DESENVOLVIMENTO MEDULAR POR ANÁLISE DA IMUNOEXPRESSÃO
DE PROTEÍNAS E MARCADORES MOLECULARES
ISABELLA GÖHRINGER
obtenção do título de Doutor, pelo programa de Doutorado em
Odontologia.
CURITIBA
2019
Elaborado pela Bibliotecária Priscila Fernandes de Assis
(CRB-9/1852)
G614 Göhringer, Isabella.
Estudo da influência do L-PRP no reparo ósseo e desenvolvimento
medular por análise da imunoexpressão de proteínas e marcadores
moleculares / Isabella Göhringer. Curitiba : Universidade Positivo,
2019. 77 f.
Tese (Doutorado) – Universidade Positivo, Programa de Pós-
graduação em Odontologia, 2019.
Orientador: Profa. Dr. Allan Fernando Giovanini.
1. Odontologia. 2. Ossos - regeneração. I. Giovanini, Allan
Fernando. II. Título.
CDU 616.314 (043.2)
Dedico este trabalho aos meus avôs (in memorian).
Vô Aloysio que, com todo seu carinho, me ensinou a amar os animais
e foi o melhor avô que
alguém poderia ter. Vô Rodolfo que me deixou de lembrança uma
coleção de lâminas histológicas
da sua própria tese.
iv
Agradecimentos
Aos meus pais, Simone e Andreas, por sempre terem me apoiado e por
servirem de exemplo
de como ser uma pessoa boa, dedicada e honesta.
Ao meu marido, Marcos, por ser meu companheiro e incentivador de
todas as horas.
Ao meu orientador, Prof Allan, meu pai acadêmico, por sempre ter
acreditado no meu
potencial e me incentivar desde a iniciação científica. Você teve
papel fundamental em todas as
minhas conquistas acadêmicas.
À amiga Rosangela Tavella, por todas as aventuras e risadas nas
disciplinas na UFPR.
À Universidade Positivo pelo ambiente propício à evolução e
crescimento pessoal e
profissional.
v
stronger than you seem,
-Winnie the Pooh
vi
Resumo
Objetivo: O objetivo deste trabalho foi avaliar o papel do L-PRP
durante o reparo ósseo e
desenvolvimento de área medular em defeitos artificiais em
calvárias de ratos tratados e não tratados
com L-PRP, através da análise da expressão imunoistoquímica das
proteínas TGF-β,
osteoprotegerina, osteocalcina, esclerostina, CD34, IGF-1, JAK2,
STAT5, IP3R e SMA.
Metodologia: Um defeito ósseo de 5mm de diâmetro x 1mm de
profundidade foi criado na calvária
de 48 ratos witsar machos. Em seguida, foram divididos em dois
grupos (L-PRP + autoenxerto de
osso particulado e apenas autoenxerto de osso particulado) e
tratados de acordo com o enxerto do seu
grupo. Os animais sofreram eutanásia em 15 e 40 dias
pós-cirúrgicos. A análise dos resultados foi
realizado através de interpretações de imunoistoquímica.
Resultados: Os resultados desta pesquisa
revelaram uma diminuição na formação da matriz óssea, e uma
alteração na diferenciação da área
medular nos grupos tratados com L-PRP. Conclusão: A presença do
L-PRP prejudicou o reparo ósseo
e produziu alterações na área medular, demonstrando condições
similares a processos fibróticos
patológicos.
vii
Abstract
Aim: The aim of this study was to evaluate the role of L-PRP during
bone repair and
medullary differentiation in artificial defect in mice calvary
treated and not treated with L-PRP
through the analysis of the immunohistochemical expression of
TGF-β, osteoprotegerin, osteocalcin,
sclerotin, CD34, IGF-1, JAK2, STAT5, IP3R and SMA. Methods: A bone
defect was created on the
calvaria of 48 wisteria mice. They were divided into four groups
(L-PRP + particulate bone autograft
and only particulate bone autograft; 15 and 40 days euthaniasia)
and treated according to their group
graft. The animals were euthanized 15 and 40 days postoperatively.
The analysis of the results was
performed through immunohistochemistry interpretations. Results:
The results of this research
revealed a decrease in bone matrix formation, and a change in
medullary differentiation in L-PRP
treated groups. Conclusion: The presence of L-PRP impaired bone
repair and produced changes in
medullary differentiation, demonstrating conditions similar to
pathological fibrotic processes.
Key-words: bone repair, L-PRP, TGF-β, SMA,
imunohistochemistry
viii
Sumário
GH Hormônio do crescimento
1
JAK2 Janus quinase 2
MEC matriz extracelular
PRP plasma rico em plaquetas
RANK recetor ativador do fator nuclear kappa B
RANKL ligante do recetor ativador do fator nuclear
kappa B
STAT5 transdutor de sinal e ativador da transcrição 5
TGF-β Fator de crescimento transformador beta
x
1
1. INTRODUÇÃO
Plasma rico em plaquetas (PRP) é um biomaterial autólogo composto
por uma pequena
quantidade de plasma enriquecido com alta concentrações de
plaquetas, o qual é obtido a partir da
centrifugação de uma fração sanguínea com consequente remoção de
hemácias (Marx, 2004). A
premissa do seu uso no âmbito ortopédico deriva da hipótese de que
as plaquetas contidas no PRP
são responsáveis pela síntese e secreção de inúmeros fatores de
crescimento, entre os quais se
destacam TGF-β, PDGF e IGF-1 (Intini, 2009; Nakata et al., 2009).
Esses fatores de crescimento são
considerados como moduladores importantes para diferenciação
celular e consequentemente
restauração de um órgão ou tecido perdido sob trauma, cirurgias e
tumores (Nikolidakis & Jansen,
2008; Nikolodakis et al, 2009).
Embora diversos autores observaram o sucesso na osteoneogênese
quando utilizam o PRP,
inúmeros estudos revelam que o agregado plaquetário também pode
também ocasionar ou mimetizar
condições patológicas (Oliveira Filho et al., 2010; Giovanini et
al., 2014).
Entre as condições patológicas distingue-se o excesso de produção
de fibras colágenas tipo
III no sítio reparador atribuído ao fenômeno trombogênico-símile
produzido pela presença de
plaquetas, ou mesmo pelo excesso do fator de crescimento TGF-β, o
qual parece ser um fator
ordinário que culmina em processos fibróticos como por exemplo as
mielofibroses (Giovanini et al.,
2011).
De fato a mielofibrose, também designada de metaplasia agnogênica,
é uma condição grave,
na qual há importante produção de fibroblastos, ou miofibroblastos
no interior medular (Tefferi,
2016). Este conteúdo celular acaba migrando e tomando toda porção
medular decrescendo a
possibilidade de hematogenese e linfogenese normal, podendo levar o
paciente a óbito.
Embora já tenha sido creditado anteriormente ao TGF-β como fator
desencadeador desta
peculiar condição patológica, outros fatores podem também ser
apontados como motivador tanto da
2
osteogênese quanto da fibrogenese da mielofibrose, entre os quais
destaca-se o fator de crescimento
semelhante a insulina-1 (IGF-1) (Daver et al., 2013, Ciaffoni et
al., 2015)
Há relatos de que este fator de crescimento é importante na
osteogênese, uma vez que promove
intenso crescimento ósseo em doenças, como a acromegalia. Contudo,
este fator também pode ser
responsável por ativar e intermediar receptores dependentes de
cálcio, o qual comporta-se como
segundo mensageiro à transcrição de actina de músculo liso e
consequentemente favorecendo a
diferenciação da mielofibrose (Giovanini et al., 2011).
Outro receptor que trabalha neste sentido parece ser o receptor
inositol trisfosfato 1,4,5
também conhecido como IP3R. Este é um complexo membrânico
glicoproteico que atua como canal
de Ca2+ ativado por inositol trifosfato (IP3). Esse receptor abre
os pertuitos membranares, as vias
intracelulares ao cálcio minimizando a deposição iônica no ambiente
extracelular, ao mesmo tempo
que trabalha como indutor da diferenciação de células musculares ou
miofibroblastos (Liu et al.,
2011), que expressam a proteína actina de músculo liso (SMA).
3
2. REVISÃO DE LITERATURA
A eficiente terapêutica para lesões ósseas extensas em locci que
foram perdidos por tumores,
cistos ou traumas, são um grande desafio na prática da cirúrgica,
ortopédica e bucomaxilofacial. A
alternativa de escolha, considerada ainda como “gold standard” para
reconstrução de um defeito ósseo
é por meio do uso de enxerto ósseo autólogo particulado (Burchardt,
1983). Sua utilização
proporciona atividade osteocondutiva no sítio de reparo, sem
exercer atividade antigênica, uma vez
que ainda possui remanescente celular gênico viável, e seu
componente mineral serve como
arcabouço ou scaffold para o processo reparativo (Schroeder &
Mosheiff, 2011).
