A interação entre células tumorais e células estromais tem um papel central na progressão neoplásica. As células estromais mesenquimais multipotentes (MSCs) podem se integrar ao microambiente tumoral onde modulam o crescimento dos tumores por meio de distintos mecanismos. Entretanto, pouco se sabe sobre o papel das MSCs na metástase, a principal causa de morte em pacientes com câncer. Utilizando um modelo de melanoma murino ortotópico, nós demonstramos que MSCs obtidas da medula óssea de camundongos (MO-MSCs) ocupam o nicho perivascular nos tumores primários e aumentam 2,5 vezes a incidência de micrometástases pulmonares quando co-infundidas com células de melanoma B16. Observamos ainda que o meio condicionado das MO-MSCs não altera o potencial de colonização pulmonar das células B16 infundidas sistemicamente. Isto indica que as MO-MSCs modulam as fases iniciais da cascata metastática, durante a qual ocorrem os processos de invasão e intravasão nos vasos sangüíneos. Em correlação com estes efeitos pró-metastáticos, o secretoma das MO-MSCs induziu a transição epitélio-mesenquimal (EMT) nas células de melanoma in vitro. Após cultivo em meio condicionado das MO-MSCs, as células B16 adquiriram uma morfologia evidentemente fibroblástica. Ao mesmo tempo, houve o rearranjo dos filamentos de actina e o aumento da expressão de marcadores mesenquimais como fibronectina, vimentina, FSP1, N-caderina e ZEB2, acompanhado da repressão transcricional de E-caderina. A ativação da EMT pelo secretoma das MO-MSCs resultou na aquisição de propriedades metastáticas nas células de melanoma. Após cultivo em meio condicionado de MO-MSCs, as células B16 tiveram seu potencial de ancoragem à fibronectina reduzido, ao passo que houve o aumento na mobilidade e no potencial de invasão em matrizes tridimensionais. Utilizando inibidores competitivos de ATP contra o receptor tirosina-cinase Met, demonstramos que a aquisição de todas as propriedades metastáticas avaliadas e a ativação da EMT nas células de melanoma é mediada pela ativação da via HGF/Met. Estes dados destacam o papel das MOMSCs no microambiente tumoral como fonte perivascular de moléculas indutoras da EMT, cuja ativação leva a aquisição de traços metastáticos nas células de melanoma. Além disso, a inibição da via HGF/Met pode neutralizar os efeitos das MO-MSCs sobre as células tumorais, contribuindo para a repressão de propriedades fundamentais que sustentam a progressão e a disseminação neoplásica. Estas informações são importantes para o desenvolvimento seguro das MO-MSCs como ferramenta terapêutica e demonstram a importância da sinalização entre MSCs e células tumorais na disseminação metastática. Mais especificamente, estas observações reforçam a inibição da via HGF/Met como uma abordagem promissora para o tratamento da metástase. / The crosstalk between tumor cells and stromal cells can profoundly impact tumor progression. Multipotent mesenchymal stromal cells (MSCs) have been reported to integrate the tumor microenvironment where they are described to modulate tumor growth by distinct mechanisms. However, little is known about the impact of MSCs on metastasis, the main cause of death in patients with cancer. Using an orthotopic mouse melanoma model, we showed that mouse bone marrow-derived MSCs (BMMSCs) occupy the perivascular niche within primary tumors and increased by 2.5-fold the incidence of lung micrometastases after co-infusion with B16 melanoma cells. Also, MO-MSCs conditioned medium did not affect the lung colonization ability of systemically infused B16 cells. This indicates that MO-MSCs induces the initial steps of the metastatic cascade, during which the invasion and intravasion occurs. Correlating with these metastatic effects, the BM-MSCs\' secretome activated the epithelial-to-mesenchymal transition (EMT) in B16 cells in vitro. After culture in BMMSCs\' conditioned medium, B16 cells acquired an evident fibroblastic morphology. Simultaneously, we observed the rearrangement of actin filaments and the upregulation of mesenchymal markers such as fibronectin, vimentin, FSP1, Ncadherin and ZEB2. In agreement with the loss of epithelial phenotype, BM-MSCs\' secretome also suppressed E-cadherin expression in B16 cells. The activation of EMT by BM-MSCs leaded to the acquisition of metastatic traits in melanoma cells. After culture in BM-MSCs\' conditioned medium, B16 cells displayed reduced anchorage to fibronectin and increased motility and invasiveness in threedimensional matrix plugs. Inhibition of Met receptor with competitive ATP inhibitors demonstrated that the induction of EMT and the resultant acquisition of metastatic traits are driven by activation of HGF/Met signaling pathway. Taken together, these evidences highlight the role of BM-MSCs as a perivascular source of EMT-inductive signals, whose activation leads to acquisition of metastatic traits in melanoma cells. Furthermore, inhibition of HGF/Met signaling pathway can neutralize the effects of BM-MSCs on tumor cells, thereby allowing the repression of fundamental properties which support tumor progression and metastasis. This information is useful to safely develop BM-MSCs as therapeutic tool and demonstrate the relevance of the signaling between MSCs and tumor cells during metastasis. More specifically, it reinforces that inhibition of Met signaling can be a promissory approach for the treatment of metastasis.

