i
UNIVERSIDADE POSITIVO
DOUTORADO EM ODONTOLOGIA
EFEITO DO ALENDRONATO NO DESENVOLVIMENTO DA FACE: UM
ESTUDO HISTOMÉTRICO E IMUNOHISTOQUÍMICO
SHAIENE PATRICIA GOMES
CURITIBA 2019
i
UNIVERSIDADE POSITIVO
DOUTORADO EM ODONTOLOGIA
EFEITO DO ALENDRONATO NO DESENVOLVIMENTO DA FACE: UM
ESTUDO HISTOMÉTRICO E IMUNOHISTOQUÍMICO
SHAIENE PATRICIA GOMES
Tese apresentada à Universidade
Positivo como requisito parcial para obtenção do título de Doutor, pelo programa de Doutorado em Odontologia.
Orientador: Prof. Dr. Allan Fernando Giovanini
CURITIBA 2019
ii
Dados Internacionais de Catalogação na Publicação (CIP) Biblioteca da Universidade Positivo - Curitiba – PR
Elaborada pela Bibliotecária Damaris Cardoso de Oliveira Vieira (CRB-9/201803/P)
G633 Gomes, Shaiene Patricia.
Efeito do alendronato no desenvolvimento da face : um estudo histométrico e imunohistoquímico / Shaiene Patricia Gomes. ― Curitiba : Universidade Positivo, 2019.
57 f. : il.
Tese (Doutorado) – Universidade Positivo, Programa de Pós-graduação em Odontologia, 2019.
Orientador: Prof. Dr. Allan Fernando Giovanini.
1. Odontologia. 2. Alendronato - odontologia. 3. Palato.
I. Giovanini, Allan Fernando. II. Título. CDU 616.315-007(043.2)
iv
Este trabalho de pesquisa foi realizado no Laboratório de Histopatologia da Universidade
iv
DEDICATÓRIA
v
AGRADECIMENTOS
Primeiramente, agradeço a minha família: meus pais e irmã, que sempre me
apoiaram incondicionalmente, que seguramente são os que mais compartilham da
minha alegria.
Agradeço ao meu grande exemplo de esforço e dedicação, Prof. Allan Giovanini.
Tenho muito orgulho de ter sido sua aluna durante todos esses anos. Obrigada por
acreditar em mim. Jamais esquecerei seus eternos ensinamentos e seus preciosos
conselhos.
Manifesto aqui a minha gratidão a todos os colegas da empresa em que trabalho,
Straumann Group – Neodent.
As minhas amigas Heloisa, Jocieli, Karol e Patricia, que sempre me transmitiam
força para esta caminhada.
vi
EPÍGRAFE
“Talvez não tenha conseguido fazer o melhor, mas lutei para que o melhor fosse feito.
Não sou o que deveria ser, mas Graças a Deus, não sou o que era antes.”
vii
Gomes SP. Efeito do alendronato no desenvolvimento da face: um estudo histométrico e imunohistoquímico [Tese de Doutorado]. Curitiba: Universidade Positivo; 2019.
RESUMO
Pouco se sabe sobre os efeitos do alendronato de sódio (ALN) no desenvolvimento da
rafe palatina e da cavidade nasal no período neonatal. Alguns estudos apontam que esse fármaco
pode interferir no crescimento cartilaginoso, favorecendo o desenvolvimento desse tecido. Nessa
perspectiva, o objetivo deste estudo foi avaliar, por meio da imunoexpressão de IHH, o
desenvolvimento e expansão da rafe palatina em ratos neonatos de 2 e 7 dias de vida. Além disso,
foi averiguado a imunopresença de IHH, FGF-23 e Na+K+ATPase no 7o dia de desenvolvimento do septo e das conchas nasais, bem como a imunopresença da bomba Na+K+ATPase no epitélio
do trato respiratório. Foram utilizados 32 ratos neonatos, os quais foram alocados aleatoriamente
em dois grupos, o grupo controle (C) e o teste (ALN). Os grupos foram divididos em dois
subgrupos para eutanásia em 2 e 7 dias de vida. Após a eutanásia, foram realizadas as análises
imunohistoquímicas por meio da expressão das proteínas IHH, FGF-23 e Na+K+ATPase, bem
como foi averiguado o aspeto histológico da topografia anatômica. No segundo dia, os aspectos
histológicos do palato do grupo controle demonstraram a presença de tecido conjuntivo fibroso
circundado por tecido cartilaginoso hialino. Neste mesmo período, o IHH foi expresso em células
escassas no mesênquima da cartilagem, bem como em alguns dos condrócitos. No sétimo dia,
foram observadas células condrocíticas intensas separadas por tecido conjuntivo denso, porém o
IHH era escasso. No segundo dia, os espécimes que receberam ALN revelaram padrões similares
quando comparados ao grupo controle. No entanto, várias células que compõem o mesênquima
conectaram à intensa imunoexpressão do IHH. No sétimo dia, no grupo teste, observou-se área
intensa de condrócitos hipertróficos, formando áreas isogênicas bem caracterizadas, em relação
viii
seis camadas de cartilagem hialina e um mesênquima em todo o comprimento do palato médio.
Nesse período, a presença de deposição óssea nesses espécimes não foi identificada.
Conclui-se que o alendronato promoveu expansão condroide da rafe palatina em ratos
neonatos, associado ao aumento da imunoexpressão do IHH nas áreas condroides da rafe
palatina. Em conchas nasais, aumentou a imunoexpressão do IHH e aumentou a expansividade,
associado a supressão do FGF-23, o qual é um opositor funcional do IHH. Ainda, os espécimes
que receberam alendronato tiveram a bomba Na+K+ATPase inibida no epitélio respiratório,
tanto no revestimento da cavidade nasal quanto no seio maxilar.
ix
Gomes SP. Effect of Alendronate on the development of the face: histometric and immunihistochemical study [Tese de Doutorado]. Curitiba: Universidade Positivo; 2019.