É digno de nota que a obtenção de material de auto enxerto é
limitado, e também proporciona
ao paciente maior morbidade cirúrgica. Assim, uma a combinação do
transplante autógeno e uso de
biomateriais liberadores de fatores de crescimento, que aumentem a
propriedade osteoindutiva e
osteocondutora são especulados na literatura como alternativa de
tratamento. Entre os biomateriais
autogênicos e que tem sido inferido como fator de melhora da
regeneração óssea é o uso de plasma
rico em plaquetas e leucócitos (L-PRP) (Marx et al., 1998; Wallace
& Froum, 2003; Marx, 2004; De
Long et al., 2007; Intini, 2009).
Foi descrito que diversos agregados plaquetários na realidade
continham não apenas
plaquetas, mas também leucócitos, e estes leucócitos poderiam
influenciar o reparo de lesões (Everts
et al., 2008). Um sistema de classificação completo foi proposto
por Dohan Ehrenfest et al. (2009),
baseando-se no conteúdo leucocitário e arquitetura de fibrina de
vários agregados plaquetários.
Dentre eles, está o plasma rico em plaquetas e leucócitos
(L-PRP).
A tese sobre o uso do L-PRP como fator osteogênico é baseada na
hipótese de que as plaquetas
presentes no concentrado autógeno, quando ativado, sintetizam e
secretam uma extensiva fonte de
fatores de crescimento liberados. Muitos desses fatores de
crescimento, como por exemplo fator de
crescimento transformante- beta (TGF-β) e fator de crescimento
semelhante à insulina-1 (IGF-1)
supostamente agem estimulando a quimiotaxia e diferenciação de
vários estágios de osteoneogênese
4
(Eppley et al.,2004; 19- Freymiller & Aghaloo, 2004; Mooren et
al., 2010).
Mesmo sob esta peculiar hipótese, os resultados obtidos na
literatura não são unânimes, e
algumas pesquisas têm revelado falta de benefícios ou de
consistência no reparo ósseo induzido pelo
L-PRP, gerando conflitos sobre o o papel clínico/biológico ndeste
tratamento (Nikolidakis & Jansen,
2008; Intini, 2009; Mooren et al., 2010; Balaguer et al., 2010;
Oliveira Filho et al., 2010; Giovanini
et al., 2011), bem como têm gerado alterações na diferenciação
medular.
Intrigantemente, a divergência ocorrida no reparo L-PRP, tanto na
osteogênese quanto no
reparo intramedular, parece recair sobre a ação do TGF-β, o qual
possui efeitos pleiotrópicos. Essa
multifuncionalidade seria capaz de induzir diferentes interações
com cada componente celular
durante o reparo tecidual, conduzindo assim, diferentes vias de
sinalização, razão pelo qual não
apenas o TGF-β, mas também o IGF-1 podem alterar de forma a excitar
ou suprimir uma resposta
celular que de fato altera a diferenciação em sítio artificial de
reparo (de Oliva et al., 2009; Giovanini
et al., 2010).
De fato, a formação de tecido ósseo viável durante um processo de
reparo advém de um
processo complexo, cuja etapa final consiste na produção, maturação
e mineralização da matriz
extracelular (MEC) produzida em locci ósseos injuriados. Durante a
osteonegênese, as células que
compõem a matriz extracelular normalmente reconhecem e respondem a
estímulos extracelulares,
por meio de programas de sinalização gênica intracelular. Entre a
sinalização anabólica óssea, há
necessidade de uma ativação de proteinas morfogenéticas do osso
(BMP) e inativação do gene SOST,
o qual codifica a síntese de uma proteína denominada de
esclerostina (Winkler 2003).
A esclerostina é uma glicoproteína monomérica com um domínio
semelhante a família
Cerebrus/DAN, antagonistas da BMPs e Wnts, as quais constituem as
proteínas fundamentais da
diferenciação óssea (Balemans et al., 2001; Brunkow et al., 2001;
Veverka et al., 2009).
Funcionalmente, a esclerostina exerce efeitos anti-anabólicos,
diminuindo a proliferação e
5
diferenciação dos osteoprogenitores e promovendo a apoptose dos
osteoblastos. Curiosamente a
expressão da esclerostina, parece ser ativada pela presença do
TGF-β1, o qual é abundante no
concentrado plaquetário (Loots, 2012).
Independente da via anabólica ou catabólica da razão
BMP/esclerostina, a diferenciação dos
osteoblastos também é mantida endocrinologicamente por meio da
atuação direta ou indireta do
hormônio do crescimento (GH). Neste sentido, a via Janus quinase 2
(JAK2) - transdutores de sinal
ativadores de transcrição B (STAT5B) parece constituir um eixo
central no mecanismo de sinalização
direta de GH, ou por meio de sinalização autócrina/parácrina via
IGF-1. Assim, a ativação persistente
de JAK2/STAT5B e a inibição da degradação de STAT5B mostraram
aumento da diferenciação
osteoblástica e atividades de STAT5B/osteoproteínas.
Contudo, estudos genéticos revelam a importância da via JAK2-STAT5B
na estimulação de
outros fatores de transcrição e na expressão de outros marcadores
de diferenciação, em especial a
diferenciação das células da medula óssea.
Nessa semântica, Xu et al., (2018) demonstraram a evolução
osteoporótica via JAK2/STAT5,
por meio de indução do fator de crescimento fibroblástico 23
(FGF23).
O FGF23 é uma proteína sintetizada e secretada pelos osteócitos em
resposta a vários
estímulos, incluindo paratormônio (PTH), vitamina D, cálcio e
fósforo, sendo expresso em diversos
tecidos, como tecido osseo, vasos na medula ossea e linfonodos (Liu
et al., 2003).
Este hormônio é capaz de suprimir a expressão dos cotransportadores
de sódio-fosfato (NaPi-
2a e NaPi-2c), assim afetando a atividade do hormônio da
paratireóide, induzindo a excreção urinária
de fosfato. Camundongos transgênicos que superexpressam FGF23 tem
hipofosfatemia devido à
supressão de co-transportadores renais de NaP, bem como níveis
séricos reduzidos de calcitriol e
defeitos de deposição de minerais esqueléticos na forma de
osteomalácia, osteopenia e osteoporose.
O FGF23 também pode influenciar a atividade sistêmica da vitamina D
suprimindo a
expressão renal de 1α hidroxilase, o que resulta em diminuição da
produção de calcitriol. Esse efeito
reduz a atividade do calcitriol aumentando a síntese da enzima
catabólica 24-hidroxilase 24 e a
6
atividade reduzida de vitamina D resultante pode induzir secreção
do hormônio paratireóide e
fosfatúria concomitante (Yokota, 2010).
O efeito da secreção do paratormônio, via PTH, pode gerar
hipercalcemia. O cálcio livre,
resultado deste fenômeno, pode ativar receptores de cálcio, em
especial o receptor do fosfatidilinositol
-3 (IP3R), fazendo com que este íon adentre aos limites
intracelulares Wong, 2006).
O resultado dos níveis intracelulares de cálcio pode ser
multifatorial. Corroborando com essa
premissa, IP3R pode aumentar Ca2+, o qual medeia o comportamento
celular via Ca2+/calmodulina.
É notório que Ca2+/calmodulina regula a expressão de
osteoprotegerina, uma proteína
importante que não apenas confere ao tecido ósseo uma proteção anti
atividade osteoclástica, como
também parece ser responsável pela terminal diferenciação de
osteoblasto em osteócito, o que em
tese, aumenta a atividade de formação de matriz óssea.
Em antítese, há também relatos de que o cálcio intracelular altera
a conformação do DNA,
fato que também o condiciona como fator de tradução direta para
proteínas intracelulares, incluindo
a produção de actina de músculo liso (SMA), assim promovendo a
diferenciação de células
miofibroblásticas em detrimento à osteoblastos (Chen, 2014).
7
3. PROPOSIÇÃO
Este estudo tem o objetivo de avaliar o papel do L-PRP durante o
reparo ósseo em defeitos
criados artificialmente em calvária de ratos tratados e não
tratados com L-PRP por meio da análise
da expressão imunoistoquímica das proteínas do TGF-β1,
osteoprotegerina, osteocalcina e
esclerostina no desenvolvimento ósseo. Ainda verificar o
desenvolvimento medular por meio da
imunoexpressão do CD34, IGF-1, JAK2, STAT-5, IP3R e actina de
músculo liso.
8
4. MATERIAL E MÉTODOS
Este estudo foi aprovado pelo Comitê de Ética em uso de animais de
pesquisa (protocolo
109/09), da Universidade Positivo, estabelecido na cidade de
Curitiba, Estado do Paraná, Brasil. No
delineamento deste estudo, blocos de parafina de estudo anterior
foram reavaliados e neste trabalho
os espécimes foram submetidos ao processamento imunoistoquímico
para averiguação das proteínas
TGF-β, osteoprotegerina, osteocalcina e esclerostina no
desenvolvimento ósseo e CD34, IGF-1,
JAK2, STAT-5, IP3R e actina de músculo liso no desenvolvimento
medular.
Foram selecionados 40 ratos wistar machos hígidos, com peso entre
400 e 500g e idade entre
5-6 meses. Os ratos foram mantidos em uma sala com temperatura
controlada (22oC) e em ciclo de
claro-escuro, por 12 horas.
