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.
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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.
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stronger than you seem,
-Winnie the Pooh
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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.
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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
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Sumário
GH Hormônio do crescimento
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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
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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
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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).
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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
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(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
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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
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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).
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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.
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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.
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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.
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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.
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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.
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of transforming growth factor beta1 (TGF-β1) on the early bone-healing around oral implants inserted
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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.
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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.
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