ABSTRACT
Little is known about the effects of sodium Alendronate on the development of the palatal
raphe and nasal cavity in the neonatal period. Some studies have indicated that this drug may
interfere with cartilaginous growth and promote the development of cartilage. This study evaluated
the development and expansion of the palatal raphe in newborn rats 2 and 7 days old using HIH
immunoexpression. In addition, on the 7th day of life, the presence of IHH, FGF-23 and
Na+K+ATPase was determined in the nasal concha and cartilage, and the presence of
Na+K+ATPase immunopresence, in the respiratory tract epithelium. Thirty-two neonatal rats were
used, which were randomly assigned to two groups, the control group (C) and the test (ALN). The
groups were divided into two subgroups for euthanasia at 2 and 7 days of age. After euthanasia,
immunohistochemistry was used to analyze the expression of IHH, FGF-23 and Na+K+ATPase
proteins, and histology was used to describe the topography. On the second day, the histological
aspects of the palate in the control group revealed the presence of fibrous connective tissue
surrounded by hyaline cartilaginous tissue. At the same time, HHI was expressed in a few cells in
the mesenchyme of the cartilage, as well as in some of the chondrocytes. On the 7th day, many
chondrocytic cells were separated by dense connective tissue, but IHH was scarce. Specimens of
animals that received ALN had similar patterns when compared to the control group. However,
several cells that make up the mesenchyme were associated with the intense IHH
immunoexpression. On the seventh day, in the test group, chondroid staining revealed that a large
area of hypertrophic chondrocytes formed well-characterized isogenic areas. Circumscribing most
chondrocytes, there were four to six layers of hyaline cartilage and a mesenchyme all along the
x
It is concluded that alendronate promoted chondroid expansion of palatine raphe in
neonatal rats, associated with the increase of HIH immunoexpression in the chondroid areas of the
palatine raphe. In nasal concha, it increased the immunoexpression of HHI and increased the
expansivity, associated with suppression of FGF-23, which is a functional opponent of IHH. Also,
specimens receiving alendronate had the Na + K + ATPase pump inhibited in the respiratory
epithelium, both in the nasal cavity lining and in the maxillary sinus.
xi
SUMÁRIO
INTRODUÇÃO GERAL ... 1
PROPOSIÇÃO GERAL ... 7
MANUSCRITOS ... 8
MANUSCRIPT 1...8
MANUSCRIPT 2...18
CONSIDERAÇÕES FINAIS ... 30
REFERÊNCIAS ... 31
APÊNDICE 1 - METODOLOGIA ... 37
ANEXO I ... 43
1
INTRODUÇÃO GERAL
Entre as descobertas relevantes realizadas nos últimos anos, destacam-se as que lidam
com o desenvolvimento e evolução do crânio e dos tecidos com os quais estão intimamente
associados. Possivelmente, a descoberta predominante entre eles é o reconhecimento gradual,
e agora aceitação generalizada, do papel essencial desempenhado pela crista neural
embrionária, como material fonte de muitos tecidos cranianos adultos, como também da
deformidade craniana [1-4]. De fato, o papel potencialmente predominante das células advindas
da crista neural no desenvolvimento craniano e sua evolução só começou a ser estudado na
década de 1950 e não foi amplamente reconhecido por 30 anos, seguindo com publicação
marcante de Gans & Northcutt [5].
A crista neural é a principal fonte celular das células cartilaginosas do crânio. Isso inclui
maxila, septo nasal e mandíbula [6-9]. No entanto, a embriologia da face, principalmente na
compreensão do desenvolvimento da cavidade nasal, é ainda mal compreendida em estudos
clássicos, nos quais há lacunas e controvérsias encontradas sobre as diferentes origens
embriológicas dos ossos nasais, cartilagens e envelopes de tecidos moles [10, 22, 23].
Estudos abordaram o papel da sinalização do fator de crescimento fibroblástico (FGF)
durante o desenvolvimento inicial do palato por meio da análise de embriões de camundongos
[11,12]. Em geral, as atividades de sinalização estão sujeitas a um rígido controle
espaço-temporal e, em muitos casos, muito ou pouco de uma coisa boa pode ser prejudicial para um
órgão em desenvolvimento. Isso é bem ilustrado em anomalias causadas pela sinalização
desregulada de hedgehogs [13] e FGF [14,15].
Com exceção do membro em desenvolvimento, órgãos constituídos por um epitélio e
um mesênquima expressam os membros da família hedgehog, Sonic Hedgehog (SHH) ou
2
são encontrados em ambas as camadas de tecido, indicando atividades de SHH e IHH à
distância de suas fontes [16].
A proteína IHH está envolvida na diferenciação, proliferação e maturação dos condrócitos, especialmente durante a ossificação endocondral. Dessa forma, os condrócitos sofrem apoptose e são substituídos por matriz óssea secretada por osteoblastos para formar osso trabecular, devido a osteocalcina positivo, que é um marcador de neoformação óssea, cujas funções incluem uma que tem relação com a ligação do cálcio à matriz óssea, além de determinar o nível de atividade dos osteoblastos e formação óssea [17].
Como grande parte do mesênquima que forma a região craniofacial é derivado das células da crista neural, a proteína Indian hedgehog é responsável pelo crescimento, expansão e migração para o desenvolvimento ósseo e a apoptose de células importantes para o
crescimento craniofacial [18].
Porém, existem alguns fatores que podem comprometer essas atividades celulares:
No que se refere ao processo de formação cartilaginosa, o fator de fibroblástico (FGF)
inibe o crescimento de condrócitos e forma um arcabouço de tecido conjuntivo que,
posteriormente, quando associado à calcificação, limita o crescimento ósseo [19].
No que se refere ao processo de formação óssea, a utilização de bisfosfonatos (BPs),
suprimem a reabsorção óssea, induzindo a inativação dos osteoclastos e inibindo sua função e
maturação. Os bisfosfonatos são drogas sintéticas com configuração similar e não hidrolisável
de pirofosfato inorgânico [20].