4.1 Obtenção do Plasma rico em plaquetas rico em plaquetas e
leucócitos (L-PRP)
Com uma seringa contendo 0,35 mL de citrato de sódio de 10% foi
coletada uma amostra de
3,2 mL de sangue, por meio de punção cardíaca de cada animal. O
conteúdo coletado foi centrifugado
a 200 × g durante 20 minutos em temperatura ambiente, para separar
o plasma contendo as plaquetas
de eritrócitos (Beckman J-6 M indução unidade centrífuga; Beckman
Instruments Inc., Palo Alto,
CA). Uma fração de plasma foi extraída da porção superior do
líquido sobrenadante e o restante foi
novamente centrifugado por 10 min a 400 × g para separar as
plaquetas. O plasma pobre em plaquetas
foi removido do nível superior do sobrenadante, deixando o PRP e
uma pequena camada
sobrenadante. A camada mais superficial e o PRP (0,36 mL) foram
aspirados com uso de pipeta de
precisão e posteriormente ativados com utilização de uma mistura de
cloreto de cálcio a 10% (0,05
mL), sendo adicionados ao PRP previamente preparado e misturados
por aproximadamente 1 minuto
até formar gel.
9
Na sequência, as plaquetas no L-PRP foram contadas depois de
centrifugadas com uma
máquina de contagem de plaqueta, da marca Coulter STK, Beckman
Coulter, Chicago, IL.
Após o procedimento, a média de 2826,44x103 (±69,81x103)
plaquetas/µL foi atingida, sendo
que a integridade morfológica e a concentração de mais de cinco
vezes e o enriquecimento do PRP
foram confirmados, enquanto os valores iniciais mensurados foram de
617,66× 103 (±69,81x103)
plaquetas/μL. O L-PRP utilizado em cada animal pertencentes aos
grupos tratados com L- PRP é
fabricado a partir do seu próprio sangue.
4.2 Procedimento cirúrgico
Inicialmente os ratos foram divididos em 2 grupos, de acordo com o
material a ser inserido
no defeito ósseo (grupo osso autógeno e osso autógeno associado ao
L-PRP), e em seguida foram
divididos novamente em dois grupos, desta vez de acordo com o tempo
de eutanásia (15 e 40 dias).
Os ratos foram anestesiados com uma injeção via intramuscular de
xilazina 5mg/kg e
quetamina 70mg/kg. Na sequência, antes da cirurgia, foi realizada a
tricotomia da região cirúrgica e
preparada com antisséptico, limitando a área de trabalho com
barreiras estéreis e efetuou-se uma
incisão de 5 cm dermoperiostal para a exposição da superfície da
calvária. Foram criados defeitos
circulares com diâmetro de 5 mm x 1 mm de profundidade com broca
trefina (Biomedic Research
Instruments Inc., Silver Sprigns, Maryland) irrigada em abundante
solução salina.
Os fragmentos ósseos removidos das calvárias foram particulados e
reutilizados como
autoenxertos nos grupos - autoenxerto ou ainda autoenxerto
associado com PRP. Nos grupos
denominados autoenxertos utilizou-se o tratamento por enxerto de
0,01mL do osso autógeno
particulado. Concomitantemente, os ratos do grupo autoenxerto
associado ao PRP receberam 0,01
mL de osso particulado associado com 150 μL de PRP. Após este
procedimento, os tecidos foram
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reposicionados e suturados (fio de seda 4-0 , Ethicon, São Paulo,
SP, Brazil). Todos os animais
receberam 1 injeção intramuscular profilática de penicilina
G-benzatina 24.000 UI e dose diária de
200mg/kg de paracetamol via oral.
4.3 Eutanásia dos Animais e Sistema de Processamento dos
Tecidos
Nos períodos pré determinados de 15 e 40 dias, os ratos foram
eutanasiados em câmara de
CO2, conforme protocolo do Biotério da Universidade Positivo.
Após a eutanásia, imediatamente os fragmentos de calvária dos ratos
foram removidos em
blocos com auxílio de utilização de broca de uso odontológico
tronco-cônica invertida. Cada bloco
cirúrgico necropsiado foi depositado em frasco contendo solução de
formol a 10% por 48 horas,
posteriormente descalcificadas em solução de ácido fórmico e
citrato de sódio a 10% por um período
aproximado de um mês.
Na sequência dos procedimentos, as amostras cirúrgicas foram
lavadas em água corrente.
As amostras foram cuidadosamente hemiseccionadas paralelamente ao
eixo sagital e seguiram
para inclusão e incorporação à parafina em cassetes de histologia
para confecção de blocos
histológicos.
Em cada bloco contendo o material necropsiado, cortes seriados de
3μm ao centro dos defeitos
de cada animal foram realizados com auxílio de micrótomo (RM2155,
Leica Microsystems, GmbH,
Nussloch, Alemanha). Os cortes foram estendidos em lâminas de vidro
para na sequência serem
corados pelo método de tricrômico de Masson, para análise
histomorfométrica e histomorfológica.
11
4.4 Processamento de Imunoistoquímica
Na análise imunoistoquímica, cortes de 3μm de espessura foram
obtidos e estendidas em
lâminas silanizadas. Cada amostra foi desparafinizada em solução de
xilol por 30 minutos em
temperatura de 60oC, secas em papel de filtro e posteriormente
submetidas à hidratação em cadeia
descendente de álcool, iniciando-se por 5 minutos em solução de
álcool absoluto, passando-se por
soluções alcoólicas em 90 GL, 80 GL e 60 GL por 5 minutos
cada.
Cada lâmina contendo a amostra seguiu para recuperação antigênica e
rompimento das pontes
fixadores pela formalina por meio de imersão a solução de pepsina
1% (pH 7,2) durante uma hora, à
temperatura de 37°C em estufa.
Na sequência, os dispositivos com amostras histológicas foram
imersos em 3% de peróxido
de hidrogênio por 30 minutos para eliminar a atividade de
peroxidase endógena, na sequência fez-se
a incubação com tampão fosfato salino (1%) - (PBS; pH 7, 4).
As amostras foram incubadas com os anticorpos primários anti-TGF-β1
(Santa Cruz
Biotechnology, EUA), anti-osteoprotegerina (Santa Cruz
Biotechnology, EUA), anti-osteocalcina
(Santa Cruz Biotechnology, EUA), e anti-esclerostina (Santa Cruz
Biotechnology, EUA). Utilizou-
se um sistema de detecção de ligação de anticorpo (LSAB plus –
DAKO) para detectar anticorpos
primários e a reação imune se revelou com a solução de tetracloreto
diaminobenzidine (Sigma, St.
Louis, MO), produzindo um precipitado marrom no local do antígeno.
As amostras foram
contracoradas com hematoxilina de Harris. Um controle negativo foi
realizado para todas as amostras,
usando isotipos IgG policlonais de Ratos (2 μg/mL, Abcam), durante
um período de dez minutos à
temperatura ambiente com um anticorpo primário.
12
4.5 Análise de Imagens
As imagens das seções histológicas e imunoistoquímica foram
capturadas com uma câmera
digital marca Samsung (Coreia do Sul) e um microscópio de luz
(ZEISS) com ampliação original
200´. As imagens digitais foram coletadas e salvas com a resolução
de 600 dpi, produzindo uma
imagem virtual de 117 × 80 cm. Não tendo sido possível capturar
todos os defeitos na imagem no
nível de ampliação usada o pesquisador construiu uma imagem digital
de todo o defeito, combinando
duas imagens menores com base nas estruturas de referência
histomorfológica, especialmente, das
trabéculas ósseas depositadas, além de vasos sanguíneos.
As mensurações histomorfométricas foram realizadas usando o
software Image Tool 2,00
(Universityof Texas), sendo os dados contados manualmente,
expressos por área ou pelo número de
células por mm2, dependente da proteína respectivamente de matriz
extracelular ou nuclear.
Nos dados obtidos foram marcadas as células e a matriz com um
sistema de detecção
automático de cores (Amarela/marrom) utilizando o software Image J.
Os perímetros da matriz óssea
histológica foram depositados, as áreas de maior nicho da medula
óssea foram rastreadas e
computadas, células e matrizes positivas foram contadas e
identificadas manualmente.
Uma imagem de 1 mm foi usada para calibrar todas as medições. Os
dispositivos foram
analisados para cada um dos parâmetros e uma média de 3 medições
para cada parâmetro foi calculada
para cada amostra. No sentido de homogeneizar a análise dos dados,
a totalidade foi transformada em
percentuais para facilitar a análise comparativa entre grupos (área
mm2 e células/mm2).
4.6 Análise Estatística
Os parâmetros histomorfométricos, área de matriz óssea e osso
medular formados e a
frequência da presença das células imunomarcadas, foram avaliados
no período de acompanhamento,
13
principalmente, as semelhanças mais significativas entre os dois
grupos. A imunoistoquímica foi
determinada por escore, que consistiu no seguinte critério (Tabela
1):
Símbolo % células imunopositivas
Tabela 1: Determinação de símbolo a partir daporcentagem de células
imunopositivas.
