UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
AVALIAÇÃO DA LIBERAÇÃO IN VITRO DE CITOCINAS E FATORES DE CRESCIMENTO POR MEMBRANAS DE FIBRINA RICA EM PLAQUETAS E
LEUCÓCITOS
Niterói 2017
UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
AVALIAÇÃO DA LIBERAÇÃO IN VITRO DE CITOCINAS E FATORES DE CRESCIMENTO POR MEMBRANAS DE FIBRINA RICA EM PLAQUETAS E
LEUCÓCITOS
EMANUELLE STELLET LOURENÇO
Dissertação apresentada à Faculdade de Odontologia da Universidade Federal Fluminense, como parte dos requisitos para obtenção do título de Mestre, pelo Programa de Pós-Graduação em Odontologia.
Área de Concentração: Clínica Odontológica
Orientador: Prof. Dr. Gutemberg Gomes Alves
Niterói 2017
L892 Lourenço, Emanuelle Stellet
Avaliação da liberação in vitro de citocinas e fatores de crescimento por membranas de fibrina rica em plaquetas e leucócitos / Emanuelle Stellet Lourenço; orientador: Prof. Dr. Gutemberg Gomes Alves. – Niterói: [s.n], 2017. 47 f.: il.
Inclui tabelas.
Dissertação (Mestrado em Clínica Odontológica) – Universidade Federal Fluminense, 2017.
Bibliografia: f. 33-37.
1. Reparo tecidual. 2. Fibrina. 3. Fatores de crescimento. 4. Citocinas. I. Alves, Gutemberg Gomes [orient.]. II. Título.
CDD617.6
Prof(a). Dr(a). Gutemberg Gomes Alves
Instituição: Universidade Federal Fluminense - UFF
Decisão: _________________________Assinatura: ________________________ Prof(a). Dr(a). Monica Diuana Calasans Maia
Instituição: Universidade Federal Fluminense - UFF
Decisão: _________________________Assinatura: ________________________
Prof(a). Dr(a). Paulo Emilio Correa Leite
Instituição: Universidade Federal do Rio de Janeiro - UFRJ
Dedico essa dissertação a minha família: meus admiráveis pais, Edileia e Manuel e ao meu querido irmão Yago, que sempre me apoiaram e incentivaram durante todos os momentos
da minha vida. Muito obrigada por tudo, amo vocês.
Redigir este texto de agradecimento foi uma das coisas mais complexas que fiz nesse trabalho. Em um espaço tão pequeno, ter que exprimir em poucas palavras a sensação de infinita gratidão. Agradeço, primeiramente a Deus, que me permitiu vivenciar essa fase singular e conviver com pessoas maravilhosas, que contribuíram com meu crescimento profissional e pessoal. Aos meus pais, minha referência de fortaleza, dedicação e amor, por todo o carinho
e apoio em toda trajetória da minha vida e carreira. Ao meu irmão e amigo Yago, por todo apoio e incentivo nas horas em que o desânimo e cansaço pairavam sobre mim. Ao meu noivo Marcelo, amigo e companhia de todas as horas, por sempre me
incentivar a realizar meus sonhos e objetivos Ao meu orientador e amigo Gutemberg Alves, pelos ensinamentos, incentivo,
dedicação e paciência. Agradeço infinitamente em ter acreditado em minha capacidade quando eu mesma duvidava. À professora Monica Calasans, pelos ensinamentos, apoio e carinho durante toda a caminhada. Ao professor Paulo Emilio, pela colaboração no desenvolvimento do trabalho com
tamanha maestria. Ao amigo Carlos Mourão, pelo trabalho árduo, dedicação e entusiasmo na realização das diversas etapas deste trabalho.
acompanhadas de muitas risadas. A todos que contribuíram de alguma forma para a concretização desse sonho, minha
eterna gratidão.
Lourenço ES; Mourão CFAB; Leite PEC; Maia MDC; Alves GG. Avaliação da liberação in vitro de citocinas e fatores de crescimento por membranas de Fibrina Rica em Plaquetas e Leucócitos [Dissertação]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2017.
Membranas de fibrina rica em plaquetas (PRF) são arcabouços baseados em fibrina, carreadores de células imunológicas e plaquetas, de larga utilização para fins terapêuticos. Neste trabalho, foram avaliadas sua estabilidade in vitro e sua capacidade de liberação gradual de citocinas e fatores de crescimento. Membranas produzidas em centrífugas de rotor horizontal a partir do sangue de 14 doadores foram avaliadas morfologicamente por microscopia eletrônica de varredura e microscopia de fluorescência, e sua estabilidade avaliada por registro fotográfico após incubação em meio de cultura por até 28 dias. A liberação de 27 citocinas e fatores de crescimento foi monitorada através de um imunoensaio multiparamétrico. As membranas de PRF apresentaram estrutura tridimensional complexa, com alta densidade células nucleadas. Grande liberação de fatores de crescimento (PDGF, FGFb e VEGF) foram detectadas nas primeiras 24 horas, seguida de decaimento tempo-dependente, mas ainda mantendo concentrações consideráveis após 3 semanas. Citocinas anti-inflamatórias (IL-10, IL-4 e IL1-RA) e Pró-inflamatórias (IL-6, TNF-alfa, IL-1b, IFN-g) apresentaram diferentes picos de liberação, mantendo altas taxas até 21 dias. Algumas citocinas (IL-2, IL-7, IL-9 e IL-17) tiveram concentrações não detectáveis. Quimiocinas de grande relevância no reparo tecidual (RANTES, G-CSF) também foram produzidas em grande quantidade por todo o período experimental. Os resultados apontam para uma grande complexidade nos possíveis efeitos biológicos do PRF a médio prazo, que podem contribuir para uma melhor compreensão de suas aplicações clínicas em diferentes cenários.
Lourenço ES; Mourão CFAB; Leite PEC; Maia MDC; Alves GG Evaluation in vitro of release of cytokines and growth factors by leucocyte-rich and platelet-rich fibrin membranes [Dissertation]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2017.