O alendronato é o representante mais importante dos bisfosfonatos que contêm
nitrogênio e apresenta ampla magnitude na inibição da reabsorção óssea in vivo quando
3
REVISÃO DA LITERATURA
O morfodesenvolvimento craniofacial é um processo complexo que exige migração,
proliferação, diferenciação e apoptose celular a partir de um desenvolvimento sincronizado
entre células derivadas da crista neural cranial, ectoderma neural, mesoderma e endoderma axial
[22,23]. O resultado desse processo é a fusão de proeminências (processos) anatômicos,
formando as estruturas de todo osso maxilar, palato e rafe palatina, a partir de um
desenvolvimento condroide, bem como de coanas, cartilagem nasal, cornetos, e da estrutura dos
seios nasais e maxilares.
Todo o desenvolvimento da face inicia com a migração das células da crista neural, um
processo que começa no oitavo dia do embrião (E8) e finda em dois dias [22], o que em
humanos, corresponde ao E19 e E38 [23].
Após a migração da crista neural, o mesênquima calvarial se origina do mesoderma
paraxial e da crista neural cranial migrada [24]. As áreas limites entre a crista neural e o osso
derivado do mesoderma sofrerão mudanças estruturais. Essas células da crista neural
originam-se no tubo neural dorsal e, à medida que migram, são atraídas para a área frontomedial por meio
de quimio-tropismo em consequência da expansão da epiderme para formar o processo
frontonasal. O mesênquima primordial frontal forma, então, os ossos frontais, que se
diferenciam nas placas ósseas primárias [25], e o processo continua nas placas ósseas até que
elas se encontrem, resultando na formação de uma sutura.
Esse desenvolvimento facial propriamente dito tem sua gênese em meados do 10,5E no
camundongo e ratos, o que equivale à sexta semana de gestação humana. Após esse fenômeno,
os processos nasais mediais que se originam do processo frontonasal fundem-se entre si e,
4
pelo lábio superior e o palato primário. Analogamente, a fusão dos processos mandibulares
bilaterais ao longo da linha média produz o lábio inferior e a mandíbula inferior [23].
Em torno do E11 no rato, o sinal mais precoce de iniciação do palato secundário se
manifesta como consequências bilaterais, primórdios das estruturas palatinas, que emergem da
parte interna dos processos maxilares e se estendem anterosuperiormente [23]. Esse
crescimento polarizado assegura a aproximação das proeminências palatinas opostas e a sua
aderência ao longo do epitélio da borda medial, criando um epitélio transiente de múltiplas
camadas que posteriormente sofrerá apoptose para a formação da linha média. O
desaparecimento progressivo desse tecido epitelial permite a fusão do palato secundário ao
longo da linha média [23,27]. Em conseguinte, o palato secundário também se funde com o
palato primário anteriormente e com o septo nasal dorsalmente, formando a estrutura palatal
propriamente dita [23,27, 28].
Após o término da palatogênese, a cavidade oronasal precoce é subdividida em uma
cavidade oral e uma nasal, um pré-requisito funcional para a formação das vias aéreas
superiores, bem como o inicio anatômico de uma via digestória, aqui representada pela cavidade
oral. Assim, é relevante ressaltar que a diferenciação de células mesenquimais produz os
processos palatinos dos ossos maxilar e palatino do palato duro. Quanto às áreas centrais da
rafe palatina, a cartilagem nasal advém propriamente da crista neural [30].
A regulação via fator de crescimento é um dos principais mecanismos que coordenam
e padronizam a migração, formação e ossificação dos ossos craniofaciais. As vias de sinalização
mais comumente estudadas advém do fator de crescimento transformante (TGF-β) e a
sinalização da proteína morfogenética óssea (BMP) [31-35].
A importância dessas vias foi discutida em estudos que demonstram que a
desregularização da sinalização de TGF-β, por exemplo, altera significativamente a formação
da ossificação e o destino das suturas (fusão vs. patência) [35-37]. Embora o periósteo esteja
5
suturais [38,39]. Em conjunto, as sinalizações dos fatores de crescimento, os caminhos de
diferenciação celular por meio da regulação de genes da família Hedgehog (HH) na
morfogênese das áreas derivadas da crista neural parece ser de clara importância.
Alendronato
O alendronato é uma droga pertencente a família dos bisfosfonatos, cuja estrutura
mimetiza os análogos de pirofosfatos inorgânicos. É considerado uma das terapias de primeira
escolha no tratamento de entidades patológicas ósseas que afetam o metabolismo ósseo
(osteoporose e doença de Paget) e metástases ósseas [56].
Seu mecanismo nos osteoclastos interfere na geranil-geranilação, uma condição
responsável ímpar para a geração de proteínas preniladas, que são responsáveis pela interação
da camada de fosfolipídio com as proteínas reguladoras. O resultado dessa perda proteica
culmina na inibição de proteínas de superfície de membrana e receptores de membrana,
suprindo a excitação quimiotática e a inativação e maturação celular, como ocorre nos
osteoclastos [57].
As GTPases preniladas são proteínas de sinalização que regulam uma variedade de
processos celulares importantes para a função celular e a síntese de reposição celular, pois
atuam em conjunto com fatores de crescimento dependentes que são receptores acoplados à
membrana plasmática [58]. Entre esses fatores, alguns possuem destaque no desenvolvimento
ósseo, tais como o fator de crescimento transformante B1 (TGF-B1), o fator de crescimento
vascular endotelial (VEGF) e as proteínas Na+K+ATPase, que podem, de forma direta ou
indireta, alterar, inibir, ou excitar a transcrição de proteínas ósseas.
Os efeitos do alendronato durante o desenvolvimento do tecido ósseo e cartilaginoso
pós-natal não são bem conhecidos. Os ossos craniofaciais detêm origem no mesênquima e na
6
desenvolvimento de ossos chatos, como os ossos faciais e o crânio [23]. Esse processo envolve
a diferenciação direta das células mesenquimais em osteoblastos, o que resulta na formação
óssea. Durante esse processo, uma nova matriz óssea é sintetizada e mineralizada por
osteoblastos, ao mesmo tempo que há a formação de cartilagem. Ocorre nesse momento a
proliferação de condrócitos e a sua diferenciação em cartilagem, que é depois substituída por
osso [23]. Assim, este estudo averiguou os efeitos do alendronato de sódio no desenvolvimento
7
PROPOSIÇÃO GERAL
Os objetivos desta tese apresentada no formato de manuscritos foram:
Manuscrito 1
Avaliar os efeitos do uso de 2,5 mg/kg de alendronato de sódio durante o
desenvolvimento e a expansão da rafe palatina em ratos neonatos, nos períodos de 2 e 7 dias.