Em nosso trabalho anterior mostramos que dentre as citocinas analisadas, os níveis do mRNA de LIF nas células estromais pediátricas, de SMD e de SMD-LMA foram maiores quando comparados às células estromais de crianças saudáveis. No presente estudo, observamos um aumento tempo dependente nos níveis da proteína LIF após adição de SFB em todas as células analisadas (células estromais de crianças saudáveis, de SMD e de SMD-LMA). O envolvimento de p38, ERK e JNK na expressão LIF nestas células foi determinado pelo uso de inibidores dos membros das proteínas quinase ativadas por mitógenos: ERK (PD98059), p38 (SB302580) e JNK (SP600125) os quais inibiram a produção de LIF nas células estromais de crianças saudáveis, após estas serem estimuladas por SFB. No entanto, os níveis da expressão de LIF-induzido por soro nas células estromais de SMD e de SMD-LMA tratadas com SB302580 (p38) foram significativamente diminuídos, em comparação com a inibição observada no tratamento com PD98059 e SP600125 (p <0001, teste ANOVA). Em adição analisamos as formas fosforiladas de p38 e ERK, após 48hs na ausência ou na presença de soro por diferentes tempos. Níveis de atividade de ERK e do p38 foram inicialmente elevados na ausência de soro. A atividade de p38 foi sustentada após tratamento com SFB, entretanto, ERK apresentou uma variação de atividade durante o tratamento. Sugerimos que a sinalização das MAPKs (p38, ERK e JNK), em resposta a fatores de crescimento presentes no soro, parece desempenhar um papel importante na expressão da LIF em células estromais de crianças saudáveis, mas a sinalização do p38 parece ser funcionalmente mais importante nas mielodisplasias ou naquelas associadas à LMA / Our previous report showed that among the cytokines analysed, LIF mRNA levels in stromal cells from pediatric MDS and MDS-AML were higher as compared to those found in healthy stromal cells. In the present study, we have observed an increased protein LIF levels in a time dependent manner after FCS stimulation in all stromal cells analysed (MDS, MDS-AML and healthy children) and the involvement of p38, ERK and JNK pathways in the LIF expression in these cells was determined. In stromal cells from two healthy children, LIF production was equally inhibited in a dose dependent manner after FCS stimulation by mitogen-activated protein kinase (MAPKs) members inhibitors: ERK (PD98059), p38 (SB302580) and JNK (SP600125). However, in MDS and MDS-AML stromal cells, the levels of LIF-induced by serum, were significantly decreased by SB302580, as compared with the inhibition observed by treatment with PD98059 and SP600125 (p <0,001, ANOVA test). In addition we have analysed the presence of p38 and ERK phosphorylated forms in stromal cells, after 48hs of serum starvation or in the presence of FCS for different times. Activated ERK and p38MAPK levels were initially elevated in the absence of serum. p38MAPK activation was sustained after treatment with FCS, whereas ERK presented a variation of the activated forms during treatment. We suggest that the signalling of the MAPKs (p38, ERK and JNK) in response to growth factors present in the serum, seems to play an important role in the LIF expression by stromal cells of healthy children, but p38 MAPK signalling appears to be functionally more important in MDS and MDS-AML

INTRODUÇÃO: Apesar dos primeiros relatos sobre o uso clínico de células estromais derivadas do tecido adiposo (ADSC) sugerirem que esta abordagem seja factível e efetiva na reconstrução de partes moles, não existem na literatura ensaios clínicos randomizados e com inclusão de controles. Assim sendo, foi objetivo deste estudo investigar se um novo protocolo para o isolamento de ADSC e seu uso em conjunto com os enxertos de gordura é de fato capaz de aumentar a sobrevivência destes enxertos. MÉTODOS: Pacientes com microssomia craniofacial (n=18) foram selecionados e randomicamente distribuídos para o grupo EC (com suplementação de ADSC) ou o grupo EE (sem suplementação de ADSC). Para o grupo EC, ADSC imediatamente isoladas a partir de metade do volume lipoaspirado foram utilizadas para o enriquecimento de células progenitoras do enxerto. A quantidade de tecido adiposo transplantado foi determinada pela simetria com o lado não afetado, não sendo realizado qualquer tipo de supercorreção. O número total de células viáveis isoladas antes e após a suplementação dos enxertos de gordura foi calculado, e através de citometria de fluxo estas células foram examinadas quanto à presença de marcadores de superfície para células mesenquimais. Tomografia computadorizada foi realizada para avaliar ambas as hemifaces no pré-operatório e no pós-operatório de seis meses. A espessura do revestimento de partes moles em quatro diferentes pontos de referência e o volume das hemifaces foram calculados. Morbidade geral e variáveis transoperatórias foram registradas. RESULTADOS: Para ambos os grupos o volume médio injetado foi de aproximadamente 28 mL. O número médio de células viáveis isoladas antes e após a suplementação dos enxertos foi 5,6 x 105 e 9,9 x 105 células por mL de tecido adiposo (p=0,015). Análise de citometria de fluxo revelou que ADSC foram positivas para marcadores de células mesenquimais (>95% para CD 73 e CD 105). No pós-operatório de seis meses, o índice de espessura média nos pontos de 1 a 4 foi de 0,91 (p=0,002), 1,02 (p=0,01), 1,05 (p=0,001), e 1,00 (p=0,04) no grupo EC, e 0,80 (p=0,58), 0,95 (p=0,18), 0,85 (p=0,009), e 0,84 (p=0,59) no grupo EE. Para o grupo EC o volume de gordura mantido após seis meses foi de 84 % e para o grupo EE foi de 51 % (p=0,003). Nenhuma complicação foi detectada. CONCLUSÕES: Estes resultados sugerem que o isolamento e a suplementação de ADSC é efetivo, seguro e superior à lipoenxertia convencional para o aumento de partes moles da face em pacientes portadores