14
ON OSTEOCONDUCTIVITY OF AUTOGRAFT IN PRESENCE OF LEUKOCYTE-
PLATELET-RICH PLASMA (L-PRP)
PURPOSE: The aim of this study was to evaluate the osteoconductive
effect of an autograft, in the
presence or absence of the L-PRP, using histomorphometric analysis
of the bone formed, comparing
the results in the presence of TGF-β1, OPG, OC and sclerostin
detected by immunohistochemistry.
METHODS: Two bone defects were produced in the calvaria of 40 rats.
The defects were treated
with autograft and autograft combined with L-PRP. The animals were
euthanized at 15 and 40 days
post-surgery. Data was analyzed by histomorphometric and
immunohistochemical interpretation.
RESULTS: The results revealed that the presence of bone matrix was
significantly smaller in the
defects treated with L-PRP. These results coincided with changes of
the immunolocalization of the
TGF-β1. CONCLUSION: The use o L-PRP suppresses osteoneogenesis
since it increases
simultaneously TGF-β and sclerotin expression, and decreases the
expression of OPG and OC.
Key-words: L-PRP, TGF-β, sclerostin, osteoprotegerin,
osteocalcin
INTRODUCTION
After Marx (2004) described an important results demonstrating
larger and faster bone
formation using application of autogenous blood fraction rich in
platelets as a biomaterial, this
compound named platelet-rich plasma became a promising alternative
for the treatment craniofacial
bone defects. The favorable use of PRP for osteogenesis has been
justified by the hypothesis that
platelets produce growth factors, especially TGF-β1, which
modulates not only the osteoproliferation
15
are responsible for bone matrix formation.
Despite this remarkable hypothetical action, it has been
demonstrated that beneficial results
of PRP on osteogenesis is not unanimous. A relevant speculation in
the divergent results produced
by PRP has been assigned in the heterogenous production of PRP.
While the original definition of
PRP uses a only pure mixture of plasma and platelets, the majority
of studies published in literature
reveals that a common fabrication of PRP has expanded to include a
variety of hematological final
products; including leukocytes (L-PRP).
In this context, the real action of L-PRP remain unclear. Several
studies have demonstrated
successful of L-PRP due to presence of TFG-β1 and its capability of
osteodifferntiation induction.
However, several studies in animals, have revealed failure of bone
formation associated to the use of
L-PRP in bone sites.
Two proteins are considered to be very important markers for bone
formation: osteoprotegerin
and osteocalcin. Osteoprotegerin is an amino acid synthesized
mainly by osteoblasts. It is a member
of the TNF receptor super-family, attributed to bone homeostasis by
acting as a competitive receptor
for RANKL, preventing its binding to RANK, thus decreasing the
action of osteoclasts (Hofbauer &
Heufelder, 2001). Osteocalcin is also a protein synthesized by
mature osteoblasts. It is responsible
for regulation of bone mineralization through bonding of calcium to
the ECM (Wei & Karsenty,
2015).
Based in these perspective, we evaluated the immunoexpression of
TGF-β1, osteoprotegerin,
osteocalcin and sclerotin in bone sites treated with autograft in
presence ou absence of L-PRP.
16
MATERIAL AND METHODS
Forty, 5 to 6 month-old male Wistar rats (Rattus norvegicus
albinus) weighing 400 to 500g
and no previous disease were used following a protocol (109/09)
approved by the Institutional Board
for Animal Care and Use. The rats were kept in a room with a
controlled temperature (22oC) and
maintained under a 12-h light-dark cycle. The protocol of PRP
production and quantification as well
as the surgical procedures performed in this study are described
below.
L-PRP preparation
An amount of 3.2 mL of autogenous blood was collected from each
animal through cardiac
puncture into a syringe containing 0.35 mL of 10% sodium citrate.
The blood collected from each
animal was centrifuged at 200×g for 20 min at room temperature in
order to separate the plasma and
platelets from the erythrocytes (Beckman J-6M Induction Drive
Centrifuge; Beckman Instruments
Inc., Palo Alto, CA, USA). The plasma fraction was collected from
the top of the supernatant. The
remaining portion was centrifuged once more at 400×g for 10 min at
room temperature to separate
the platelets. The plasma fraction was removed from the upper level
of the supernatant, leaving the
PRP and buffy coat. Both the buffy coat and PRP (0.35 mL) were
re-mixed and activated with a
mixture of 10% calcium chloride (0.05 mL/mL of PRP). They were then
added to the previously
prepared PRP and mixed for 1 min until they formed a gel.
The platelets and leukocytes on the PRP were counted after
centrifugation using a Coulter
STKS hematology-counting machine (Beckman-Coulter, Chicago, IL,
USA).
Surgical Procedure
The rats were anesthetized by intramuscular injection of xylazine
(5 mg/kg) and ketamine (70
17
mg/kg). The surgical region was shaved and aseptically prepared
with sterile barriers in order to limit
the surgical field. A 5-cm dermo-periosteal incision was made along
the midline to expose the
calvarium surface with complete removal of the periosteum in order
to remove the fibroblast and
periosteum stem cells and their possible proliferation into the
artificial defect. An artificial defect of
5×1 mm (diameter × depth) was created with a trephine (Biomedical
Research Instruments Inc., Silver
Spring, MD, USA) under abundant saline solution irrigation in each
rat.
Bone fragments removed from each rat's own calvarium were
particulated and used as
autograft. Particulation of calvarium bone fragment was obtained
using an exclusive periodontal
instrument for bone particulation developed by Neodent (Curitiba,
PR, Brazil). Images of the particles
were captured with a digital camera and analyzed with Image J
software (National Institutes of Health,
Bethesda, MD, USA) to determine the average particle size. An image
size of 1 mm was used to
standardize all measurements. The average bone particle size of
autograft was 0.95±0.03 mm2.
The animals were randomly assigned to 4 groups (n=10), according to
the material used in
each defect - filled with 0.01 mL of autograft or 0.01 mL of
autograft plus 0.15 mL PRP, and
according to euthanasia date (15 or 40 days). Soft tissues were
repositioned and sutured to achieve
primary closure (4-0 silk, Ethicon, São Paulo, SP, Brazil). Each
animal received a prophylactic
intramuscular injection of 24,000 IU of penicillin G benzathine and
a daily dose of 200 mg/kg/day of
liquid acetaminophen administrated orally.
Euthanasia Procedure and Tissue Processing
On the 15th and 40th post-surgery (n=12/group) the animals were
euthanized by brief
exposure in a CO2 chamber. The calvarium of each animal was
necropsied using an inverted cone
bur. The fragments obtained were fixed in 10% buffered formalin for
48 h and decalcified in 20%
formic acid and sodium citrate for 7 days. The specimens were
washed with tap water, dehydrated,
18
cleared in xylene and embedded in paraffin. Serial 3-µm-thick
sections parallel to the mid-sagittal
suture were cut from the center of each defect using a microtome
(RM2155, Leica Microsystems
GmbH, Germany) and stained with Giemsa to observe the qualitative
and quantitative histological
characteristics.
Immunohistochemistry Processing
The specimens were deparaffinized and subjected to antigen
retrieval 1% pepsin solution (pH
1.8) for 1h at 37oC for all the antibodies. The slides containing
the histological pieces were immersed
in 3% hydrogen peroxide for 30 min to remove endogenous peroxidase
activity, followed by
incubation with 1% phosphate-buffered saline (pH 7.4; PBS). The
sections were incubated overnight
with the primary antibody anti-TGF-β1, anti osteoprotegerin,
anti-osteocalcin and anti-sclerostin. The
labeled streptavidin biotin antibody-binding detection system
(Universal HRP immunostaining kit;
Diagnostic Biosystems, Foster City, CA, USA) was employed to detect
the primary antibodies. The
immune reaction was revealed with diaminobenzidine tetrachloride
chromogen solution (Diagnostic
Biosystems), which produced a brown precipitate at the antigen
site. The specimens were
counterstained with Harris hematoxylin for 30 s. A negative control
was made for all samples using
rabbit polyclonal isotype IgG (2 µg/mL, Abcam, ab 27472) for 10 min
at room temperature as a
primary antibody. For each specimen, three slides were used for
incubation with each antibody.
Image Analysis
The images of both the histological and immunohistochemistry slices
were taken with a digital
camera (Samsung, Seoul, South Korea) connected to a light
microscope with 200× original
magnification. Each digital image was captured and saved with 600
dpi resolution, producing a virtual
picture of 115.88×81.93mm. Because it was not possible to have the
entire bone defect in a single
19
image at the used magnification, a digital image of the whole
defect was built by combining two
smaller images based on reference histological structures. The
significant similarities among all
groups were determined by score:
Symbol % immunopositive cells
RESULTS
For platelets, the average number in the whole blood and in the PRP
was 638.62±54.12×103
and 2791.81±312.28×103 platelets/µL, respectively (p<0.05). For
leukocytes, the average number in
the whole blood and in the PRP was 9.07±0.32×103 and 3.01±0.71×103
leukocytes/mL (p<0.05).
Repair in defects treated with autograft: On the 15th day
post-surgery, bone fragments were
shown to have scarce new bone formation from the autograft bone
among the granulation tissue (GT),
which was comprised of leukocytes and myeloid cells and intense
collagen deposition. On the 40th
day post-surgery, the areas where the artificial bone defect was
created revealed the formation of
haversian compact bone (HCB). Further, areas composed of fibrous
tissue were present, however
they were restricted in well-formed bone marrow that surrounded the
HCB tissue.