Ainda, avaliar a imunopresença do IHH durante a formação e ossificação da linha média.
Manuscrito será submetido ao periódico: Brazilian Dental Science. Manuscrito
formatado de acordo com as normas específicas do periódico (acessado em: 05/03/2019).
Manuscrito 2
Avaliar a imunopresença de IHH, FGF-23 durante o desenvolvimento das conchas
nasais, bem como a imunopresença da bomba Na+K+ATPase no epitélio do trato respiratório
após 7 dias de uso de 2,5 mg/kg de alendronato de sódio.
Manuscrito será submetido ao periódico: Brazilian Dental Science. Manuscrito
8
MANUSCRITOS
MANUSCRIPT 1
ANALYSIS OF THE IMMUNOEXPRESSION OF IHH IN THE DEVELOPMENT OF THE MIDPALATAL SUTURE OF NEONATAL RATS TREATED WITH
ALEDRONATE1
ABSTRACT
Postnatal palatal growth arrest or deficiencies in the midpalatal area are common
sequelae of physical injury. As alendronate may induce chondroid expansion, this study
evaluated the immunoexpression of IHH in the area of the palatal raphe and compared results
with the histological characteristics of the midpalatal area at 2 and 7 days of life of newborn
rats receiving alendronate. Thirty-two newborn rats were included in this study: 16 in a control
group, and 16 in a group that received intraperitoneal applications of 2.5 mg/kg/day of
alendronate. The animals were euthanized on the 2nd or 7th day, and their heads prepared for
histological and immunohistochemical analyses. Specimens of animals that received
alendronate had larger chondroid matrix deposition in areas where there was proliferation and
isogenic formation of hypertrophic chondrocytes, and the analyses revealed immunoexpression
of IHH. In addition, higher levels of IHH were detected in the mesenchymal tissue of the palatal
raphe, forming an area of resting chondrocytes and a perichondral topography resulting from
the differentiation of mesenchyme into chondrocytes. Alendronate may be used in the treatment
of orthopedic diseases, especially to promote the growth of the midpalatal area.
KEYWORDS
Alendronate, palatal raphe, IHH
1 Manuscrito será submetido ao periódico: Brazilian Dental Science. Manuscrito formatado de acordo
9
INTRODUCTION
Postnatal craniofacial disorders due to physical injury are a common concern in clinical
practice, because the resulting midpalatal growth disorders may have serious consequences,
such as craniosynostosis and midface hypoplasia, conditions that not only affect facial harmony,
but that also contribute to the compression of the growing encephalon, especially in children
[1].
These pathological disorders invariably result from changes in the usual signaling
pathways that activate proteins for the differentiation of cells that originate in the neural crest
and the mesenchyme. They may also be caused by the activation of growth factors that act in
the suppression of the usual pathways in the formation of the midpalatal suture [2].
The HH family of proteins has a function in these processes of palatal and facial
development. Specifically, Indian hedgehog (IHH) expression has been associated with the
induction of cell proliferation and differentiation, found mainly on the osteogenic fronts of the
calvarial, maxillary and mandibular bones. In addition, IHH seems to play an important role in
the site of plate growth and cartilage formation, and in early craniofacial development,
especially in the growth of midpalatal areas [3].
Therefore, dugs that stimulate differentiation of chondrocytes and osteogenesis may be
used for the treatment of disorders, such as craniosynostosis and midface hypoplasia. The
consecutive use of alendronate may be an option for chondrocyte proliferations in midpalatal
areas, contributing for chondrocyte maturation and ossification of this anatomical site [4].
This study evaluated the immunoexpression of IHH in the midpalatal suture area, and
10
MATERIAL AND METHODS
Animals
This study followed the Principles of Laboratory Animal Care (NIH Publication 85-23,
revised in 1985) and Brazilian laws on animal use, and was approved by the Ethical Committee
for Animal Research of the institution were it was conducted (Protocol #266).
Ten female rats (Rattus norvegicus albinus, Holtzman) aged 2-3 months and weighing
approximately 242 to 260 g were kept under controlled temperature (22±2°C) and humidity
(55%±5%), with a 12 h/12 h light/dark cycle and food and water ad libitum. Using a cotton
swab soaked in saline solution (pH 7.0), vaginal oncotic cytology samples were collected from
all female rats every day. The cytological material was spread on a glass slide and stained using
the Papanicolaou technique to determine the phase in the estrus cycle, according to the
characteristics of epithelial cells and the presence or absence of inflammatory cells.
Female rats in the estrus phase were mated overnight with male rats, with two females
per male in each cage. Early in the next morning, oncotic cytological vaginal smears were
collected from female rats again, stained using Shorr staining, and examined under a light
microscope to verify the presence of sperm. Presence of sperm in vaginal smears was indicative
of first day of pregnancy.
Treatment
Pregnant rats were housed singly in polypropylene cages until after the birth of the pups.
Afterwards, the newborn rats were randomly assigned to one of two groups: a control group, in
11
rats received 2.5 mg/kg/day of alendronate trihydrate (Biolife, Curitiba, Brazil; lot number:
14042132C) (n=16). The saline and alendronate solutions were administered intraperitoneally
daily until euthanasia, which occurred on the 2rd and 7th day after birth for eight animals in
each treatment group at each time point. Euthanasia was performed by brief exposure to
isoflurane.
Histology
Immediately after euthanasia, the heads of newborn rats were removed and the surgical
specimens were immersed in a 4% formaldehyde fixative solution, prepared from
paraformaldehyde and 0.1 M sodium phosphate, at a pH of 7.2. After decalcification for 3
weeks in 7% edetate disodium (EDTA) containing 0.5% formaldehyde in 0.1 M sodium
phosphate (pH 7.2), the specimens were dehydrated in graded concentrations of ethanol, cleared
in xylene and embedded in paraffin. Serial 3-µm-thick histological sections were obtained from
each specimen in the anterior-posterior direction according to the coronal anatomic plane. Some
sections were stained with hematoxylin and eosin, Masson trichrome, and Alcian blue to
examine histomorphologic and histomorphometric aspects of bone and chondroid matrix, and
other sections were deposited on silanized slides (Sigma-Aldrich Chemie, Steinheim, Germany)
for the immunohistochemical detection of IHH.