de microssomia craniofacial / INTRODUCTION: Although first reports of the clinical use of adipose-derived stromal cells (ADSC) suggest that this approach may be feasible and effective for soft tissue reconstruction, there is a lack of randomized, controlled clinical trials in the literature. Hence, this study aimed to investigate whether a novel protocol for isolation of ADSC and their use in combination with fat tissue could really improve the long-term retention of the grafts. METHODS: Patients with craniofacial microsomia (n=18) were selected and randomly assigned to group EC (with supplementation of ADSC) or group EE (without supplementation of ADSC). For group EC, ADSC freshly isolated from half of the aspirated fat sample were used to enrich for progenitor cells in the graft. Amount of transplanted adipose tissue was determined by trying to obtain symmetry with the unaffected side without any overcorrection. Number of viable cells isolated before and after the supplementation of the grafts was calculated, and these cells were examined for mesenchymal cell surface markers using flow cytometry. Computed tomography was performed to assess both hemifaces preoperatively and at six months postoperatively. Soft tissue thicknesses at four different reference points and the hemifaces volume were calculated. Overall morbidity and surgical variables were recorded. RESULTS: For both groups mean injected volume was 28 mL. Average number of viable cells isolated before and after supplementation of the grafts was 5,6 x 105 and 9,9 x 105 cells per mL of fat tissue (p=0,015). Flow cytometry analysis revealed that the ADSC were positive for mesenchymal cell markers (>95% for CD 73 and CD 105). At six months postoperatively, average thickness ratio at points 1 to 4 was 0,91 (p=0,002), 1,02 (p=0,01), 1,05 (p=0,001), and 1,00 (p=0,04) in group EC, and 0,80 (p=0,58), 0,95 (p=0,18), 0,85 (p=0,009), and 0,84 (p=0,59) in group EE. For group EC surviving fat volume at six months was 84 % and for group EE it was 51 % (p=0,003). No complications were detected. CONCLUSIONS: These results suggest that this strategy for isolation and supplementation of ADSC is effective, safe and superior to conventional lipoinjection for facial recontouring in patients with craniofacial microsomia

Orientador: Karina Gonzales Silvério Ruiz / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T13:33:07Z (GMT). No. of bitstreams: 1 Albiero_MayraLaino_M.pdf: 1767389 bytes, checksum: 14c8bc6c823503f78c08850abc5ed95b (MD5) Previous issue date: 2013 / Resumo: O objetivo do presente estudo foi avaliar se a exposição das células mesenquimais indiferenciadas do ligamento periodontal (PDLMSCs) ao lipopolissacarídeo da Porphyromonas gingivalis (Pg) e da Escherichia coli (Ec), levaria a alterações biológicas que comprometessem as propriedades relacionadas ao fenótipo mesenquimal indiferenciado. Para testar essa hipótese, inicialmente foi verificado se as cinco populações de células mesenquimais indiferenciadas purificadas para o antígeno de superfície CD105 (células CD105+) expresssavam os receptores Toll-like 2 e 4 (TLR2 e 4). Em seguida, as células foram cultivadas na presença do LPS da Pg e da Ec, e avaliadas quanto a sua viabilidade e proliferação pelo ensaio do MTS, expressão dos genes para citocinas inflamatórias IL-1?, IL-6, IL-8 e TNF-? (técnica do PCRq), imunomarcação para STRO-1 e expressão do gene identificador de pluripotencialidade - Oct-4, e capacidade de diferenciação osteoblástica/cementoblástica através dos ensaios para identificação de nódulos minerais e expressão dos genes para RUNX2, ALP e OCN. Os resultados mostraram que todas as populações celulares apresentaram fenótipo mesenquimal indiferenciado com marcação positiva para STRO-1, além de se mostrarem positivas para os receptores TRL2 e 4. O ensaio de MTS revelou que a exposição às três concentrações de LPS da Pg e da Ec (100 ng, 1 ?g e 10 ?g/ml) não comprometeu a viabilidade celular, mantendo todas as populações de PDLMSCs proliferativas ao longo dos 10 dias de cultivo. Em paralelo, verificou que LPS da Pg não alterou os níveis de RNAm para citocinas estudadas enquanto que, o LPS da Ec promoveu um aumento significativo na expressão de IL-6 e IL-8, quando aplicado nas concentrações de 100 ng e 1 ?g/ml. Adicionalmente, os resultados revelaram que a exposição celular aos LPS bacterianos, ambos na concentração de 1?g/ml, não alterou o fenótipo mesenquimal indiferenciado destas populações, uma vez que a expressão do gene OCT-4 e a marcação positiva para STRO-1 mantiveram-se semelhantes às células do grupo controle. Além disso, as células mantiveram a capacidade de diferenciação em fenótipo osteoblástico/cementoblástico, confirmado pela produção de nódulos minerais (ensaio de vermelho de alizarina) e expressão dos genes para RUNX-2, ALP e OCN semelhante ao grupo controle (células cultivadas em meio osteogênico). Somente foi observado um aumento significativo (p<0,05) na produção de nódulos minerais quando as células foram cultivadas na presença de 1?g/ml do LPS de Ec. Contudo, esse aumento da matriz mineral não está relacionado ao aumento nos níveis de RNAm para os genes relacionados ao fenótipo osteogênico comparado ao grupo controle. Dentro das condições experimentais avaliadas, concluí-se que a exposição das PDLMSCs ao LPS da Porphyromonas gingivalis e da Escherichea coli não alterou as propriedades biológicas destas células, mantendo assim, as características que conferem a estas a condição de mesenquimal indiferenciada / Abstract: The aim of this study was to evaluate if the exposure of mesenchymal stem cells of the periodontal ligament (PDLMSCs) to lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) and Escherichia coli (Ec), would lead to biological changes that compromised the properties related to undifferentiated mesenchymal