Platelet rich fibrin (PRF) membranes are fibrin-based, immunological cell and platelet-based scaffolds that are widely used for therapeutic purposes. In this work, PRF in vitro stability and its ability to gradually release cytokines and growth factors were evaluated. Membranes produced from the blood of 14 donors were morphologically evaluated by scanning electron microscopy and fluorescence microscopy, and their stability was evaluated by photographic recording after incubation in culture medium for up to 28 days. The release of 27 cytokines and growth factors was monitored through a multiparametric immunoassay. The PRF membranes presented complex three-dimensional structure with high density of nucleated cells. Large release of growth factors (PDGF, FGFb and VEGF) was detected in the first 24 hours, followed by time-dependent decay, but still maintaining considerable concentrations after 3 weeks. Anti-inflammatory cytokines (IL-10, IL-4 and IL1-RA) and pro-inflammatory cytokines (IL-6, TNF-alpha, IL-1b, IFN-g) presented different release peaks, maintaining high rates of elution for up to 21 days. Chemokines of great relevance in tissue repair (RANTES, G-CSF) were also produced in large quantities throughout the experimental period. The results point to a great complexity in the possible biological effects of PRF in the medium term, which may contribute to a better understanding of its clinical applications in different scenarios.
Keywords: Wound healing – fibrin – growth factors - cytokines .
1 - INTRODUÇÃO
Concentrados plaquetários têm sido largamente empregados em procedimentos clínicos e cirúrgicos que necessitem de reparo tecidual (ANITUA; PINO; ORIVE, 2017). Por apresentarem grande potencial regenerativo em tecidos moles e mineralizados, são material de interesse para diversas áreas da saúde (BORIE et al., 2015), incluindo tratamentos de feridas crônicas (PICCIN et al., 2016), procedimentos ortopédicos (DOHAN et al., 2014) e tratamentos de osteoartrite e defeitos infraósseos (JOSHI JUBERT et al., 2017). Na odontologia, seu uso está associado ao tratamento de lesões de furca e melhoria nos resultados após cirurgias plásticas periodontais (CASTRO et al., 2017a), bem como nos procedimentos de elevação do soalho do seio maxilar e preservação de rebordo alveolar na implantodontia (CASTRO et al., 2017b), e no ganho de tecido mineralizado após procedimentos cirúrgicos de enucleação de cistos (DAR et al., 2016) e extrações de terceiros molares (VARGHESE; MANUEL; KUMAR, 2017).
O vislumbrar da aplicação clínica de materiais derivados de sangue periférico teve início na década de 70 (ROSS et al., 1974), quando foram observados efeitos destes sobre proliferação e migração celular, que estariam associados diretamente ou indiretamente com a liberação de componentes ativos posteriormente identificados como fatores de crescimento, capazes de estimular resposta a lesões teciduais. Dentre os diversos protocolos existentes de concentrados plaquetários, destacam-se as membranas de fibrina rica em plaquetas (Platelet Rich Fibrin, PRF) pertencem à segunda geração de agregados plaquetários, cujo protocolo caracteriza-se pela centrifugação única de amostras de sangue periférico, sendo isento de adição de anticoagulantes e aditivos para desencadear a polimerização da fibrina (DOHAN et al., 2006b). Devido à sua polimerização ocorrer de forma mais branda e natural, ocorre o confinamento de plaquetas, leucócitos e fatores de crescimento na matriz, conferindo estabilidade estrutural por longos períodos de tempo (AHMED; DARE; HINCKE, 2008). Por ser dotado destas características, o PRF configura-se também como arcabouço para migração e proliferação celular, com alta aplicabilidade clínica, protocolo simplificado e baixo custo.
Os bons resultados clínicos da fibrina rica em plaquetas também são diretamente relacionados à liberação gradual de fatores de crescimento produzidos
por plaquetas (KIM; KIM; KIM, 2014), tais como VEGF (Vascular Endothelial Growth Factor), PDGF (Platelet Derived Growth Factor), FGF (Fibroblast Growth Factor) e TGF (Transforming Growth Factor), que promovem regeneração através de processos como angiogênese, e o estimulo migração e proliferação de osteoblastos, fibroblastos e células mesenquimais (DE PASCALE et al., 2015). No entanto, além de fatores de crescimento, diversas citocinas pró e anti-inflamatórias também podem ser produzidas por leucócitos presentes na membrana de PRF, podendo impactar tanto positivamente quanto negativamente sobre o desfecho clínico de seu uso em diversos tratamentos, permanecendo como assunto controverso na literatura (ANITUA et al., 2015; DOHAN et al., 2006a). De todo modo, pode-se inferir que o desfecho clínico do uso de PRF deve ser afetado pela cinética da liberação de fatores de crescimento e citocinas, uma vez que seus efeitos desejáveis e indesejáveis surgem em diferentes momentos do reparo tecidual.
Outro assunto controverso trazido na literatura é a padronização do protocolo de PRF. Segundo DOHAN e colaboradores, a membrana de PRF é produzida a partir da centrifugação de uma alíquota de 9 ml de sangue periférico por 10 minutos, em centrífuga de rotor de angulação fixa, sendo preconizada a centrífuga da Intra-Lock (marketed with CE/FDA clearance as Xpression kit, Intra-Intra-Lock, Boca-Raton, FL, USA). Em um trabalho recente, foi abordada a interferência da vibração, oriunda de diferentes padrões de centrifugação, na qualidade de membranas de PRF produzidas, onde foram observadas a deterioração de componentes celulares e a formação anômala da rede de fibrina em membranas obtidas através de outras centrífugas(Dohan Ehrenfest et al., 2017). Sendo assim, DOHAN e colaboradores ressaltam que a membrana de PRF, além de possuir um determinado protocolo, possui uma (biological signature) que consiste numa membrana com arquitetura natural de fibrina com capacidade de aprisionamento de células viáveis e liberação gradual de fatores de crescimento. Entretanto, uma alternativa encontrada na literatura (Dohan et al., 2006a) e bastante utilizada em clínicas e hospitais, é a produção de PRF em centrífugas de rotor horizontal, originalmente empregadas em setores de hematologia, sendo equipamento de fácil acesso para diversos profissionais da saúde.