Immunohistochemistry
From each specimen, 3-µm-thick sections were used for the immunohistochemical
detection of proteins. For antigen retrieval, deparaffinized sections were immersed in 10 mM
sodium citrate buffer (pH 6.0) and microwaved in 3× 5-minute cycles to detect IHH. After
12
were incubated for 30 min at room temperature with 2% bovine serum albumin (BSA;
Sigma-Aldrich Chemie, Steinheim, Germany). The sections were then incubated overnight at 4º C with
primary antibody anti-IHH (200 mg/mL, Santa Cruz Biotechnology, CA, sc-271101) at a
dilution of 1:200. A labeled streptavidin/biotin antibody binding detection system (Universal
HRP immunostaining kit; Diagnostic Biosystem, Foster City, CA) was used to detect the
primary antibodies. After washing in 0.05 M Tris-HCl buffer (pH 7.2), the sections were
incubated for 30 min at room temperature in biotinylated anti-rabbit/mouse/goat
immunoglobulin (LSAB-plus kit; Dako, CA). Sections were counterstained with Harris
hematoxylin. For the negative control group, the primary antibodies were omitted, and the
sections were incubated with nonimmune serum.
Image Analyses
Images of both histological and immunohistochemical sections were captured with a
digital camera (Samsung, South Korea) under a light microscope at x100 and x200
magnifications. The digital images were captured and saved at 300 dpi resolution (image size:
115 × 75 cm).
The amount of bone matrix deposited in the body of the maxillary bone, as well as the
amount of chondroid tissue in the midpalatal area, was determined on each histological sample.
The software Image J was used to record the histomorphometric data about bone matrix and
chondroid matrix deposition.
Histomorphometric measurements were made using, the microscopic images and
Adobe Photoshop for Mac. A line tangent to the lower edge of the nasal septum cartilage was
drawn on each image. The maxillary area below this line was the focus of this study and the
point at which all measurements were made. The lateral borders were established by lines drawn
13
limit was the epithelial lining of the mucosa. Figure 1A shows the area examined in each
specimen.
Each micrograph was transferred to the Image J software (https://imagej.nih.gov/ij), and
the total area and the areas of bone matrix and of chondroid tissue stained with hematoxylin
and eosin were carefully selected and measured. Cells positive for IHH were scored as: - 0% to
1%; + 1% to 25%; ++ 25% to 50%; +++ 50% to 75%; and ++++ >75%.
A 1-mm slide micrometer was used to calibrate all measurements. The average of three
measurements for each parameter was calculated for each specimen. After that, all data were
expressed in percentages.
Statistical Analysis
Data for histomorphologic and immunohistochemical analyses were collected along the
monitoring time. The Shapiro-Wilk test was used to determine normality, followed by the
Kruskal Wallis nonparametric test to detect significant differences between groups. The level
of significance was set at p<0.05.
RESULTS
All histomorphometric data and immunohistochemical scores are showed in Table 1,
The main findings of specimen analyses are described here. On the 2rd day, the histological
findings for the midpalatal area of the control group indicated the presence of a fibrous
connective tissue surrounded by hyaline cartilaginous tissue. The hyaline cartilage had two to
three layers of chondrocytes surrounded by hypertrophic chondrocytes. At this time point, few
14
there was intense separation of chondrocyte cells by dense connective tissue. Bone matrix was
visible among the hyperplastic chondrocytes. However, IHH was scarce on the 7th day.
On the 2nd day, the specimens that received alendronate had similar patterns to those of
the control group. However, several cells of the mesenchymal connective tissue had an intense
immunoexpression of IHH. In contrast, there were several IHH+ chondrocytes and few isogenic
and proliferative groups of hypertrophic IHH+ chondrocytes. On the 7th day, a large area of
hypertrophic chondrocytes formed characteristic isogenic IHH+ areas, differently from the
chondroid stained areas. Four to six layers of hyaline cartilage and one layer of IHH+
mesenchyme circumscribed most chondrocytes throughout the length of the midpalatal area. At
this time point, no bone deposition was detected in these specimens.
Figure 1: A and B - Larger presence of IHH in specimens that received alendronate;
simultaneous presence isogenic and proliferative hypertrophic chondrocytes. C and D -
Immunohistochemical presence of IHH in specimens that received alendronate and in control
specimens. A 2nd day Test C 2nd day Control B 7th day Test D 7th day Control
15
Table 1: Histomorphometric and immunohistochemical findings of both groups.
Findings Day Control Alendronate p
Area of chondroid tissue
(mm2) 2 1.34 0.08 1.42 0.11 0.523 7 1.42 1.01 1.73 0.77 0.032 Area of hyperplasic chondrocytes 2 1.12 0.02 1.33 0.21 0.437 7 1.42 1.01 1.69 0.55 0.028 IHH in chondrocytes 2 + ++ 7 + ++++ IHH in mesenchymal tissue 2 + +++ 7 - ++++ DISCUSSION
In this study, the use of alendronate changed the histological aspect of the formation of
the midpalatal suture. In specimens that received alendronate, there was an increase in the
immunoexpression of IHH, formation of a net of isogenic groups of hyperplasic chondrocytes,
resulting in a lower area of mineralization and an increase in hypertrophic and proliferative
chondrocytes. The mesenchymal tissue that formed the palatal raphe was also classified as
16
These results suggested that the use of alendronate induces a sequence of events in the
endochondral metaphyseal area, as well as the differentiation of mesenchymal cells distributed
into a resting zone. Alendronate promotes chondral protection in the midpalatal suture area and
shapes it. It is responsible for the expansion of the chondroid area and simultaneously delays
the mineralization of the midpalatal area [5]. All these sequences of events seem to be
associated with chondrocytes and the increase in IHH expression.