Repair in defects treated with autograft associated with L-PRP: On
the 15th day post-surgery,
implanted bone fragments were detected among the granulation
tissue. This GT was comprised of
collagen fibers surrounding scarce inflammatory cells, especially
represented by mononuclear cells.
On the 40th day post surgery, the microscopic analysis of
reparative sites in specimens that received
20
autografts associated with L-PRP demonstrated the presence of a
trabecular bone (TB) that
surrounded a medullary area (MA) comprised of fibrous and adipocyte
tissue.
Immunohistochemical results
A brief description of the immunohistochemical characteristics
found for each group is
provided below.
TGF-β1
On the 15th day post-surgery, TGF-β1 was present in all specimens,
as seen in Fig. 1. In the
specimens that received L-PRP, the percentage of the positive area
was significantly higher when
compared to specimens treated only with autograft. In both groups,
the presence of TGF-β1 was
positive in cells surrounding the autograft (AG) and peripheral to
blood vessels present in the
granulation tissue (GT). Specially in the specimens that received
L-PRP, immunoexpression was also
seen in the cells and in the extracellular matrix that comprised
the GT. On the 40th day post-operative,
the pattern of TGF-β1 immunohistochemical expression in both groups
remained similar to the 15th
day post-surgery; however, the percentage of TGF-β1 decreased as
soon as the bone matrix or
medullary area (MA) formed, as can be seen in sites treated with
autograft in Fig. and by autograft
associated with L-PRP.
21
Fig 1. Immunoexpression patern of TGF-β1 among all 4 groups. A and
C correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
Osteoprotegerin (OPG) and osteocalcin (OC)
The presence of OPG and OC+ cells was observed in all groups on the
15th day post-surgery
for the group treated only with autograft and for the group that
received autograft associated with L-
PRP (Fig. 2 and Fig. 3). A higher quantity of OPG and OC+ cells was
found in the control group
when compared to the groups that receive the L-PRP. Similar
findings were observed at 40 days post-
operation. However, the quantity of OPG and OC+ cells decreased as
soon as the bone matrix or
22
medullary area (MA) were formed, as demonstrated in the sites
treated by autograft and by autograft
associated with L-PRP in Fig.2 and Fig. 3, respectively.
Fig. 2- Pattern of osteoprotegerin immunoexpression between groups.
A and C correspond to the
control group (autograft), respectively between 15 and 40 days,
while B and D demonstrate the OPG
pattern in the group receiving L-PRP+autograft.
23
Fig 3. - Pattern of osteocalcin immunoexpression between groups. A
and C correspond to the control
group, respectively between 15 and 40 days, while B and D
demonstrate the OC pattern in the group
receiving L-PRP.
Sclerotin
The presence of sclerotin was observed mainly on specimens that
received L-PRP. in this
group the patter of sclerotin+ cells were demonstrated an spread
pattern among the defect while on
24
control group the presence protein was scarce. This
immunohistochemical pattern was similar both
15 and 40th day postoperative time period (Fig. 4).
Fig. 4 - Pattern of sclerostin immunoexpression between groups. A
and C correspond to the control
group, respectively between 15 and 40 days, while B and D
demonstrate the sclerostin pattern in the
group receiving L-PRP.
25
Table 1 - Score of immunopositivity for TGF-β1, OPG, OC e
Sclerostin among the groups
Time period Group Protein Protein Protein Protein
TGF-β1 OPG OC SCLEROSTIN
L-PRP ++++ + + +++
Discussion
It is known previously that the autograft is considered a gold
standard for bone grafts due to
its important osteogenic and osteoconductive potentials. This
hypothesis comes from the observation
that autograft has the capacity to stimulate new bone formation
either by recruitment of
osteoprogenitor mesenchymal stem cells or by regenerating itself
through production of new bone
(Zhang et al., 2008). However the amount of autogenous bone for
grafting is limited (Banwart et al.,
1995; De Long et al., 2007; Giovanini et al., 2010); thus, the
combination of autograft with L-PRP
has been considered a likely alternative for increasing bone
quantity, as L-PRP possesses chemotactic
and mitotic action (Marx et al., 1998; Wallace & Froum, 2003;
Aghaloo et al., 2004; Marx, 2004; De
Long et al., 2007; Intini, 2009). Despite of the appealing reason,
several manuscripts have revealed
failure of osteoconductor potential when the L-PRP is used.
In this context Giovanini et al. (2014) has suggested that although
use of L-PRP induces
osteoproliferation, when the biomaterial is applied associated with
autograft, the presence of TGF-β1
could inhibit the terminal differentiation of osteoblasts
suppression the bone matrix development.
26
In fact, bone formation is a complex event in which the final steps
are the production,
maturation and mineralization of the extracellular matrix in
injured bone sites. In this pathway the
differentiation of preosteoblasts into mature osteoblasts is
essencial and goes through distinct stages
and is under the control of transcription factors. During
differentiation from preosteoblast to mature
osteoblasts, the mineralization potential has been correlated in
osteoprogenitors cells that express
osteoprotegerin (OPG), indicating that OPG promotes matrix
maturation in preosteoblast.
On other hand, when osteoblast undergo transformation into
osteocytes, they increase the
expression of some proteins through the modulation of the
transforming growth factor-β (TGF-β)
dependent pathway, such sclerostin.
Sclerostin is an osteocyte-derived glycoprotein that inhibits
Wnt/β-catenin signaling and
activation of osteoblast function, thereby inhibiting bone
formation. It plays a vital role in the
regulation of skeletal growth, and its expression has been
associated to osteopenia and osteoporosis.
Herein we demonstrated that the L-PRP improved the expression of
sclerotin, condition that
concided to lower bone matrix formation in artificial bone
sites.
A study by Martina Gruber et al. (2017) has demonstrated an
important correlation between
TGF-β and sclerotin in post treatment of periodontitis, where
occurred limitation of bone
development in area treated, agreeing with our results.
It is highlighted that the connection between TGF-β1 and sclerotin
is complex. Loots (2012)
suggested that the TGF-β1 activates the receptor of membrane, named
Ecdysone Receptor (ECR5),
which activates second messengers that excite the SOSt gene. In
turn, this gene is responsible for
transcription and tradition of protein named sclerotin, that
usually suppress bone genes and
consequently osteoproteins, as demonstrated herein through
decreased of OPG and OC.
27
CONCLUSION
It may be inferred that use of L-PRP suppresses osteoneogenesis
since it increases
simultaneously the TGF-β and Sclerotin, and decreases the
expression of OPG and OC.
REFERENCES
1. Marx RE. Platelet-rich plasma: evidence to support its use. J
Oral Maxillofac Surg 2004;62: 489-
496.
2. Hofbauer LC, Heufelder AE. Role of receptor activator of nuclear
factor- KB ligand and
osteoprotegerin in bone cell biology. J Mol Med
2001;79:243-53.
3. Wei J, Karsenty G. An overview of the metabolic functions of
osteocalcin. Rev Endocr Metab
Disord 2015; 16:93–98.
4. Zhang X, Awad HA, O'Keefe RJ, Guldberg RE, Schwarz EM. A
perspective: engineering
periosteum for structural bone graft healing. Clin Orthop Relat Res
2008;466:1777-1787.
5. Banwart JC, Asher MA, Hassanein RS. Iliac crest bone graft
harvest donor site morbidity. A
statistical evaluation. Spine (Phila Pa 1976)
1995;20:1055-1060.
6. De Long WG Jr, Einhorn TA, Koval K, McKee, Smith W, Sanders R,
Watson T. Bone grafts and
bone graft substitutes in orthopaedic trauma surgery. A critical
analysis. J Bone Joint Surg Am
2007;89:649-658.
7. Giovanini AF, Deliberador TM, Gonzaga CC, de Oliveira Filho MA,
Göhringer I, Kuczera J,
Zielak JC, de Andrade Urban C. Platelet-rich plasma diminishes
calvarial bone repair associated with
1603.
8. Wallace SS, Froum SJ. Effect of maxillary sinus augmentation on
the survival of endosseous dental
implants. A systematic review. Ann Periodontol
2003;8:328-343.
9. Aghaloo TL, Moy PK, Freymiller EG. Evaluation of platelet-rich
plasma in combination with
anorganic bovine bone in the rabbit cranium: a pilot study. Int J
Oral Maxillofac Implants 2004;19:59-
65.
10. Intini G: The use of platelet-rich plasma in bone
reconstruction therapy. Biomaterials 2009;30:
4956-4966.
11. Giovanini AF1, Grossi JR, Gonzaga CC, Zielak JC, Göhringer I,
Vieira Jde S, Kuczera J, de
Oliveira Filho MA, Deliberador TM. Leukocyte-platelet-rich plasma
(L-PRP) induces an abnormal
histophenotype in craniofacial bone repair associated with changes
in the immunopositivity of the
hematopoietic clusters of differentiation, osteoproteins, and
TGF-β1. Clin Implant Dent Relat Res.
2014;16:259-272.