Procurando contribuir para a compreensão da capacidade de atuação biológica do PRF em processos de regeneração tecidual, neste trabalho objetivamos avaliar in vitro as características morfológicas e estabilidade estruturais de
membranas de fibrina rica em plaquetas produzidas em centrífugas de rotor horizontal, bem como sua capacidade de liberação de fatores de crescimento e citocinas pró e anti-inflamatórias durante as primeiras semanas após sua produção.
2 - MATERIAL E MÉTODOS
O presente trabalho foi desenvolvido de acordo com os princípios preconizados para a experimentação com seres humanos determinados na Declaração de Helsinki. O protocolo de pesquisa foi aprovado pelo Comitê de Ética em Pesquisa HUAP-UFF (CAAE:22763513.6.0000.5243). Os participantes foram informados sobre os procedimentos e objetivos do estudo, e assinaram o respectivo termo de consentimento.
2.1 - Preparo das membranas de Fibrina Rica em Plaquetas
Sangue periférico foi coletado de 14 doadores participantes da pesquisa, saudáveis e com idades entre 20 e 56 anos, sem histórico de uso de medicação anticoagulante. Para a produção de cada membrana foi utilizada uma alíquota de 9 ml de sangue, coletada em tubos sem adição de qualquer substância (DryVacutube, Biocon®, Brasil), imediatamente centrifugada a 400 g em centrífuga com rotor horizontal (B-40, RDE®, Brasil) por 10 minutos. Ao término da centrifugação foi possível identificar claramente a sedimentação de células vermelhas, separadas do soro sanguíneo. Após a polimerização da matriz de fibrina, o material era retirado do tubo e uma leve compressão era aplicada com auxílio de gaze estéril, dando origem a uma estrutura gelatinosa e resistente, identificada como a membrana de Fibrina Rica em Plaquetas.
2.2 - Caracterização Das Membranas de Fibrina Rica em Plaquetas
2.2.1 - Microscopia Eletrônica de varredura
Imediatamente após sua produção, as membranas (N=6) foram segmentadas em três fragmentos de comprimento equivalente, denominados como superior, médio e inferior, sendo o fragmento inferior correspondente à localização da área de buffy coat ou camada leuco-plaquetária. Cada porção foi fixada com solução de Karnovsky e pós-fixada com solução de cacodilato de sódio 0,2M e tetróxido de ósmio 1%, sendo finalmente desidratada em soluções de álcool (variando de 15% a 100%) e hexametildisilazina (HMDS). Os materiais foram metalizados com ouro e observados a 15 kV com um microscópio eletrônico de varredura (JEOL JSM-6490 LV, JEOL, Japão).
2.2.2 – Microscopia de fluorescência
De modo a avaliar a distribuição de células nucleadas ao longo das membranas, foi realizada a observação das mesmas por microscopia de fluorescência. Amostras divididas nos fragmentos denominados como superior, médio e inferior (buffy coat) foram fixadas por tratamento em solução de paraformaldeído a 4% por 15 minutos. A marcação de núcleos celulares foi feita com exposição a DAPI (4’,6-diamidino-2-fenilindol) diluído a 1: 5000 em tampão fosfato em solução salina (PBS, Phosphate Buffered Saline). As amostras foram observadas com objetiva de 20X em microscópio de fluorescência invertido (Axio.Observer A1, Zeiss, Alemanha) e fotografadas com uma câmera digital (Axiocam Rev.3 MRc, Zeiss, Alemanha). A quantificação de células nucleadas foi feita com ajuda do software Image-Pro Plus 6.0 (Media Cybernetics, EUA). Para cada seção das membranas, os núcleos foram contados por contraste em cinco campos subjacentes e não sobrepostos, sendo a densidade de células calculada pela média de núcleos por campo.
2.3 - Eluição in vitro de citocinas e fatores de crescimento
Com o intuito de analisar a dinâmica de liberação de citocinas e fatores de crescimento ao longo do tempo, as membranas de fibrina rica em plaquetas (N=8) foram cultivadas por até três semanas após sua confecção. Para isso, membranas foram incubadas em triplicata em placas de cultivo de 6 poços (TPP, EUA), em presença de 4 ml de meio DMEM (Dulbecco's Modified Eagle's Medium, GIBCO, EUA), sem uso de antibióticos, em atmosfera úmida a 37°C e 5% de CO2. Alíquotas
dos extratos foram coletadas nos tempos de 1, 4, 7, 14, e 21 dias de cultura e armazenadas em freezer a -80°C.
2.4 - Teste de estabilidade
A avaliação da estabilidade estrutural foi realizada através do acompanhamento da evolução das amostras mediante registro fotográfico. As membranas foram incubadas em placas de 6 poços em presença de 4 ml de meio DMEM sem antibiótico e atmosfera úmida a 37°C e 5% de CO2. O registro
fotográfico foi feito nos períodos de 1, 7, 14, 21 e 28 dias. As fotos foram avaliadas com o software Image Pro Plus 6.0, e o comprimento total das membranas em cada tempo experimental foi estimado.
2.5 - Dosagem de citocinas e fatores de crescimento
As concentrações de citocinas e fatores de crescimento liberados pelas membranas foram mensuradas através da análise de meios condicionados. As membranas foram cultivadas nos tempos de 1, 7, 14 e 21 dias, e posteriormente, os extratos foram armazenados em freezer -80°C. Para a detecção das biomoléculas
foi utilizado um imunensaio multiparamétrico baseado em micropérolas magnéticas marcadas com a tecnologia XMap (LuminexCorp, EUA), através de um kit comercial (27-plex panel, Biorad Inc.,, EUA) capaz de quantificar IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, CCL11, FGF-b, CSF3, CSF2, IFN-γ, CXCL10, CCL2, CCL3, CCL-4, PDGF, CCL5, TNFα e VEGF. A quantificação das pérolas magnéticas e dosagens foi realizada com um sistema Bio-Plex MAGPIX (Biorad Inc., EUA).Os resultados foram analisados usando Xponent v. 3.0 software (Luminexcorp, EUA).