phenotype. To test this hypothesis, initially it was checked whether the five populations of mesenchymal stem cells purified by surface antigen CD105 (CD105+ cells) expressed the Toll-like receptors 2 and 4 (TRL2 and 4). Then, cells were cultured in the presence of LPS from Pg and Ec, and evaluated for viability and proliferation by the MTS assay, expression of proinflammatory cytokines IL-1?, IL-6, IL-8 and TNF-? (PCRq technique), immunostaining for STRO-1 and OCT-4 gene expression, an identifier of pluripotency, and osteoblast/cementoblastic differentiation capacity through assays to identify minerals nodules, as well as the gene expression of RUNX2, ALP and OCN. All these assays were carried out also in the presence of LPS from Escherichia coli (Ec), which is not considered a periodontal pathogen. The results showed that all cell populations with undifferentiated mesenchymal phenotype had positive staining for STRO-1, and also showed to be positive for the receptors TLR2 and 4. The MTS assay revealed that exposure to the three concentrations of Pg and Ec LPS (100 ng, 1?g and 10?g/ml) did not affect cell viability, keeping all PDLMSCs populations proliferative over 10 days of culture. In parallel, it was found that the LPS Pg did not alter mRNA levels for the cytokines studied, while the Ec LPS caused a significant increase in IL-6 and IL-8, when applied concentrations of 100ng/ml and 1?g/ ml. Additionally, the results revealed that cellular exposure to both LPS in the concentration of 1?g/ml, did not alter the undifferentiated mesenchymal phenotype of these populations, since the expression of gene OCT-4 and positive STRO-1 staining remained similar to cells in the control group. In addition, these cells retained their ability to differentiate into osteoblastic/cementoblastic phenotype confirmed by production of mineral nodules (alizarin red assay) and expression of the genes for RUNX-2, ALP and OCN similar to cells control group (cultured in osteogenic medium only). It was only observed a significant increase (p<0.05) in the production of mineral nodules when cells were cultured in the presence of 1?g/ml Ec LPS. However, this increase in the mineral matrix it is not related to increased levels of mRNA for genes related to osteogenic phenotype compared to the control group. Within the experimental conditions, it can be concluded that exposure of PDLMSCs to LPS of Porphyromonas gingivalis and Escherichia coli did not alter the biological properties of these cells thereby, maintaining the characteristics that confer to these cells the condition of mesenchymal stem cells / Mestrado / Periodontia / Mestra em Clínica Odontológica

Orientadores: Luiz Carlos Zeferino, Silvia Helena Rabelo dos Santos / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T06:30:30Z (GMT). No. of bitstreams: 1 Westin_MariaCristinadoAmaral_D.pdf: 3122088 bytes, checksum: 3d2afed3690cd3566bbb3fe26bb492a3 (MD5) Previous issue date: 2013 / Resumo: Introdução: O carcinoma escamoso do colo uterino é precedido pela neoplasia intraepitelial cervical grau 3 (NIC 3). A invasão tumoral envolve a degradação da matriz extracelular e membrana basal do epitélio por enzimas proteolíticas denominadas metaloproteinases (MMPs). Os inibidores teciduais das metaloproteinases (TIMPs) também interferem no processo de invasão. Angiogênese é condição indispensável para a progressão tumoral. Objetivo: Analisar a expressão de MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2 e VEGF-A na NIC 3 e carcinoma do colo uterino. Sujeito e Métodos: Estudo do tipo comparativo observacional constituído de três grupos:- Grupo 1: 55 casos com diagnóstico de NIC 3, Grupo 2: 30 casos com NIC 3 e carcinoma associados e Grupo 3: 46 casos com carcinoma. A expressão protéica foi pesquisada separadamente nas células tumorais e estromais por reação imunoistoquímica. Para estabelecer a porcentagem de células imunopositivas utilizou-se software morfométrico. Análise Estatística: Aplicou-se o Teste T-pareado ou de Mann-Whitney ou Wilcoxon Signed Rank. Resultados: Em todos os grupos, a expressão tumoral de MMP-14 foi maior que a estromal. Inversamente, a expressão de TIMP-2 foi maior nas células estromais que nas tumorais, em cada grupo diagnóstico. A expressão de MMP-9 foi maior nas células estromais que nas tumorais, com exceção do componente invasor do Grupo 2. A expressão estromal de TIMP-1 foi maior que a tumoral no carcinoma e, ao contrário, sua expressão foi maior nas células tumorais da NIC 3. A expressão de VEGF-A foi maior apenas nas células tumorais da NIC 3. Comparando a expressão dos marcadores entre os grupos, foram encontradas as maiores diferenças entre grupos extremos, ou seja, entre NIC 3 e carcinoma. A expressão de MMP-2 nas células estromais foi maior no componente NIC 3 do Grupo 2 que no NIC 3 do Grupo 1. A expressão de VEGF-A nas células estromais do carcinoma foi maior que nas células estromais da NIC 3. Conclusões: Os resultados deste estudo sugerem que a expressão de TIMP-1 aumenta nas células do estroma e diminui nas células tumorais quando a NIC 3 progride para carcinoma invasor. MMP-9 e TIMP-2 tiveram expressão similar na NIC 3 e no carcinoma, o que limita inferências sobre seu papel na progressão neoplásica. O padrão imunoistoquímico da expressão das MMPs, TIMPs e VEGF-A na NIC 3 e no carcinoma invasivo, quando estas lesões estavam associadas, foi semelhante. A expressão do VEGF-A foi maior nas células tumorais do que nas estromais da NIC 3, porém quando esta lesão progride para carcinoma invasivo sua expressão aumenta nas células do estroma e não se altera nas tumorais. A expressão de MMP-14, MMP-2, TIMP-1 e VEGF-A aumentou com a gravidade da neoplasia / Abstract: Introduction: Squamous cell carcinoma of the cervix is preceded by cervical intraepithelial neoplasia grade 3 (CIN 3). Tumor