12. Gruber M, Gruber R, Agis H. Transforming growth factor -B1
increases sclerostin in fibroblasts
of the periodontal ligament and the gingiva. Matters Achive
2017;1-4.
13. Loots GG, Keller H, Leupin O, Murugesh D, Collette NM, Genetos
DC. TGF-β regulates
sclerostin expression via the ECR5 enhancer. Bone
2012;50:663-639.
29
MANUSCRITO 2
EFFECT OF PLATELET RICH PLASMA (L-PRP) ON EXPRESSION OF CD34,
JAK2/STAT-5;
IGF-1, FGF23, IP3R, TGF- β, CALMODULIN AND SMOOTH ACTIN MUSCLE
DURING
MEDULLARY DIFFERENTIATION IN ARTIFICIAL BONE DEFECTS
PURPOSE: The aim of this study was to evaluate the effect of L-PRP
during medullary
differentiation in bone defects through the analysis of the
expression of CD34, JAK2, STAT-5, IGF-
1, FGF23, IP3R, TGF-β, calmodulin and smooth actin muscle. METHODS:
A bone defect was
produced in the calvaria of 40 rats, measuring 5mm in diameter and
1mm in depth. The defects were
treated with autograft and autograft combined with L-PRP. The
animals were euthanized at 15 and
40 days post-surgery. Data was analyzed by immunohistochemical
interpretation. RESULTS: These
results suggest that bone repair was impaired due to the increase
in TGF-β expression. Also, it is
notable that the expression of medullary differentiation markers
were overexpressed in the L-PRP
autograft group in both time periods, when compared to the
autograft group. CONCLUSION: The
use of PRP hindered bone deposition by increase in TGF-β, enhanced
chemotaxis of CD34+
progenitor cells, and demonstrated two non-canonical developmental
pathways of pathological
fibrotic processes through JAK2, STAT-5, IGF-1, FGF23, IP3R,
calmodulin and smooth actin muscle
interactions.
Key-words: L-PRP, JAK2, STAT5, IGF-1, FGF23, TGF-β, IP3R, SMA,
calmodulin
Introduction
Leukocytes-platelet rich plasma (L-PRP) is an autologous
biomaterial composed of a small
amount of plasma enriched with high platelet concentrations and
leukocytes, which is obtained from
the centrifugation of a blood fraction with the consequent removal
of red blood cells (Marx 2004).
30
The premise of its orthopedic use derives from the hypothesis that
platelets contained in L-PRP are
responsible for the synthesis and secretion of numerous growth
factors, including TGF-β, PDGF and
IGF-1 (Intini 2009, Nagata et al., 2009). These growth factors are
considered as important modulators
for cell differentiation and consequently restoration of an organ
or tissue lost under trauma, surgery
and tumors (Nikolidakis et al., 2008; Nikolidakis et al.,
2009).
Although several authors raise the regenerative success when using
L-PRP, numerous studies
reveal that platelet aggregation can also raise or mimic
pathological conditions (Oliveira Filho et al.
2010, Giovanini et al., 2014).
In this context Giovanni et al. (2010) revealed intense amount of
CD34+ cells in artificial
bone sites treated with platelet concentrated. Despite the fact
that the authors suggested that the
presence of these cells supressed the osteoneogenesis, another fact
may be inferred from this results.
Usually the larger amount of CD34+ cells (>20% of medullary
cells) may be indicative of leukemia
field.
Another study demonstrated that use of L-PRP also may develop
fibrosis with excess
production of type III collagen fibers, which according to authors
this phenomenon may be due to or
thrombogenic-like phenomenon produced by the presence of platelets,
or even by the excess growth
factor TGF-β1, which seems to be an ordinary factor culminating in
pathological fibrotic processes
mimicking myelofibrosis (Giovanini et al., 2011).
In fact myelofibrosis, also called agnogenic metaplasia, is a
serious condition in which there
is significant production and disseminated infiltration of
fibroblasts, or myofibroblasts within the
medullar area (Tefferi, 2016). This cellular content ends up
migrating and taking the entire medullary
portion decreasing the possibility of normal hematogenesis and
lymphogenesis, which, in extreme
cases, may lead the patient to death.
Although it has been previously credited to TGF-β1 as being a
triggering factor of this peculiar
pathological condition, other factors may also be pointed as
motivating both osteogenesis and
fibrogenesis of myelofibrosis as well as leukemia area, including
insulin-like growth factor 1 (IGF-
31
1) (Daver et al., 2013; Ciaffoni et al., 2015). This growth factor
has been reported to be important in
osteogenesis as it promotes intense bone growth in diseases such as
acromegaly. However, this
growth factor may also be responsible for activating and mediating
calcium-dependent receptors,
which behave as a second messenger to the bone smooth muscle actin
transcription and consequently
favoring differentiation of myelofibrosis (Giovanini et al.,
2011).
One receptor working in this regard appears to be the inositol
triphosphate 1,4,5 receptor also
known as IP3R. This is a membrane glycoprotein complex which acts
as an inositol triphosphate
(IP3) activated Ca2+ channel that interact to its intracellular
protein named calmodulin. This
interaction activates various biological process including
differentiation of muscle cells or
myofibroblasts (13- Liu et al., 2011), which express the smooth
muscle actin (SMA).
It is highlighted that other pathways may culminate to leukemia and
myelofibrosis
development. Among them, the presence of Jak-2/Stat-5 seems to be a
crucial event for myelfibrosis,
as well as the intense presence of FGF23, which decrease levels of
phosphate and improves the
pathways IP3R and calmodulin.
Despite this knowledge, these proteins were not correlated to bone
repair when L-PRP is used.
So, in this present study, we evaluated the immunoexpression of
IGF-1, IP3R, calmodulin, Jak-2,
STAT-5, CD34 and SMA in specimens treated and not treated with
L-PRP.
Material and Methods
Forty 5-6-month-old male Wistar rats (Rattus norvegicus albinus)
weighing 400 to 500 g and no
previous disease were used following a protocol approved by the
Institutional Board for Animal Care
and Use. The rats were kept in a room with a controlled temperature
(22 oC) and maintained under a
12-h light-dark cycle. The protocol of PRP production and
quantification as well as the surgical
procedures performed in this study were based on Nagata et al.
(2009) and are described below.
32
PRP Production and Quantification
An amount of 3.2 mL of autogenous blood was collected from each
animal through cardiac puncture
into a syringe containing 0.35 mL of 10% sodium citrate. The blood
collected from each animal was
centrifuged at 200×g for 20 min at room temperature in order to
separate the plasma and platelets
from the erythrocytes (Beckman J-6M Induction Drive Centrifuge;
Beckman Instruments Inc., Palo
Alto, CA, USA). The plasma fraction was collected from the top of
the supernatant. The remaining
portion was centrifuged once more at 400×g for 10 min at room
temperature to separate the platelets.
The plasma fraction was removed from the upper level of the
supernatant, leaving the PRP and buffy
coat. Both the buffy coat and PRP (0.35 mL) were re-mixed and
activated with a mixture of 10%
calcium chloride (0.05 mL/mL of PRP). They were then added to the
previously prepared PRP and
mixed for 1 min until they formed a gel.
The platelets and leukocytes on the PRP were counted after
centrifugation using a Coulter STKS
hematology-counting machine (Beckman-Coulter, Chicago, IL,
USA).
Surgical Procedure
The rats were anesthetized by intramuscular injection of xylazine
(5 mg/kg) and ketamine (70 mg/kg).
The surgical region was shaved and aseptically prepared with
sterile barriers in order to limit the
surgical field. A 5-cm dermo-periosteal incision was made along the
midline to expose the calvarium
surface with complete removal of the periosteum in order to remove
the fibroblast and periosteum
stem cells and their possible proliferation into the artificial
defect. An artificial defect of 5×1 mm
(diameter × depth) was created with a trephine (Biomedical Research
Instruments Inc., Silver Spring,
MD, USA) under abundant saline solution irrigation in each
rat.
The animals were randomly assigned to 4 groups (n=12), according to
the material used in
33
each defect - filled with 0.01 mL of autograft or 0.01 mL of
autograft plus 0.15 mL PRP, and
according to euthanasia date (15 and 40 days). Bone fragments
removed from each rat's own
calvarium were particulated and used as autograft. Particulation of
calvarium bone fragment was
obtained using an exclusive periodontal instrument for bone
particulation developed by Neodent
(Curitiba, PR, Brazil). Soft tissues were repositioned and sutured
to achieve primary closure (4-0 silk,
Ethicon, São Paulo, SP, Brazil).
Each animal received a prophylactic intramuscular injection of
24,000 IU of penicillin G
benzathine and a daily dose of 200 mg/kg/day of liquid
acetaminophen administrated orally.
Images of the particles were captured with a digital camera and
analyzed with Image J
software (National Institutes of Health, Bethesda, MD, USA) to
determine the average particle size.
An image size of 1 mm was used to standardize all measurements. The
average bone particle size of
autograft was 0.95±0.03 mm2.