Para confirmar os resultados obtidos por luminometria de fluxo, um analito (FGF-b) foi também dosado nas mesmas amostras a partir de um ensaio de ELISA convencional. Alíquotas de 1, 4 e 7 dias foram analisadas quanto àa presença de FGF-b através do método ELISA (Enzyme-Linked Immunosorbent Assay) utilizando um kit comercial (Human FGF Basic ELISA KIT, AVIVA System Biology, EUA).
2.6 - Análise estatística
A comparação dos diferentes tempos experimentais foi realizada por one-way ANOVA com pós-teste de Bonferroni, considerando um erro alfa de 5%. A análise estatística foi realizada com ajuda do software Graphpad Prism 6 (Graphpad inc., EUA).
3 - CONCLUSÃO
Os presentes resultados demonstram que as membranas de Fibrina Rica em Plaquetas possuem grande estabilidade estrutural em meio líquido por até 28 dias, apresentando grande liberação inicial de fatores de crescimento, com cinética decrescente nas semanas subsequentes, acompanhada de uma produção consistente de citocinas pró e anti-inflamatórias ao longo de três semanas.
4 - AGRADECIMENTOS
Os autores agradecem o apoio financeiro da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), da Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) e do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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ANEXO I – ARTIGO CIENTÍFICO
EVALUATION OF IN VITRO RELEASE OF CYTOKINES AND GROWTH FACTORS OF FIBRIN RICH MEMBRANES IN PLATELETS AND LEUCOCYTES
Lourenço ES; Mourão CFAB; Leite PEC; Maia MDC; Alves GG.
ABSTRACT
Platelet rich fibrin (PRF) membranes are fibrin-based, immunological cell and platelet-based scaffolds that are widely used for therapeutic purposes. In this work, PRF in vitro stability and its ability to gradually release cytokines and growth factors were evaluated. Membranes produced from the blood of 14 donors were morphologically evaluated by scanning electron microscopy and fluorescence microscopy, and their stability was assessed by photographic recording after incubation in culture medium for up to 28 days. The release of 27 cytokines and growth factors was monitored through a multiparametric immunoassay. The PRF membranes presented complex three-dimensional structure with high density of nucleated cells. Large release of growth factors (PDGF, FGFb and VEGF) was detected in the first 24 hours, followed by time-dependent decay, but still maintaining significant concentrations after 3 weeks. Anti-inflammatory cytokines (IL-10, IL-4 and IL1-RA) and pro-inflammatory cytokines (IL-6, TNF-alpha, IL-1b, IFN-g) presented different release peaks, maintaining high rates of elution for up to 21 days. Chemokines of great relevance in tissue repair (RANTES, G-CSF) were also produced in large quantities throughout the experimental period. The results point to a great complexity in the possible biological effects of PRF in the medium term, which may contribute to a better understanding of its clinical applications in different scenarios.
1. INTRODUCTION
Due to their high regenerative potential, platelet concentrates have been widely used in clinical and surgical procedures that require tissue repair (BORIE et al., 2015; ANITUA; PINO; ORIVE, 2017), including treatments for chronic wounds (PICCIN et al., 2016), osteoarthritis, infra-osseal defects (JOSHI JUBERT et al., 2017), and orthopedic procedures (DOHAN et al., 2014). In dentistry, its use is associated with several procedures, including treatment of furcation lesions and improvement of periodontal plastic surgeries (CASTRO et al., 2017a). Interesting results have also been found in procedures such as the elevation of the maxillary sinus floor, and preservation of the alveolar ridge (DAR et al., 2017), extractions of third molars (VARGHESE; MANUEL; KUMAR, 2017), and cranial enucleation (DAR et al., 2016).
The first glance on the clinical application of materials derived from peripheral blood began in the 1970s (ROSS et al., 1974), with the descritpion of several effects on proliferation and cell migration induced by platelet-dependent serum factors, later identified as growth factors, capable of stimulating responses to tissue lesions. The following decades witnessed a multitude of novel techniques and materials, often identified generically as Platelet-Rich Plasma (PRP). Among those materials, Leukocyte- and Platelet-Rich Fibrin (L-PRF) membranes belong to a second generation of platelet aggregates. Favored by a rather simple protocol, its production is characterized by a single centrifugation of peripheral blood samples, circumventing the addition of anticoagulants of blood activators to trigger fibrin polymerization (DOHAN et al., 2006b). The confinement of platelets, leukocytes and growth factors in a high density fibrin network allows it to be handled like a real solid material, and contributes to structural stability for long periods of time (AHMED; DARE; HINCKE, 2008), also turning PRF into a possible scaffold for tissue engineering, with high clinical applicability, simplified protocol and low cost.
The good clinical results of platelet-rich fibrin products are directly related to the gradual release of platelet-derived growth factors (KIM; KIM, 2014), such as VEGF (Vascular Endothelial Growth Factor), PDGF (Platelet Derived Growth Factor), FGF (Fibroblast Growth Factor) and TGF (Transforming Growth Factor), which promote regeneration through processes such as angiogenesis, and stimulate the migration and proliferation of osteoblasts, fibroblasts and mesenchymal cells (DE
PASCALE et al., 2015). However, in addition to growth factors, several pro- and anti-inflammatory cytokines can also be produced by leukocytes present in the L-PRF membranes, which can impact both positively and negatively on the clinical outcome of its use in several treatments, remaining a controversial subject in the scientific literature (ANITUA et al., 2015, DOHAN et al., 2006a). However, it can be inferred that the clinical outcome of the use of PRF should be affected by the kinetics of the release of growth factors and cytokines, since their desirable and undesirable effects arise and combine at different moments of the tissue repair.