invasion involves degradation of extracellular matrix and epithelium basement membrane by proteolytic enzymes called metalloproteinases (MMPs). Tissue inhibitors of metalloproteinases (TIMPs) are also involved in the invasion process. Angiogenesis is a prerequisite for tumor progression. Objective: To analyze the expression of MMP-2, MMP-9 and MMP-14, TIMP-1, TIMP-2 and VEGF-A in CIN 3 and invasive carcinoma. Subject and Methods: This comparative observational study was consists of three groups: Group 1: 55 cases diagnosed with CIN 3, Group 2: 30 cases with CIN 3 associated with invasive carcinoma and Group 3: 46 cases with invasive carcinoma. Protein expression was investigated separately in tumor and stromal cells by immunohistochemistry and evaluated by the percentage of cells positive for immunostaining using morphometric software. Statistical Analysis: Was performed applying paired t-test or Mann-Whitney or Wilcoxon Signed Rank. Results: In each diagnostic group, expression markers were significantly higher: MMP-14 in tumor cells, and TIMP-2 in stromal cells; also MMP-9 expression was significantly higher in stromal cells, except in invasive component of group 2, and TIMP-1 had significantly higher expression in stromal cells of invasive carcinoma and in tumor cells of CIN 3. VEGF-A expression was significantly higher only in tumor cells CIN 3. Comparing the expression of markers between groups, two by two, we find the greatest differences between the extreme groups, i.e. between invasive carcinoma and CIN 3. The expression of MMP-2 was significantly greater in the stromal component CIN 3 in group 2 than in CIN 3 only. The expression of VEGF-A was significantly higher in the group stromal cell carcinoma when compared to stromal cells CIN 3. Conclusions: The results of this study suggest that the expression of TIMP-1 increases in the stromal cells and decreases in tumor cells when CIN 3 progresses to invasive carcinoma. MMP-9 and TIMP-2 had similar expression in CIN 3 and invasive carcinoma, which limits inferences about its role in neoplastic progression. The immunohistochemical pattern of expression of MMPs, TIMPs and VEGF-A in CIN 3 and invasive carcinoma, as these lesions were associated, was similar. The expression of VEGF-A was higher in tumor cells than in stromal cells in CIN 3, but when the lesion progresses to invasive carcinoma its expression increases in the stromal cells and the tumor cells does not change. The expression of MMP-14, MMP-2, TIMP-1 and VEGF-A was increased with the severity of the neoplasia / Doutorado / Oncologia Ginecológica e Mamária / Doutora em Ciências da Saúde

O crescente número de aplicações clínicas envolvendo células mesenquimais estromais multipotentes (CMMs) gera a necessidade da produção em larga escala destas células com adequada qualidade terapêutica. As CMMs são geradas atualmente através de culturas aderentes na presença de soro fetal bovino (SFB). Entretanto, apesar da eficiência na promoção do crescimento celular, o uso de SFB não é isento de desvantagens e riscos. Além do alto custo, sua utilização pode gerar risco de contaminação do produto final com agentes adventícios como vírus e príons. Por outro lado, a sua variabilidade em diferentes lotes e fornecedores dificulta a padronização do meio e reprodutibilidade do cultivo. Uma alternativa promissora para a cultura de células destinadas a terapia celular é a substituição de SFB por soro AB humano obtido a partir de plasma humano. Neste cenário, o objetivo deste trabalho foi produzir soro AB humano e avaliar a sua qualidade e eficácia como substituto do SFB na produção de células mesenquimais destinadas à terapia celular. Para a produção do soro AB humano foram avaliados tanto Plasma comum (PC>24h) (n=3) quanto Plasma isento de crioprecipitado (PCIC) (n=3). Após a produção, foi realizado o controle de qualidade dos lotes produzidos sendo verificado que os mesmos atenderam as exigências necessárias para a utilização na terapia celular. As duas fontes de plasma utilizadas para a produção do soro AB humano apresentaram características semelhantes quanto aos constituintes bioquímicos e demais parâmetros analíticos e foram eficazes na suplementação do cultivo e expansão das CMMs. A suplementação do meio com 10% de soro AB humano foi eficaz tanto no cultivo em culturas estáticas quanto em microcarregadores. Esta característica alinhase com a possibilidade de produção em larga escala, uma vez que, permitiu a expansão das CMMs de maneira similar à condição controle (suplementada com 10% de SFB) e preservou as características imunofenotípicas, funcionais e perfil citogenético pós cultivo. Além disso, o soro AB humano produzido pode ser utilizado por no mínimo doze meses após produção, quando armazenado em temperatura inferior a 20ºC negativos / The growing number of clinical applications involving multipotent mesenchymal stromal cells (MSCs) generates the need for large-scale production of these cells with appropriate treatment quality. The MSCs are now generated through adherent cultures in the presence of fetal bovine serum (FBS). However, despite the efficiency of this method, the use of FBS is not exempt of drawbacks and hazards. Besides the high cost, their use can lead to the risk of final product contamination with adventitious agents such as viruses and prions. In addition, due to its high variability of reproducibility, this methodology requires high level of standardization. A promising alternative would be to replace FBS by human AB serum from human plasma. In this scenario, our objective was to produce human AB serum and evaluate their quality and effectiveness as a FBS substitute on production of mesenchymal cells for therapy. For the production of human AB serum were evaluated both common plasma (CP > 24h; n=3) and plasma cryoprecipitate depleted (PCD; n=3). Quality controls were carried out to stablish minimal requirements of quality to be used in a cell therapy scenario. Both plasma sources of human AB serum presented similar biochemical characteristics and other analytical parameters, being effective in supplementation of the culture and expansion of MSCs. Supplementing the culture medium with 10% human AB serum was effective both in cultivation of static cultures as for microcarriers. This condition might indicate that this method would be useful for large scale production, allowing the expansion of MSCs similarly to the control condition and maintaining immunophenotypic, functional and cytogenetic characteristics. Furthermore, the human AB serum produced can be used for at least twelve months after production when stored at temperature below 20ºC negative