A factor associated with alendronate that may increase IHH action and cartilage growth
in the midpalatal suture is the potential capacity of this drug to increase TGF-β [6].
Corroborating this premise, a study of TGF-β2 conducted by Erlebacher and Derynck
(1996) found TGF-β2 overexpression, which may lead to specific increases of IHH expression
in the chondral osteoblasts, and revealed a novel pathway of action of this pluripotent growth
factor in endochondral metabolism [7].
These results also seem to be supported by the findings reported by Tekari et al. (2015),
who demonstrated that the expansion of chondrocytes seem to be strictly associated with
functional endogenous TGF-β signaling. The authors demonstrated that TGF-β receptor
inhibited the cytokine-dependent pathway, a condition that coincided with the arrest of
cartilaginous growth and chondroid differentiation [8]. Here we add that these effects may occur
via IHH.
In our study, alendronate inhibited precocious mineralization and increased IHH
expression. These changes enhanced endochondral ossification through chondrocyte expansion
induced by alendronate. According to our results, alendronate may contribute to the orthopedic
growth of the maxilla, which provides new evidence that alendronate may be an option for the
treatment of diseases affecting the midpalatal.
CONCLUSION
17
REFERENCES
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developmental defects. Dev Dyn. 2016 Mar;245(3):276-93. Epub 2015 Dec 11.
2) Twigg SR, Wilkie AO. A genetic-pathophysiological framework for craniosynostosis. Am J
Hum Genet. 2015 Sep 3;97(3):359-77.
3) Deng A, Zhang H, Hu M, Liu S, Wang Y, Gao Q, Guo C. The inhibitory roles of Ihh
downregulation on chondrocyte growth and differentiation. Exp Ther Med. 2018
Jan;15(1):789-794. doi: 10.3892/etm.2017.5458. Epub 2017 Nov 7.
4) St-Jacques, B., Hammerschmidt, M., McMahon, A.P. Indian hedgehog signaling regulates
proliferation and differentiation of chondrocytes and is essential for bone formation. Genes
Dev. 1999: 13: 2072–2086.
5) Yaffe A, Kollerman R, Bahar H, Binderman I. The influence of Alendronate on bone
formation and resorption in a rat ectopic bone development model. J Periodontol. 2003;
74(1):44-50.
6) Manzano-Moreno FJ, Ramos-Torrecillas J, Melguizo-Rodríguez L, Illescas-Montes
R, Ruiz C, García-Martínez O. Bisphosphonate modulation of the gene expression of
different markers involved in osteoblast physiology: Possible implications in
bisphosphonate-related osteonecrosis of the jaw. Int J Med Sci. 2018 Feb 12;15(4):359-367.
7) Erlebacher A, Derynck R. Increased expression of TGF-beta 2 in osteoblasts results in an
osteoporosis-like phenotype. J Cell Biol. 1996 Jan;132(1-2):195-210.
8) Tekari A, Luginbuehl R, Hofstetter W, Egli RJ. Transforming growth factor beta signaling
is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.
18
MANUSCRIPT 2
EFFECT OF SODIUM ALENDRONATE ON THE DEVELOPMENT OF NASAL
CONCHA AND MUCOSA. AN IMMUNOHISTOCHEMICAL STUDY2
ABSTRACT
Alendronate (ALN) may change or even accelerate the chondroid development in the
nasal cavity. This study evaluated the immunoexpression of IHH, FGF-23 and Na+K+ATPase
in the nasal cartilage and concha and Na+K+ATPase immunopresence in the respiratory tract
epithelium. Results were compared with the histological characteristics on the 7th day of life
of newborn rats receiving ALN. Sixteen newborn rats were included in this study: eight in a
control group, and eight in the ALN group, in which animals received 2.5 mg/kg/day of
intraperitoneal alendronate daily. The animals were euthanized on the 7th day, and their heads
prepared for histological and immunohistochemical analyses. Specimens in the test group had
a greater number of cells and a larger area of cartilage than those in the control group.
Chondrocytes with discrete to moderate IHH immunolabeling and areas of endochondral bone
deposition were detected at the apex of the hyperplastic nasal concha in the control group. The
nasal concha of the specimens that received alendronate had proliferative chondrocytes and
hyperplastic areas, with intense IHH immunopositivity. FGF-23 positivity was high, whereas
FGF-23 was scarce in the ALN specimens. ALN promoted proliferation of nasal cartilage and
increased the number of chondrocytes per area, which also increased the size of the nasal
concha. Alendronate may affect the histophysiology of the nasal cavity, because it increases the
size of the nasal concha. At the same time, it suppresses Na+K+ATPase in respiratory epithelia.
KEYWORDS
Alendronate, nasal concha, IHH, FGF-23, Na+K+ATPase.
2 Manuscrito será submetido ao periódico: Brazilian Dental Science. Manuscrito formatado de acordo
19
INTRODUCTION
Alendronate (ALN) is a bisphosphonate drug used in clinical practice to minimize the
deleterious effects of pathologies that promote intense bone loss. The use of bisphosphonate
therapy in pediatric patients was suggested in 1998 when the cyclic administration of
intravenous bisphosphonates in children with osteogenesis imperfecta resulted in reduction in
bone resorption, increase in bone density, and reduction in fracture incidence [1]. This drug has
also been used in the treatment not only of comorbidities, but also of endochondral growth
deficiencies in children, as alendronate seems to promote bone and cartilage growth [2].
In addition to the endochondral area of the metaphysis and epiphysis of long bones,
several anatomical areas are composed of chondrocytes and may present with deficiencies
during growth. An important area that may have its anatomy affected by ALN use is the nasal
cartilage and concha [3]. In fact, ALN may disturb protein immunoexpression in this site, either
by stimulating or suppressing the chondroid expansion, or even by changing the respiratory
epithelium in the region [4].
One of the proteins responsible for cartilage growth is the Indian hedgehog (IHH). This
protein is an important marker of cartilaginous expansion and development of hyperplastic
chondrocytes. The area of development of this cartilage is regulated by the fibroblast growth
factor-23 (FGF-23), a protein that reduces and suppresses chondrogenesis [5].
Despite these benefits, ALN may also damage the development of respiratory epithelia
and induce ulcers in cylindrical epithelia, such as enterocytes [6, 7].