Another controversial subject brought up in the literature is the standardization of the production of leukocyte and platelet-rich fibrin products. Initially developed as an open access technique, nowadays the production of L-PRF is strongly related to the only FDA-approved CE-marked system with certified materials, marketed under the name Intra-Spin L-PRF (Intra-Lock Inc., Boca Raton, FL, USA). This system uses a fixed-angle rotor centrifuge to process a 9 ml aliquot of peripheral blood for 10 minutes. A recent study investigated the vibration interference from different centrifugation patterns in the quality of PRF membranes, where the deterioration of cellular components and the anomalous formation of the fibrin network were observed in membranes obtained with four models of commercially available table-top centrifuges (Dohan Ehrenfest et al., 2017). However, the literature still lacks on reports of the impact of the production of leukocyte- and platelet- rich fibrin derivatives by the use of horizontal centrifugation with swing-out rotor centrifuges, which are very accessible and of wide use in clinics and hospitals.
In this context, the aim of this study was to evaluate the in vitro morphological characteristics and structural stability of leukocyte and platelet- rich fibrin membranes produced with horizontal rotor centrifuges, as well as their capacity of releasing growth factors, as well as pro- and anti-inflammatory cytokines, during the first weeks after production.
2. MATERIALS AND METHODS
The present work was developed according to the principles recommended for experimentation with human beings determined in the Declaration of Helsinki. The research protocol was approved by the Research Ethics Committee HUAP-UFF
(CAAE: 22763513.6.0000.5243). The participants were informed about the procedures and objectives of the study, and signed the respective consent form.
2.1 - Preparation of Platelet Rich Fibrin Membranes
Peripheral blood was collected from 14 healthy donors, aged between 20 and 56 years, with no history of anticoagulant medication use. For the production of each membrane, a 9 ml aliquot of blood, collected in tubes without addition of any substance (DryVacutube, Biocon®, Brazil), was immediately centrifuged at 400 g in a horizontal swing-out rotor centrifuge (B-40, RDE® , Brazil) for 10 minutes. After polymerization of the fibrin matrix, the material was withdrawn from the tube and a slight compression was applied with the aid of a sterile gauze, giving rise to a resistant gelatinous structure, identified as the Platelet Rich Fibrin membrane.
2.2 - Characterization of the Platelet Rich Fibrin Membranes
2.2.1 - Scanning Electron Microscopy
Immediately after production, six membranes were segmented into three fragments of equivalent length, identified as superior, middle and lower sections, the lower fragment corresponding to the location of the buffy coat area or leuco-platelet layer. Each portion was fixed with Karnovsky's solution and post-fixed with 0.2M sodium cacodylate solution and 1% osmium tetroxide, and finally dehydrated in alcohol solutions (ranging from 15% to 100%) and hexamethyldisilazine (HMDS). The materials were metallized with gold and observed at 15 kV with a scanning electron microscope (JEOL JSM-6490 LV, JEOL, Japan).
2.2.2 - Fluorescence microscopy
In order to evaluate the distribution of nucleated cells along the membranes, they were observed by fluorescence microscopy. Samples divided into superior, middle and lower (buffy coat) sections were fixed by treatment with 4% paraformaldehyde solution for 15 minutes. Marking of cell nuclei was done with exposure to DAPI (4 ', 6-diamidino-2-phenylindole) diluted 1: 5000 in phosphate
buffered saline (PBS). The samples were observed with a 20X objective in an inverted fluorescence microscope (Axio.Observer A1, Zeiss, Germany) and photographed with a digital camera (Axiocam Rev.3 MRc, Zeiss, Germany). Quantification of nucleated cells was done using Image-Pro Plus 6.0 software (Media Cybernetics, USA). For each section of the membranes, the nuclei were counted by contrast in five underlying and non-overlapping fields, the cell density being calculated by the mean nuclei per field.
2.3 - Elution and detection of cytokines and growth factors
To assess the dynamics of release of cytokines and growth factors, eight platelet rich fibrin membranes were cultured for up to three weeks after their preparation. For this, membranes were incubated in 6-well culture plates (TPP, USA) in the presence of 4 ml of DMEM medium (Dulbecco's Modified Eagle's Medium, GIBCO, USA), without antibiotics, in a humidified atmosphere at 37°C and 5% CO2.
Aliquots of the extracts were collected at the times of 1, 4, 7, 14, and 21 days of culture. At each experimental time, the culture medium was completely removed and stored in a freezer at -80°C, and 4 ml of fresh culture medium were added to each membrane, and incubated until the next experimental time.
The concentration of cytokines and growth factors was detected in the extracts through a multiparametric immunoassay based on XMap-labeled magnetic microbeads (LuminexCorp, USA). A commercial kit (27-plex panel, Biorad Inc., USA) was employed, capable of quantifying 1β, 10, 12 (p70), 13, 15, 10, IL-10, IL- IL-17, CCL11, FGF-b, CSF3, CSF2, IFN-γ, CXCLIL-10, CCL2, CCL3, CCL-4, PDGF, CCL5, TNFα and VEGF. Quantification of the magnetic beads and dosages was performed with a BioPlex MAGPIX system (Biorad Inc., USA). Results were analyzed using Xponent v. 3.0 software (Luminexcorp, USA).
The time-course of the alterations in the concentrations of cytokines and growth factors was calculated as a fold-change from the initial extraction (24 hours), calculated according to the formula:
Fold-Change = (Conc. Experimental Time – Conc. 24 hours) Conc. 24 hours
To confirm the results obtained by the multiplex assay, one analyte (FGF-b) was also dosed in the 24-hours elution samples by ELISA (Enzyme-Linked Immunosorbent Assay) using a commercial kit (Human FGF Basic ELISA KIT, AVIVA System Biology, USA).