As células estromais mesenquimais (CMMs) se tornaram de grande interesse para a terapia celular devido ao seu potencial de se diferenciar e reconstituir tecidos especializados. Mais recentemente, este interesse tem aumentado significativamente devido à descoberta de que as CMMs são capazes de secretar uma infinidade de mediadores para estimular a regeneração in situ de tecidos lesados. Dessa forma, CMMs podem ser consideradas tanto como um produto terapêutico em si, quanto uma biofábrica de diversas proteínas relevantes do ponto de vista terapêutico. Para atender a estas crescentes demandas, ambas as aplicações requerem o desenvolvimento de processos de expansão celular com alto rendimento, sob condições de cultivo definidas, reprodutíveis, escalonáveis, permitindo a obtenção de produtos com adequada identidade, potência, pureza, segurança e viáveis economicamente. Frente ao exposto, este trabalho teve como objetivos principais o estabelecimento de um processo de expansão de CMMs baseado em biorreatores e a caracterização do secretoma destas células visando aplicações terapêuticas. Para isto, a expansão de CMMs do cordão umbilical (MCUs) foi realizada em frascos multicamadas (MC) e nos biorreatores de leito fixo (LF), tanque agitado com microcarregadores (TA) e fibrasocas (FO). Os resultados mostraram que a taxa de proliferação específica das células foi maior (< tempo de duplicação) no biorreator de FO (36,8 ± 1,7 horas), bem como o fator de expansão (9,8 ± 1,0) e a eficiência na recuperação celular (100%). Um nível similar de produção celular foi observado para o TA, MC e LF com elevado fator de expansão celular (8,8 ± 0,39, 8,7 ± 0,90, 6,9 ± 1,3, respectivamente). No entanto, em termos de eficiência na recuperação celular (%), LF apresentou a menor taxa de recuperação dentre todos os sistemas (18% (± 0,77)), acompanhado pelo TA (61% (± 15,7)). As células mantiveram suas características imunofenotípicas e o potencial de diferenciação em adipócitos, osteócitos e condrócitos em todos os sistemas de cultivo avaliados. Foi também realizada a análise de custos (COG) e avaliação da viabilidade econômica para produção de CMMs visando tratamento da doença do enxerto contra o hospedeiro (DECH) em escala comercial, utilizando os sistemas de cultivo avaliados experimentalmente sob diferentes estratégias de reembolso. Apesar dos resultados experimentais satisfatórios para o biorreator FO, o COG revelou que este sistema tem o maior custo devido aos elevados custos dos consumíveis requeridos e do custo do equipamento. O frasco MC foi considerado como a tecnologia mais rentável e robusta no cenário avaliado e o biorreator TA obteve a segunda posição. O biorreator TA foi escolhido como o mais adequado analisando de maneira conjunta os dados experimentais obtidos, a análise dos custos dos diferentes sistemas de cultivo e a escalonabilidade de cada sistema. Assim, esse biorreator foi eficientemente utilizado para o cultivo de MCUs em condições isentas de SFB e xenoantígenos, sendo possível a produção de uma grande quantidade de células, representando um passo importante no desenvolvimento de um bioprocesso em conformidade com as normas das agências regulatórias. Por fim, com a análise do secretoma das CMMs por espectrometria de massas foi possível a identificação de uma gama enorme de proteínas interessantes (aprox. 2400) envolvidas em importantes processos biológicos. O futuro monitoramento dessas proteínas em biorreatores poderá representar um método inovador e original de produção de produtos livres de células para uso na terapia celular. / Mesenchymal stem/stromal cells (MSC) have become of great interest for cell therapy because of its potential to differentiate and reconstitute specialized tissues. More recently, such interest has significantly increased due to the discovery that MSC are capable of secreting a plethora of mediators to stimulate the in situ regeneration of injured tissues. Thus, MSC can be considered as a therapeutic product itself and as a biofactory of various relevant therapeutic proteins. To meet these increasing demands, both applications require the development of high-yield, reproducible, scalable and cost-effective bioprocesses under defined culture conditions, obtaining products with proper identity, purity and safety. Based on these, the main goal of this work was the establishment of a MSC expansion process based on bioreactors and secretome characterization of these cells targeting therapeutic applications. The MSC expansion was performed using multi-layered flasks (ML) and fixed bed (PB), stirred tank (STR) and hollow fiber (HF) bioreactors. The results showed that the proliferation rate of the cells was higher (< doubling time) in the HF bioreactor (36.8 ± 1.7 hours), as well as the expansion fold-increase (9.8±1.0) and harvesting efficiency (100%). A similar level of cell production was observed for STR, ML and PB with high fold-increase (8.8±0.39, 8.7±0.90, 6.9±1.3, respectively). However, in terms of harvesting