Euthanasia Procedure and Tissue Processing
On the 15th and 40th day post-surgery (n=12/group) the animals were
euthanized by brief
exposure in a CO2 chamber. The calvarium of each animal was
necropsied using an inverted cone
bur. The fragments obtained were fixed in 10% buffered formalin for
48 h and decalcified in 20%
formic acid and sodium citrate for 7 days. The specimens were
washed with tap water, dehydrated,
cleared in xylene and embedded in paraffin. Serial 3-µm-thick
sections parallel to the mid-sagittal
suture were cut from the center of each defect using a microtome
(RM2155, Leica Microsystems
GmbH, Nussloch, Germany) and stained with Giemsa to observe the
qualitative and quantitative
histological characteristics.
Immunohistochemistry Processing
The specimens were deparaffinized and subjected to antigen
retrieval 1% pepsin solution (pH
1.8) for 1 h at 37oC for all the antibodies. The slides containing
the histological pieces were immersed
in 3% hydrogen peroxide for 30 min to remove endogenous peroxidase
activity, followed by
incubation with 1% phosphate-buffered saline (pH 7.4; PBS). The
sections were incubated overnight
with the primary antibody anti-IGF-1 (Santa Cruz Biotechnology,
USA), anti-IP3R (Santa Cruz
Biotechnology, USA), anti-calmodulin (Santa Cruz Biotechnology,
USA), anti-Jak-2/Stat-5 (Santa
Cruz Biotechnology, USA), anti-CD34 (Santa Cruz Biotechnology, USA)
and anti-SMA (Santa Cruz
Biotechnology, USA). The labeled streptavidin biotin
antibody-binding detection system (Universal
HRP immunostaining kit; Diagnostic Biosystems, Foster City, CA,
USA) was employed to detect the
primary antibodies. The immune reaction was revealed with
diaminobenzidine tetrachloride
chromogen solution (Diagnostic Biosystems), which produced a brown
precipitate at the antigen site.
The specimens were counterstained with Harris hematoxylin for 30 s.
A negative control was made
for all samples using rabbit polyclonal isotype IgG (2 µg/mL,
Abcam, ab 27472) for 10 min at room
temperature as a primary antibody. For each specimen, three slides
were used for incubation with
each antibody.
Image Analysis
The images of both the histological and immunohistochemistry slices
were taken with a digital
camera (Samsung, Seoul, South Korea) connected to a light
microscope with 200× original
magnification. Each digital image was captured and saved with 600
dpi resolution, producing a virtual
picture of 115.88×81.93 cm. Because it was not possible to have the
entire bone defect in a single
image at the used magnification, a digital image of the whole
defect was built by combining two
smaller images based on reference histological structures.
35
Symbol % immunopositive cells
Platelet and Leukocyte Counts
For platelets, the average number in the whole blood and in the PRP
was 638.62±54.12×103
and 2791.81±312.28×103 platelets/mL. For leukocytes, the average
number in the whole blood and
in the PRP was 9.07±0.32×103 and 3.01±0.71×103 leukocytes/mL.
The percentage of the quantitive data for each immunohistochemical
protein is given in Table
1. A brief description of the immunohistochemical characteristics
found for each group is provided
below.
IGF-1
The presence of IGF-1 was observed in all groups, as seen in Fig.
1. IGF-1 was mainly
36
observed in the L-PRP+autograft group in both time periods, while
in the control group the expression
was scarce specially on day 40.
Fig 1. Immunoexpression patern of IGF-1 among all 4 groups. A and C
correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
TGF-β1
On the 15th day post-surgery, TGF-β1 was present in all specimens,
as seen in Fig. 2. In the
specimens that received L-PRP, the percentage of the positive area
was significantly higher when
compared to specimens treated only with autograft (Table 1). In
both groups, the presence of TGF-
β1 was positive in cells surrounding the autograft (AG) and
peripheral to blood vessels present in the
granulation tissue (GT). Specially in the specimens that received
L-PRP, immunoexpression was also
seen in the cells and in the extracellular matrix that comprised
the GT. On the 40th day post-operative,
the pattern of TGF-β1 immunohistochemical expression in both groups
remained similar to the 15th
day post-surgery; however, the percentage of TGF-β1 decreased as
soon as the bone matrix or
37
medullary area (MA) formed, as can be seen in sites treated with
autograft in and by autograft
associated with L-PRP.
Fig 2. Immunoexpression patern of TGF-β1 among all 4 groups. A and
C correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
CD34
CD34+ cells were observed in all time periods and groups inside the
medullary area (Fig. 3).
The expression of the cells was similar in the autograft groups in
both time periods. In the L+PRP
groups, the expression of the CD34 cells diminished some in the
40th day, but still overexposed when
compared to the same time period in the the graft group.
38
Fig 3. Immunoexpression patern of CD34+ among all 4 groups. A and C
correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
FGF23
The presence of FGF23 was mainly observed in the L-PRP groups in
both time periods (Fig.
4). The expression of this protein was scarce in the autograft
group, specially on the 40th day post-
op.
39
Fig 4. Immunoexpression patern of FGF23 among all 4 groups. A and C
correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
IP3R
IPR3R were observed in all groups and all times. The expression
pattern remained similar in
40
both time periods, as seen in Fig. 5.
Fig 5. Immunoexpression patern of IP3R among all 4 groups. A and C
correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
CALMODULIN
The expression of calmodulin was observed mainly on the specimens
which received L-
PRP+autograft, showing a similar pattern in both time periods. On
the other hand, in the autograft
group, it is noteworthy that an increase of calmodulin occurred in
the 40th day group, when compared
41
to the same group on the 15th day post-op (Fig. 6).
Fig 6. Immunoexpression patern of calmodulin among all 4 groups. A
and C correspond to the
autograft group, 15 and 40 days respectively. B and D correspond to
the L-PRP + autograft group, 15
and 40 days respectively.
JAK2
In the L-PRP group, JAK2 was intensely expressed on the 15th day
and remained highly
expressed also on the 40th day time period. On the other hand, in
the autograft group, the expression
of this protein diminished on day 40 compared to day 15 (Fig.
7).
42
Fig 7. Immunoexpression patern of JAK2 among all 4 groups. A and C
correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
STAT-5
The presence of STAT-5 was observed mainly in the L-PRP group, in
both time periods. On
the control group, a higher quantity of STAT-5 was observed on the
15th day, declining over time,
becoming very scarce.
43
Fig 8. Immunoexpression patern of STAT5 among all 4 groups. A and C
correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
SMOOTH MUSCLE ACTIN (SMA)
The presence of smooth muscle actin was observed in all both graft
groups and time period
(Fig. 9). A higher quantity of SMA was found in the L-PRP+autograft
group in both time periods
when compared to the other graft group. On the autograft group, we
can observe the expression
decrease during the time periods. However, the presence of this
marker was exacerbated in the 40th
day L-PRP group when compared to the 15th day group.
44
Fig 9. Immunoexpression patern of SMA among all 4 groups. A and C
correspond to the autograft
group, 15 and 40 days respectively. B and D correspond to the L-PRP
+ autograft group, 15 and 40
days respectively.
TABLE 1 - demonstrates the score of proteins among the
specimens
TIME PERIOD PROTEIN CONTROL L-PRP
DAY 15 IGF-1 ++ ++++
TGF-β1 + ++++
CD34 + +++
FGF-23 - +++
IP3R + +++
CALMODULIN ++ +++
JAK-2 - ++++
STAT-5 - ++++
SMA + ++++
Discussion
When bone repair process occurs, sequential steps are noticed, and
primarily include an
acute inflammatory phase, where the fundamental effect biological
is represented by migration of
granulocytes and macrophages in regenerative sites and also occurs
platelet aggregation and
46
activation for posteriorly the mesenchymal cell proliferation and
differentiation, and the final phase
being the terminal osteoblasts differentiation in order to form the
mineral matrix.
In fact, when platelet aggregation occurs, the platelet
degranulation is responsible for
secretion contain a variety of active growth factors, which can
have a significant impact on both
proliferation and regulation of mesenchymal cells. Based on this
premisse several authors have
speculated that use of platelet concentrated, that includes PRP and
its variation could be an important
autologous biomaterial that contribute for a faster and more
efficient repair. Despite this interesting
hypothesis, some authors do indeed point out that the use of PRP
may increase the osteoconductive
effect. However antagonistic results, or even results that
demonstrate that the use of platelet
concentrate develops pathological conditions, are also described in
the literature.
In order to establish an optimal association between pattern of
amount PRP and autogenous
bone inserted in the artificial bone defects, Nagata et al. (2009)
indicated that the association between
0.1 mL autografts with 100 μL PRP (5 times enriched) may be the
most favorable ratio to achieve
efficient PRP action and consequently bone repair.
Different that was proposed by these authors, herein we
demonstrated that use of platelet
concentrate associated with 0,1 mL of autograft bone not only does
not improve the osteconductive
effect, but produces a pathological effect that mimics
myelofibrosis.
This peculiar pathological condition occurs simultaneously to
intense presence of blasts
CD34+ cells and posterior presence of alpha-smooth muscle actin, as
well as positivity for FGF23,
TGF-β, IGF-1, IP3R, calmodulin, Jak-2 and Stat-5.