2.5 – Membrane Stability test
The structural stability evaluation was performed by monitoring the evolution of the samples through photographic recording. Membranes were incubated in 6-well plates in the presence of 4 ml DMEM medium without antibiotic and humid atmosphere at 37°C and 5% CO2. The photographic record was made in the periods
of 1, 7, 14, 21 and 28 days. The photos were evaluated with Image Pro Plus 6.0 software, and the total length of the membranes at each experimental time was estimated.
2.6 - Statistical analysis
The statistical analysis was performed using Graphpad Prism 6 software (Graphpad Inc., USA). One-way ANOVA with post-hoc Bonferroni test was employed to compare the cell content in the fluorescent assay and the mean length of membranes at each time point in the structural stability test. In the cytokine/growth factors assay, a paired t-test was performed comparing the concentrations at each time point with the first elution (24 hours). All statistical tests considered an alpha error of 5%.
3. RESULTS
After the centrifugation step, it was possible to observe subdivisions of the material inside the tube, consisting of a red blood cell´s layer at the base of the tube, a fibrin clot in the intermediate region, and an obvious platelet poor plasma portion at the top of the tube (Fig 1A). The compression process of the membranes produced from the fibrin clot gave rise to a resistant, yellowish and malleable material (Fig. 1B).
After processing, the membranes were separated into upper, middle and lower portions, whose ultrastructure was observed by scanning electron microscopy (SEM)
(Figure 2). One can observe the formation of a dense fibrin network with overly closed frames, remembering the appearance of gauze threads. The entrapment of cells in the middle of the fibers is quite evident. An increased cell density was observed in the lower segment (Fig. 2C). No evidence was observed related to cell death or abnormal morphology. The analysis of the cell distribution along the membrane sections by fluorescence microscopy confirms the large presence of nucleated cells in the fibrin structure (Fig. 3), presenting mononuclear and polymorphonucleated cells. These cells are present throughout the membrane, however with increasing numbers along its length, notably having a higher cell density at the buffy coat area (Fig 3C), as confirmed by the quantification of cells with DAPI-labeled nuclei (Fig. 3D).
Figure 4 shows the time-dependent structural stability of the membranes when immersed in culture medium, following their evolution from the first 24h (Fig. 4A) to the 28th day of incubation (Fig. 4B). The estimation of the total length of the membranes at each time (Fig. 4C) shows that there is no significant change in membrane length over 28 days.
The culture supernatant was evaluated for the content of 27 analytes that could be eluted from the membranes in the culture medium. Table 1 shows that the detection of most of these molecules already occurred in the first day of elution. It can be observed that a range of pro-inflammatory and anti-inflammatory cytokines have been released, being accompanied by growth factors, with high concentrations of PDGF-BB and VEGF. Other molecules were detected at lower dosages than the results expressed in other studies (Passaretti et al., 2014), such as IL-2 (<4.5 pg / ml), IL-13 (6.5 ± 7.8 pg/ml ), IL-15 (2.8 Â ± 0.3 pg/ml), IL-4 and RANTES (both at less than 1.2 pg/ml). In order to validate the results obtained through flow luminometry, the FGFb concentration was also evaluated by the enzyme-linked immunosorbent assay (ELISA) method, detecting a quite similar to the concentration revealed in the flow luminometer test (13.5 ± 3.3 pg/ml versus 19.0 ± 3.1 pg/ml).
Figure 5 shows the changes in the release of these molecules over the subsequent three weeks of culture. It became evident that each analyte presents a peculiar release profile, most presenting an expressive increase of the concentrations, but at different moments of the elution process. In the case of the FGFb, VEGF and PDGF growth factors, the large release found in the first 24 hours was followed by a reduction in concentrations in the subsequent weeks, always
significantly lower (p <0.05) after 21 days. A similar pattern of release was obtained for inflammatory cytokines such as IL-12, IL-1b, TNF-a, and chemokines GM-CSF and IP-10, although with very different rates of increase for each analyte.
The cytokines IL-1RA and IL-4, with potential anti-inflammatory activity, showed their release peaks at the end of the first week, followed by a gradual reduction, but remaining at levels even higher than the first day of elution. A similar process was observed for the proinflammatory cytokines IFN-γ, eotaxin and IL-6, the latter presenting a tenfold increase in the release at the 21st when compared to the first day of elution. RANTES, MIP-1a and MCP-1 also had elution peaks within the first week, but with maintenance of high release rates throughout the experimental time.
4. DISCUSSION
Fibrin membranes with the presence of platelets and leukocytes are considered very promising tools for regenerative therapies, providing a cell scaffold that acts as a source of factors contributing to several positive results demonstrated by in vivo and clinical studies (Tatullo et al. 2012; Borie et al., 2015). The release of growth factors, pro- and anti-inflammatory cytokines by cells present in the fibrin matrix has been pointed out as a vital factor in the regenerative process (Passaretti et al., 2014; Kobayashi et al., 2016a). However, there is controversy regarding the structure, cell content and release of bioactive molecules by membranes produced with similar protocols, while employing different centrifugation systems.
In the present work, membranes were produced according to the principles diffused by DOHAN and collaborators (Dohan et al., 2006b), based on the activation of the coagulation cascade due to the platelet shock against the walls of glass tubes, in the absence of anticoagulants. However, we assessed the impact of employing performed a used a swing-out horizontal rotor centrifugal device was used widely used in hospital settings in the hematology area, being easily accessible in clinical and surgical procedures (SERRA-RENOM AND SERRA-MESTRE, 2016), allowing the applicability of the technique in question in daily activities in clinics and hospitals.