efficiency (%), PB bioreactor presented the lowest retrieval rate across all the technologies (18% (±0.77)), followed by STR (61% (±15.7)). The cells retained their functional properties after culture in all the culture systems evaluated. This study was then extended through the use of a bioprocess economics tool for the evaluation of the economic feasibility of producing MSC-based treatment for acute graft vs. host disease (aGvHD) at commercial scale, using the culture systems experimentally evaluated under different reimbursement strategies. Despite the advantageous experimental results of HF bioreactors, the COG analysis has revealed that this is the least cost effective cell culture system to be used, due to its high consumable and equipment costs. ML flasks ranked first as the most cost effective and robust technology in this scenario and microcarrier-based technologies (STR) ranked in second position. The STR bioreactor was chosen as the most suitable for MSC expansion analyzing the experimental data, COG analysis and scalability of each culture system. Thus, STR bioreactor was efficiently tested for MSC expansion under serum and xeno-free conditions and it was possible to produce a large amount of cells. The development of a scalable microcarrier-based stirred culture system using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates. Finally, with the MSC secretome analysis by mass spectrometry it was possible to identify a wide range of interesting proteins (approx. 2400) involved in important biological processes. The future monitoring of these proteins in bioreactors may represent a novel and unique method of producing cell-free products for use in cellular therapy.

L’objectif de cette thèse était d’améliorer les connaissances fondamentales sur le tissu adipeux, organe qui est au cœur de la pratique des chirurgiens plasticiens. En effet, ce tissu peut être transplanté de façon autologue afin de combler une perte de substance (rôle volumateur des adipocytes) mais également servir à des fins de régénération tissulaire en lien avec les cellules de la fraction vasculaire stromale (FVS) et tout particulièrement des cellules stromales mésenchymateuses (CSM). Ces cellules s’obtiennent après lipoaspiration du tissu par une digestion enzymatique de la graisse obtenue. Il s’avère que les connaissances disponibles sur ces CSM sont essentiellement issues d’études in vitro après une phase de culture cellulaire plus ou moins longue et ainsi les propriétés in vivo sont mal connues. Ce travail a donc consisté à caractériser l’hétérogénéité du compartiment stromal natif du tissu adipeux obtenu après digestion enzymatique. Nous avons isolé deux populations stromales natives distinctes : les ASC (CD34+), en grande majorité, et des cellules péricytaires (CD146+). Ces 2 types cellulaires différaient dans leurs phénotypes, leurs potentiels de clonogénécité et leurs propriétés immunomodulatrices in vitro et in vivo. Nous avons ensuite comparé la digestion enzymatique du tissu aux techniques de digestion mécanique utilisable au sein de nos blocs opératoires. Nous avons démontré que ces nouvelles techniques permettaient bien de produite les cellules de la FVS dont des CSM, cellules particulièrement intéressante pour des gestes de régénération tissulaire. De plus, l’ensemble des techniques de laboratoire acquise au cours de ce travail nous ont permis d’investiguer le rôle des techniques de lipoaspiration utilisé en chirurgie plastique sur le tissu adipeux. Nous avons démontré par cytométrie de flux et par immunofluorescence in situ qu’une partie de la trame microvasculaire était conservée. L’ensemble de ces résultats viennent s’ajouter aux données cliniques démontrant que la lipoaspiration du tissu est un geste permettant d’être plus conservateur pour le tissu et pourrait expliquer des taux de complications moindre après chirurgie de contour de la silhouette. / The aim of this work was to improve the knowledge on adipose tissue, organ that is at the heart of the practice of plastic surgeons. Indeed, this tissue can be transplanted autologously in order to fill a defect (volumizing role of the adipocytes) but also to be used for tissue regeneration in connection with the cells of the stromal vascular fraction (SVF) and especially the mesenchymal stromal cells (MSCs). These cells are obtained after liposuction of the tissue by enzymatic digestion of the extracellular matrix. It turns out that the knowledge available on these CSM is essentially derived from in vitro studies after a cell culture phase and thus the in vivo properties are poorly known. This work consisted in characterizing the heterogeneity of the native stromal compartment of adipose tissue obtained after enzymatic digestion. We isolated two distinct native stromal populations: the ASC (CD34 +), for the most part, and the pericyte cells (CD146 +). These 2 cell types differed in their phenotypes, their clonogenecity potentials and their immunomodulatory properties in vitro and in vivo. We then compared the enzymatic digestion of the tissue with the techniques of mechanical digestion usable within our operating room. We have demonstrated that these new techniques made it possible to produce the cells of the FVS including MSC, cells particularly interesting for regenerative surgery. In addition, all the laboratory techniques acquired during this work allowed us to investigate the role of liposuction techniques used in plastic surgery on adipose tissue. We have demonstrated by flow cytometry and confocal microscopy, that part of the microvasculature framework is conserved after liposuction. All of these results are in addition to clinical data demonstrating that liposuction of the tissue is a gesture to be more conservative for the tissue and could explain lower rates of complications after contour surgery.

