoma of Human and Pan. Anat Rec (Hoboken). 2016 Dec;299(12):1704-1717.
*De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of Medical Journal Editors – Vancouver Group. Abreviatura dos periódicos em conformidade com o PubMed.
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