The Transforming growth factor β (TGF-β) constitute a pleiotropic
cytokine, which is
produced and secreted by activated platelets. This cytokine impacts
cell proliferation, growth,
migration, and apoptosis. Also TGF-β induces cell cycle arrest in
normal cells, or produces
transcription factor that culminates in cellular
differentiation.
47
The effect of TGF-β on osteprogenitor cells seems be well defined.
Spinella Jeagles et al.
(2001) demonstrated that, in the presence of this cytokine,
chemotaxis of osteoprogenitor cells to
regenerative sites occurs in fact, but the authors revealed that
the TGF-β suppresses terminal
differentiation of osteoprogenitor for osteoblasts. This condition
also was demonstrated by our group,
revealing the suppression of BMP-2 and wnt-10b in regenerative
sites when PRP was applied.
In 2014, Giovanini et al. also demonstrated that use of PRP also
inhibited the presence of
others expression of cluster of differentiation from CD34. This
phenomenon suggest that the PRP
may constitutes an area of leukemization, that develops to for
myelofibrosis through of different
pathways known in literature.
The usual pathway for mielofibrosis is associated to presence of
TGF-β. It is well stablished
that TGF-β induces directly the transcription of alpha-smooth
muscle actin when its presence occurs
both in cytoplasm and nucleus of progenitor cells.
However herein we have suggested that TGF-β may induces fibrosis
trough different non-
canonical pathway.
The first non-canonical pathway is correlated to activation of the
expression of FGF-23
protein by TGF-β. The hormone fibroblast growth factor 23 (FGF23)
is produced by osteocytes and
osteoblasts, and its action inhibits renal phosphate reabsorption
by stimulating the internalization of
NaPi-IIa. This biological condition provokes supression of
25-hydroxyvitamin D3 1-alpha-
hydroxylase, the renal key enzyme for the synthesis of 1,25(OH)2D3
or calcitriol for caption of
calcium and phosphate. It is noteworthy intense loss of phosphate
(through urinary via) promotes an
imbalance between calcium and phosphate, promoting increase of
calcium ionic on organism (Liu,
2013).
This intense presence of Ca2+, without the correct amount of PO42-,
does not form mineral
matrix, and this free cation may interact with specific receptor in
the cellular membrane, and penetrate
48
to cell cytoplasm and nucleus whose results it is calcium
interaction to proteins, promoting gene
transcription and cellular differentiation, specially
myofibroblasts (Puri, 2020).
This biological event may be suggested herein, since occurs intense
immunohistohcemical
presence of IP3R, that is a specific receptor for calcium, at the
same time that occurs detection of
calmodulin, a specific protein for calcium receptor. Thus, these
results may give evidence that
myelofibrosis may be produced through platelet presence either
directly through production of TGF-
β or indirectly since TGF-β activates FGF-23 whose results is
smooth actin muscle transcription due
to presence of calcium and calmodulin.
A second non canonical event may be associated to co-expression
between TGF-β and IGF-
1, that it is a abundant growth factor produced by platelets
(Mousaie et al.,2019). Since TGF-β is a
natural inhibitor of osteogenesis, may be suggested that this
cytokine inhibit the effect
osteoprogenitor of IGF-1, but not exclude the action of IGF-1 on
the repair site.
In fact, the fundamental receptor for IGF-1 is a membrane receptor
called janus kinase 2
(JAK2). In normal situations, IGF-1 interacts with JAK2, which
phosphorylates the intracellular
microenvironment, resulting in the engagement of the JAK-signal
transducer and activator of
transcription-5 (STAT-5) signaling pathway (Lanning &
Carter-Su, 2006). Transcriptional factors
from the STAT family are recruited to the phosphorylated receptor
and get phosphorylated on the Tyr
residue by JAK2 whose results is the development of hematopoietic
lineage as soon as the Jak-2 is
degraded.
In pathological situations, there is the presence of a mutant clone
of Jak-2 called V617F
mutation (Nikolova et al., 2019). This mutant protein disrupts the
autoinhibitory JH2 pseudokinase
domain, leading to constitutive activation of JAK2 kinase activity
and STAT-mediated activation of
transcription, whose final effect is the transcription of smooth
actin muscle, and consequently
myelofibrosis.
49
Although the clone tagged herein detect a mutation-free clone of
JAK-2, it may be inferred in
the present study that the intense co-expression between TGF-β and
IGF-1 may prolong the half-life
of JAK-2, leading to a condition that mimics the mutant effect of
the protein. These events would
definitely produce the STAT protein clone leading to a condition
that simulates agnogenic metaplasia,
leading to increased of myofibroblast in the regenerative sites.
This event is indicated herein since
there is concomitant TGF-β+/ IGF-1+, associated to simultaneous
presence of JAK2+/STAT-5+ and
intense and diffuse presence of SMA+ cells after 45 days of use of
the platelet concentration.
The extrapolations of the results of the present study to clinical
situations suggest that the use
of PRP (concentration 5× enriched) may be unfavorable, since, when
used with autogenous bone, it
produced excess of fibrosis through of presence of canonical
pathways of TGF-β+, or even trough
two non-canonical steps of fibrosis.
However, it is noteworthy that this cross-sectional study has some
limitations. The first
analysis was performed only after 15 days and no conclusions for
immediate effects of PRP could be
elucidated. The immunohistochemistry staining identifies proteins
present in the bone matrix and
cells, regardless of the time when they were expressed. In
addition, this study focused only on
craniofacial bone repair and the present results may not be
speculated to appendicular or axial bone
repair, since craniofacial bones are derived from distinct
embryological sources, and they also
demonstrate different functional properties and exhibit differences
in protein composition.
Despite this limitation, the data presented in this present study
may give a important evidence
about the inefficient action of PRP on repair.
Conclusion
The use of PRP hindered bone deposition by increase in TGF-β,
enhanced chemotaxis of
CD34+ progenitor cells, and demonstrated two non-canonical
developmental pathways of
50
actin muscle interactions.
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5. CONSIDERAÇÕES FINAIS
Apesar das limitações desta pesquisa podemos concluir que o L-PRP
apresentou um efeito
prejudicial do reparo ósseo de defeitos em calvárias de ratos,
devido ao aumento de expressão de
TGF-β e esclerostina, sumprimindo a expressão de osteocalcina e
osteoprotegerina. Além disto, foi
demonstrado por duas vias não-convencionais o desenvolvimento de
processos fibróticos patológicos
através da análise das interações entre TGF-β, JAK2, STAT5, IGF-1,
FGF23, IP3R, calmodulina e
actina de músculo liso.
6. REFERÊNCIAS
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Oral Maxillofac Surg 2004;62: 489-
496.
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therapy. Biomaterials 2009;30:
4956-4966.
3. Nagata M, Messora M, Okamoto R, Campos N, Pola N, Esper L,
Sbrana M, Fucini S, Garcia V,
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graft/platelet-rich plasma on
bone healing in critical-size defects: an immunohistochemical
analysis in rat calvaria. Bone
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and its application in oral surgery:
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5. Nikolidakis D, Meijer GJ, Oortgiesen DA, Walboomers XF, Jansen
JA. The effect of a low dose
of transforming growth factor beta1 (TGF-β1) on the early
bone-healing around oral implants inserted
in trabecular bone. Biomaterials 2009;30:94-99.
6. Oliveira Filho MA, Nassif PA, Malafaia O, Ribas Filho JM, Ribas
CA, Camacho AC, Stieven Filho
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plasma on the bone repair using non-
critical defects in the calvaria of rabbits. Acta Cir Bras
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Vieira Jde S, Kuczera J, de
Oliveira Filho MA, Deliberador TM. Leukocyte-platelet-rich plasma
(L-PRP) induces an abnormal
55
histophenotype in craniofacial bone repair associated with changes
in the immunopositivity of the
hematopoietic clusters of differentiation, osteoproteins, and
TGF-β1. Clin Implant Dent Relat Res.
2014;16:259-272.
8. Giovanini AF, Gonzaga CC, Zielak JC, Deliberador TM, Kuczera J,
Göringher I, de Oliveira Filho
MA, Baratto-Filho F, Urban CA. Platelet-rich plasma (PRP) impairs
the craniofacial bone repair
associated with its elevated TGF-β levels and modulates the
co-expression between collagen III and
α-smooth muscle actin. J Orthop Res. 2011;29:457-463.
9. Tefferi A. Primary myelofibrosis: 2019 update on diagnosis,
risk-stratification and management.
Am J Hematol. 2018;93:1551-1560.
10. Daver N, Shastri A, Kadia T, Quintas-Cardama A, Jabbour E,
Konopleva M, O’Brien S, Pierce
S, Zhou L, Cortes J, Kantarjian H, Verstovsek S. Modest activity of
pomalidomide in patients with
myelofibrosis and significant anemia. Leuk Res.
2013;37:1440-1444.
11. Ciaffoni F, Cassella E, Varricchio L, Massa M, Barosi G,
Migliaccio AR. Activation of non-
canonical TGF-β1 signaling indicates an autoimmune mechanism for
bone marrow fibrosis in
primary myelofibrosis. Blood Cells Mol Dis. 2015;54:234-241.
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calcium signalling under flow.
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