For KOBAYASHI et al. (2012), the possible impact of the rotor angulation change may be the concentration of the platelet deposition layer on the PRF
membrane. As the rotor angle of the centrifuge changes to a more horizontal position, the action of force G will focus on the tube so as to concentrate the platelet layer in a smaller area without altering the cell distribution in the membrane (maintaining the concentration of Platelets mostly in the buffy coat area). Other protocols with considerable G-force changes (based on rotation rather than angulation) allowed the development of other platelet aggregate products such as A-PRF (Ghanaati et al., 2014). However, according to CHOUKROUN (2007) it is common practice among plastic surgeons, maxillofacial surgeons and otolaryngologists to produce PRF with the use of Coleman fat centrifuges, which are not necessarily fixed angle. Applying this methodology of production from a horizontal rotor, but maintaining strictly the same force G (400 g) suggested by the Choukroun protocol (Dohan et al., 2006a), the membranes produced in this work presented great similarity in their structural constitution and (Dirk et al., 2006). In the present study, the results of the present study were similar to those reported in the literature.
The ultrastructure of the fibrin membrane was evidenced by scanning electron microscopy (SEM), revealing a dense and intricate, gauze-like mesh, very characteristic of the PRF elastic and resistant membranes previously described (DOHAN et al., 2010). ), And with high similarity to collagen structures formed in vivo during the natural healing process (Wu et al., 2012). The absence of alteration in the size of these membranes when kept in culture medium for up to 28 days after their formation evidences the preservation of the structural integrity, confirming the stability of the PRF in aqueous medium reported by SCHÄR et al. (2015). This is a desirable feature for frameworks for tissue engineering (Galav et al., 2016), giving a longer basis for cell migration and proliferation for tissue remodeling purposes, as biodegradable barriers for guided tissue regeneration procedures (Bansal et al. Eren et al., 2016) and guided bone regeneration.
It is worth noting that cell entrapment is a marked attribute on the PRF membrane, as evidenced by fluorescence microscopy, and it is possible to detect a high density of nucleated cells within the structure. The initial density of nucleated cells proved to be quite typical of this type of membrane, being distributed unevenly by its extension, concentrating a larger number of cells in the buffy coat area (Mouhyi et al., 2010). Therefore, the presence of viable leukocytes, as well as a stable fibrin network for weeks, contributes to this material also remains a reservoir for the
release and controlled growth factors and cytokines (BORIE et al., 2015; DOHAN et al. . In fact, the literature reports the permanence and release of these substances in a period between 1 and 4 weeks after the surgical intervention with application of PRF in different tissues (Borie et al., 2015).
the release time of these molecules and the clinical effects observed in PRF treatments. In this regard, in vitro studies have attempted to evidence the presence of these substances in conditioned media exposed to PRF for reduced periods (Passaretti et al., 2014; Anitua et al., 2015, Kobayashi et al., 2016a), which often do not reflect the total time repair of some types of tissues, such as in bone lesions (Tatullo et al., 2012). In this sense, the present work was developed with the aim of contributing to a better understanding of the kinetics of the release of growth factors and pro-and anti-inflammatory cytokines by fibrin membranes rich in platelets and leukocytes in the weeks following their production.
In order to understand how this release occurs, the in vitro analysis of elution, that is, the release of these molecules in liquid medium, has been developed in several studies investigating PRP, PRF and other platelet aggregates. QIAO et al. (2017), through ELISA analysis, were able to detect the release of important factors (PDGF, IGF, TGF, VEGF and FGF-b) immediately after membrane production. The study of PASSARETTI et al. (2014) presented a broader search of cytokines and growth factors, evidencing high concentrations of IL-6, IL-8 and RANTES released in the 24-hour period. NISHIMOTO et al. (2015) used a similar method for extraction of PDGF-BB and TGF-beta1, but following the release levels for up to 1 hour, revealing low concentrations at the initial times after PRF production. The authors also observed a difference in concentration of these analytes as a function of membrane follow-up, where the highest levels were manifested in the buffy coat area, similar to the most cellularized fragment of the present work, and suggesting a role of leukocytes in this Production of growth factors.
On the other hand, the TGFb-1, PDGF-AB and TSP-1 release of VEGF, TGFb-1 and TGFb-1 were observed by PRF membranes and found a peak release of VEGF in the first 4 hours, PDGF-AB and TSP-1 after 24 hours of exposure, followed by gradual reduction of release for up to 7 days in culture. KOBAYASHI et al. (2016b) found a relatively similar pattern when investigating the release of PDGF-AA, PDGF-AB, PDGF-BB, TGFB1, VEGF, EGF, and IGF for a period of 10 days, finding release peaks in the first 15 Minutes of incubation, followed by gradual
reduction. In this sense, the present work, when monitoring these and other molecules for up to 21 days, contributes to show that the release of several growth factors and cytokines can continue to be maintained in concentrations of biological relevance for weeks after their preparation. The evaluation of a large range of molecules was possible by the methodological choice of a multiparametric system based on XMap technology (LuminexCorp, USA), which allows analyzing 27 different analytes in a single sample, increasing the reliability of the results with respect to comparisons between the Relative concentrations of the different analytes, and reduced the chance of conflicting results by experimental variability and manipulation errors.
As expected for PRF membranes, considerable high levels of VEGF, bFGF, and PDGF-BB were detected in the first 24 hours at concentrations that appear to be higher than those reported in other studies (Dohan et al., 2006b; Passaretti et al. , 2014, Schär et al., 2015). In this sense, methodological differences such as the volume of medium used for elution, dosage technique (ELISA, flow luminometry) as well as the parameters for normalization of results (membrane mass, volume of mine or initial blood volume) make comparison difficult Of data from different reports in the literature. However, the gradual reduction in the release of growth factors over time is confirmed in all of these studies, and in agreement with the present results. It is worth however, to note that biologically relevant concentrations of these molecules can still be released after 21 days (eg 35 micrograms / ml of PDGF), and capable of promoting tissue regeneration. In the case of VEGF, this action would occur through promotion of angiogenesis, recruiting endothelial cells, enabling the constitution of a network of vessels that will enable the cellular development of adjacent areas; PDGF-BB, considered one of the most important growth factors in platelet concentrates, acts on the migration and proliferation of mesenchymal cells and fibroblasts and, together with the action of bFGF, which promotes cell proliferation especially of mesenchymal cells, fibroblasts and osteoblasts, promote The development of neoformation of tissue (DOHAN et al., 2009).