This study evaluated the immunoexpression of IHH, FGF-23 and Na+K+ATPase in the
in the nasal concha and mucosa, and compared results with histomorphometric findings in the
20
MATERIAL AND METHODS
Animals
This study followed the Principles of Laboratory Animal Care (NIH Publication 85-23,
revised in 1985) and Brazilian laws on animal use, and was approved by the Ethical Committee
for Animal Research of the institution were it was conducted (Protocol #266).
Seven female rats (Rattus norvegicus albinus, Holtzman), aged 2 months and weighing
245- to 255 g, were used as progenitresses. The animals were kept in a room with controlled
temperature (22 ± 2°C) and humidity (55% ± 5%), at a 12-h light/dark cycle and with food and
water ad libitum.
Sixteen newborn rats were obtained from natural generation, using the method described
by de Souza et al. [8]. Eight newborn rats received sterile 0.9% saline solution (control group);
the other eight newborn rats received 2.5 mg/kg/day of alendronate trihydrate (Biolife, Curitiba,
Brazil; lot number: 14042132C) for seven days (ALN group). Both saline and alendronate
solutions were administered intraperitoneally daily until euthanasia by isoflurane overdose,
which occurred on the 7th day.
Histology
Immediately after euthanasia, the heads of newborn rats were removed and the surgical
specimens were immersed in a 4% formaldehyde fixative solution, prepared from
paraformaldehyde and 0.1 M sodium phosphate, at a pH of 7.2, for 48 h at 18º to 20º C. After
decalcification for 3 weeks in 7% edetate disodium (EDTA) containing 0.5% formaldehyde in
21
ethanol, cleared in xylene and embedded in paraffin. Serial 3-µm-thick histological sections
were obtained from each specimen in the anterior-posterior direction according to the coronal
anatomic plane. The sections were stained with hematoxylin and eosin to examine
histomorphologic and histomorphometric aspects of the chondroid matrix, and other sections
were deposited on silanized slides (Sigma-Aldrich Chemie, Steinheim, Germany) for the
immunohistochemical detection of IHH, FGF-23 and Na+K+ATPase.
Immunohistochemistry
From each specimen, 3-µm-thick sections were used for the immunohistochemical
detection of proteins. For antigen retrieval, deparaffinized sections were immersed in 10 mM
sodium citrate buffer (pH 6.0) and microwaved in 3× 5-minute cycles to detect IHH. After
cooling and inactivation of endogenous peroxidase with 5 % hydrogen peroxide, the sections
were incubated for 30 min at room temperature with 2% bovine serum albumin (BSA;
Sigma-Aldrich Chemie, Steinheim, Germany). The sections were then incubated overnight at 4º C with
primary antibody anti-IHH (200 mg/mL, Santa Cruz Biotechnology, CA, sc-271101) at a
dilution of 1:200. A labeled streptavidin/biotin antibody binding detection system (Universal
HRP immunostaining kit; Diagnostic Biosystem, Foster City, CA) was used to detect the
primary antibodies. After washing in 0.05 M Tris-HCl buffer (pH 7.2), the sections were
incubated for 30 min at room temperature in biotinylated anti-rabbit/mouse/goat
immunoglobulin (LSAB-plus kit; Dako, CA). Sections were counterstained with Harris
hematoxylin. For the negative control group, the primary antibodies were omitted, and the
22
Image Analyses
Images of both histological and immunohistochemical sections were captured with a
digital camera (Samsung, South Korea) under a light microscope at x100 and x200
magnifications. The digital images were captured and saved at 300 dpi resolution (image size:
115 × 75 cm).
The amount of chondroid matrix deposited in the nasal concha, as well as the number
of cells in this site, was determined on each histological sample. The software Image J was used
to record the histomorphometric data about bone matrix and number of cells at this site.
Histomorphometric measurements were made using, the microscopic images and
Adobe Photoshop for Mac. All areas of cartilage in the nasal concha were measured
Each micrograph was transferred to the Image J software (https://imagej.nih.gov/ij), and
the total area and the areas of chondroid matrix were carefully selected and measured. Cells
positive for IHH, FGF-23 and Na+K+ATPase were scored as: - 0% to 1%; + 1% to 25%; ++
25% to 50%; +++ 50% to 75%; and ++++ >75%. The average of three measurements for each
parameter was calculated for each specimen.
Statistical Analysis
Data for histomorphologic and immunohistochemical analyses were collected along the
monitoring time. The Shapiro-Wilk test was used to determine normality, followed by the
Kruskal Wallis nonparametric test to detect significant differences between groups. The level
23
RESULTS
After 7 days of alendronate administration, the specimens in the test group had a greater
number of cells and a larger area of cartilage than those in the control group. Chondrocytes with
discrete to moderate IHH immunolabeling and areas of endochondral bone deposition were
detected at the apex of the hyperplastic nasal concha in the control group. In contras, the nasal
concha of the specimens that received alendronate had numerous proliferative chondrocytes
and hyperplastic areas, with intense IHH immunopositivity. The delineation of the cartilaginous
areas in the control group revealed high FGF-23 positivity, whereas FGF-23 was scarce in the
specimens that received the drug (FIg.1). All histomorphometric data are showed in Table 1,
and the immunohistochemical scores, in Table 2.
Figure 1: IHH and FGF-23 immunopositivity in control and test groups. A - IHH
24
(brownish areas). C and D - FGF-23 in control and test groups; FGF-23 surrounds chondroid
area of nasal cartilage; FGF-23 is scarce in ALN group.
Table 1: Mean (min - max) value of chondroid area and cells/mm2
Parameter Control Alendronate p value
Chondroid area (mm2) 0.36 (0.29 – 0.41) 0.77 (0.66 – 0.81) p= 0.0027 Cells/mm2 117 (108 – 137) 153 (139 – 166) p=0.0152
At the same time, Na+K+ATPase was scarce in respiratory epithelium, both in nasal
epithelium and in the epithelium of the maxillary sinus in formation. In contrast, Na+K+ATPase
was positive in the control group.
Figure 2: Na+K+ATPase immunopositivity in control and test group. A -
25
epithelium of specimens that received alendronate (brownish areas). C and D: Na+K+ATPase
presence in area of maxillary sinus in control and test groups; pattern Na+K+ATPase
immunopositivity in maxillary sinus is similar to that of respiratory epithelium.
Table 2: Immunohistochemical scores of IHH and FGF-23 in chondroid area of nasal concha
and of Na+K+ATPase in respiratory epithelium.
Protein Score Control Alendronate IHH ++ ++++ FGF-23 ++ + Na+K+ATPase ++++ + DISCUSSION
The results of this study suggest that alendronate (ALN) may change or even accelerate
the chondroid development in the nasal cavity in a model of growing animals. ALN promoted
proliferation of nasal cartilage and increased the number of chondrocytes per area (mm2), which
also increased the size of the nasal concha. These results were associated with a high IHH
positivity.
In fact, Indian hedgehog (IHH) signaling seems to be indispensable for the proper
development of the endochondral areas and skeleton. In the developing cartilage, IHH is
primarily expressed on pre-hypertrophic chondrocytes and in early hypertrophic chondrocytes,
and IHH signals to both immature chondrocytes and the perichondral cells [9,10]. These
26
Cartilage growth suppression, or even its growth retardation, seems to be correlated to
the presence of other proteins that impair IHH action and limit excessive cartilage growth [10].
IHH plays a critical role in chondrocyte proliferation, and the downstream target of FGF
receptor 3 activation suppresses the growth of cartilage. Kawai (2013) found that IHH is the
downstream target of FGF23, a signaling that induces a decrease of IHH expression and
consequently promotes suppression of chondrocyte proliferation [11].
The intense presence of IHH and the simultaneous suppression of FGF-23 may explain
why ALN affects the biology of nasal concha development and improves cartilage growth.
Actually, the reason why ALN increases HHI or decreases FGF-23 remains unclear, but
there is evidence that this effect of ALN on these proteins may be indirect, through the increase of TGFβ, which is higher when ALN is used. Erlebacher & Derynck (1996) found a direct
correlation between enhance of effects of TGF beta and enhancement of IHH expression
chondrocytes [12], while Richter et al. (2016) found a suppression of FGF23 in cultures of
myocytes submitted to treatment with exogenous TGF-β [13]. Although myocytes are different
cells, from different embryological sources, our results suggest that this mechanism may be
similar in chondrocytes.
In fact, the use of ALN until the seventh day cannot demonstrate the actual deleterious
effect to the airways of the animal and dyspnea, but an exacerbated growth of nasal concha
cartilage may lead to upper airway stenosis and even obstructive pulmonary disease.
Na+K+ATPase suppression may also contribute to respiratory tract diseases.
Na+K+ATPase, a protein expressed in epithelial cells, plays a crucial role. Primarily found in
the basolateral plasma membrane, it catalyzes ATP-dependent transport of three Na+ and two K+ into the cell per pump cycle to maintain Na+ and K+ gradients across the plasma membrane.
This Na+ and K+ homeostasis is necessary to regulate the functions of the various ion and solute transporters in epithelial cells. It is also responsible for cell polarization [14]. Loss of
27 vacuolar degeneration [14].
The loss of Na+K+ATPase found in this study may be explained by the fact that ALN
inhibits farnesyl pyrophosphate synthase in the mevalonate cascade [15]. As result of this
inhibitory event, cholesterol synthesis may fail, and non-prenylated GTP-binding proteins may
accumulate in the cell cytoplasm. In the non-prenylated form, the binding-protein loses its
functional capacity to promote protein-protein interaction to cellular membrane [16,17]. When
a protein that works as membrane receptor is allowed to spread throughout the cytoplasm,
especially due to failure of its common interaction with membrane integrin, this situation
changes the tertiary or quaternary protein configuration, a condition that simulates the
denaturation of protein and that suppresses its usual functional action [18,19]
CONCLUSION
Alendronate may affect the histophysiology of the nasal cavity, because it increases the
size of the chondroid area and IHH, at the same time, suppresses Na+K+ATPase and FGF-23.
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30
CONSIDERAÇÕES FINAIS
Manuscrito 1
O alendronato 2,5 mg/kg promoveu expansão condroide da rafe palatina em ratos neonatos,
devido:
ao aumento da camada hipertrófica de condrócitos;
à transdiferenciação do mesênquima em zona de condrócitos em repouso.
Todo esse aumento ocorreu associado ao aumento da imunoexpressão do IHH nas áreas
condroides da rafe palatina.
Manuscrito 2
O alendronato aumentou a imunoexpressão do IHH e aumentou a expansividade cartilaginosa
nas conchas nasais.
Esse fenômeno ocorre também associado à supressão do FGF-23, o qual é um opositor
funcional do IHH.
Ainda, os espécimes que receberam alendronato tiveram a bomba Na+K+ATPase inibida no
31
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APÊNDICE 1 - METODOLOGIA
Animais
Os experimentos foram realizados no biotério e no laboratório de Histopatologia da
Universidade Positivo, após a aprovação na comissão de Ética no Uso de Animais da
Universidade (protocolo #266).
Para o desenvolvimento deste trabalho, nove ratas com idade de três meses, com
aproximadamente 250 g de massa corporal, e três ratos machos adultos com massa variando de
250-300g foram usados para acasalamento.
Acasalamento e obtenção da amostra
Os animais selecionados para acasalamento foram mantidos no biotério em condições
controladas de temperatura (23 ±10oC) e umidade relativa do ar (55±5%), com iluminação artificial por lâmpada fluorescente com fotoperíodos de 12 horas claro/escuro e com ração e
água (0,7 ppm/F) ad libitum.
Inicialmente, foi realizado esfregaço vaginal nas ratas para uma avaliação do período do
ciclo estral em qual se encontravam. O resultado da citologia esfoliativa nos permitiu uma
avaliação precisa da atividade do conteúdo celular representado por epitélio vaginal ou a
presença ou ausência de processo inflamatório, os quais mudam abruptamente devido as
flutuações constantes nos níveis de estradiol.
A estratificação dos critérios que foram considerados para cada período obtidos por