The interpretation of the impacts of release of bioactive molecules by fibrin membranes rich in platelets and leukocytes becomes more difficult when considering other molecules besides growth factors such as the chemokine RANTES or CCL-5. A comparative study between different platelet derivatives (Passaretti et al., 2014) showed that the release of CCL-5 is much higher in PRP than in PRF in the first 24
hours after its production. In fact, the present result for 24 hours indicates a low release of this molecule, but this is followed by an expressive increase (of more than 10 times) from the 7th to the 21st day, indicating the need for investigations for longer times for the global comprehension of Release of molecules associated with platelet derivatives. It is difficult to interpret the real impact of these high rates of release of RANTES with clinical results from the use of leukocyte-rich PRF membranes. However, the literature presents evidence of RANTES acting on angiogenesis and potential effects on the differentiation of osteoblasts in the tissue regeneration process (Czekanska et al., 2014).
In the present study, high concentrations of inflammatory cytokines such as IL-1β, IL-6, IL-5, IL-15, TNF-alpha and IFN-γ were detected at initial periods (from day 1 to day Several cases remaining with high release rate after three weeks, indicating large activation of immune cells in the material. In fact, the presence of immune system cells within the PRF membrane and consequent release of inflammatory mediators has been a subject of great controversy in the literature. Although it has great importance in immune responses, IL-6 stimulates bone resorption, being present in high concentrations in the sites affected by periodontitis. However, because it is an in vitro assay, there is no activation response from the negative feedback process to exposure of high IL-6 rates, as would possibly occur in vivo. According to DOHAN et al. (2006b) the leukocyte-rich PRF membrane is an immune nodule capable of stimulating defense mechanisms. Although proinflammatory cytokines may affect the regeneration process of lesions (De Pascale et al., 2015), they also play important immunological functions against possible infections and help to clean the perimeter of the wound. Development of healing. The high release of proinflammatory cytokines concomitant with growth factors could, for example, signal a possible osteolysis (Dohan et al., 2006b) in treatments involving bone tissues, occurring simultaneously with the proliferation of osteoblasts, fibroblasts And endothelial cells, suggesting a rapid remodeling of the PRF membrane implantation sites, with an interesting absence of classic signs of the inflammatory picture (heat, flushing, pain, edema and loss of function), as observed in the clinic (Jankovic et al. , 2012). This may be related to the also large long-term release of anti-inflammatory cytokines (IL-10, IL-1RA and IL-4), suggesting the occurrence of a self-limited inflammatory process kinetics , Minor edema, associated with immunological protection of the region since the beginning of the tissue repair).
The high concentrations of G-CSF found in the samples of this work reinforce the thesis of the promotion of tissue remodeling, since the literature evidences the process of bone regeneration via revascularization and osteogenesis in experiments in vivo by presence of G-CSF, often with Similar to that of VEGF (Ishida et al., 2010). In addition, the positive role that some proinflammatory cytokines may exert indirectly on tissue repair, such as evidence that IL-1b released by PRF membranes may be associated with the migration of mesenchymal stem cells and human endothelial cells , And in vitro studies (Schär et al., 2015).
Taken together, the results of the present work demonstrate that the structural stability of fibrin membranes rich in platelets allows it to be able to release relatively slowly molecules as growth factors for weeks after their production, and that the high amount of cells Can also contribute to this release being concomitant with the large production of pro- and anti-inflammatory cytokines and chemokines. We also show that this prolonged release does not follow a uniform pattern, but is peculiar to each molecule, ranging from total exhaustion (TNF-alpha) to the maintenance of very high release rates (G-CSF). Therefore, although further studies are needed to confirm whether this in vitro release is parallel to the physiological environment, as well as its actual impact on the clinical outcome of treatments involving PRF, the present results point to a great complexity in the potential biological effects of these Platelet derivatives in the medium term, which may contribute to a better understanding of their clinical applications in different scenarios.
5. CONCLUSION
The present results demonstrate that Plankton Rich Fibrin membranes have high structural stability in liquid medium for up to 28 days, with a large initial release of growth factors, with decreasing kinetics in subsequent weeks, accompanied by a consistent production of pro- and anti-inflammatory cytokines over three weeks.
6. ACKNOWLEDGMENTS
The authors acknowledge the financial support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à
Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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Table 1. Concentrations on conditioned medium after 24 hours elution
Results presented as mean ± S.D. (n=8 fibrin membranes).
Analyte Concentration (pg/ml) FGF-b 19,0 ± 3,1 VEGF 3.075,5 ± 1.341,7 PDGF-BB 220.495,6 ± 4.687,2 GM-CSF 3,8 ± 1,1 G-CSF 9,8 ± 1.1 IL-1β 77,8 ± 49,2 IL-2 < 4.5 IL-6 15,5 ± 21,0 IL-8 12.441,6 ± 8.074,9 IL-13 6,5 ± 7,8 IL-15 2,8 ± 0,3 IFN- 168,6 ± 15,7 TNFα 57,3 ± 17,5 IL-12p70 65,9 ± 3,4 IL-7 <4,5 IL-17A <7,1 IL-9 <6,3 IL-5 3,5 ± 0,4 IP-10 4957,5 ± 1656,0 IL-10 9,7 ± 0,4 IL -4 1.2 ± 0 IL-1RA 81,6 ± 35,8 RANTES 1,2 ± 1 MIP-1α 27,8 ± 15,1 MIP-1β 594,5 ± 20,6 MCP-1 131,3 ± 38,8 Eotaxin 84,1 ± 20,4
Figure 1. Fibrin membrane after centrifugation process. (A) Content in three layers: hematocrit, fibrin clot and acellular plasma - from the lower end to the top of the tube. (B) Fibrin membrane rich in platelets and leukocytes after being withdrawn from the tube.