Hintergrund: Der medizinische Fortschritt der letzten Jahrzehnte verbessert das Überleben insbesondere extrem kleiner Frühgeborener. Bei diesen stellt die adäquate Oxygenierung über die unreife Lunge den kritischsten Prozess in der klinischen Betreuung dar, an dessen Ende häufig eine Beeinträchtigung der postnatalen Lungenentwicklung und -reifung steht. Klinisch als Bronchopulmonale Dysplasie (BPD) imponierend, stellt diese Erkrankung die häufigste Folge der extremen Frühgeburtlichkeit dar und ist im weiteren Verlauf mit einer bedeutenden Langzeitmortalität und gesundheitsökonomischen Belastung verbunden. Außer der Vermeidung der Frühgeburtlichkeit existiert keine Therapie für BPD. Exogene Mesenchymale Stromazellen (MSC) erwiesen sich jedoch in Tiermodellen der BPD als therapeutisch ausgesprochen wirksam und stellen somit einen vielversprechenden Therapieansatz dar. Dennoch ist wenig über die Mechanismen der exogenen MSC-Wirkung in der frühgeborenen Lunge bekannt; ein Verständnis dieser ist jedoch unabdingbar für eine sichere und effektive Translation von MSC-basierten Therapien in die klinische Anwendung. Hypothese: Lungenresidente MSC sind an der normalen Lungenentwicklung beteiligt, werden durch Bedingungen, welche die zu frühe Geburt simulieren, beeinträchtigt, und tragen so zur Pathogenese der BPD bei. Exogene MSC unterstützen die lungenresidenten MSC in ihrer normalen Funktion und/oder schützen sie vor Schaden. Methoden und Resultate: Um mesenchymale Zellen aus human-fetalem Lungengewebe (FLMSC) zu isolieren, wurde eine Methode zur enzymatischen Gewebedissoziation mit anschließender selektiver Dichtegradientenzentrifugation entwickelt. Der überwiegende Mehrheit der isolierten Lungenmesenchymzellen wurde als MSC identifiziert. Damit ist mit der hier vorliegenden Arbeit erstmals die vollständige Beschreibung von humanen, fetalen Lungen-MSC gelungen. Nabelschnur (UC)MSC wurden durch enzymatischen Verdau der Wharton-Sulze gewonnen. Kultur der FLMSC in einer hypoxischen, den intrauterinen Bedingungen ähnlichen Atmosphäre resultierte in einem das Lungenwachstum stimulierenden Cytokin- und Genexpressionsmuster. Zudem produzierten die FLMSC für Lungenwachstum und -reifung unabdingbare Extrazellulärmatrixproteine. Unter Exposition gegenüber hyperoxischen Kulturbedingungen – welche die zu frühe Geburt mit anschließender Behandlung auf einer Neugeborenenintensivstation simulierten – begannen FLMSC einen Transdifferenzierungsprozess und sezernierten proinflammatorische und antiangiogene Signalmoleküle. Zudem wurde eine Reduktion der Produktion von für die Lungenentwicklung unabdingbaren Matrixproteinen beobachtet. FLMSC sendeten zudem “Danger-Signale” an andere Zellen, sobald sie Hyperoxie ausgesetzt wurden. Exogene UCMSC sezernierten in vitro große Mengen an Proteinen, welche Lungenzellen vor Schaden schützen und Lungenwachstum und -differenzierung unterstützen. Diskussion und Schlussfolgerung: Das Mesenchym der humanen fetalen Lunge am Ende der kanalikulären Entwicklungsphase besteht zum Großteil aus MSC, und nicht Fibroblasten. Das impliziert eine mesenchymale Stamm- und Progenitorzellhierarchie in der fetalen Lunge sowie bislang unbeschriebene zelluläre mesenchymale Transdifferenzierungsprozesse im weiteren intrauterinen Entwicklungsverlauf. In vitro wurde Evidenz für eine Beteiligung der endogenen Lungen-MSC an der normalen Lungenentwicklung generiert; eine Beteiligung an der Koordination von epithelialem und endothelialem Lungenwachstum und -reifung durch die endogenen MSC kann auf Grund der vorliegenden Daten angenommen werden. Nach Exposition gegenüber Hyperoxie entwickelten FLMSC einen die BPD unterstützenden Phänotyp. Exogene UCMSC besitzen das Potential, die in diesem Zustand fehlenden Faktoren bereitzustellen. Endogene pulmonale MSC sind daher potentielles Ziel und potentielle Effektorzellpopulation einer MSC-basierten Therapie für BPD. Dennoch sind weitere in vivo Experimente mit Tiermodellen der extremen Frühgeburtlichkeit unabdingbar, um die Rolle der endogenen MSC in der normalen, und insbesondere gestörten Lungenentwicklung zu verstehen und folgend potente, und vor allem sichere zellbasierte Therapeutika für unsere wohl verletzlichste Patientenpopulation – Frühgeborene – bereitzustellen. / Background: Despite great achievements in neonatal and perinatal medicine over the past decades, the immature lung remains the most critical organ to care for after premature birth. As a consequence, impairment of of postnatal lung development – bronchopulmonary dysplasia or BPD – remains the most common complication of extreme prematurity and a major healthcare burden. There is no therapy for BPD, except prevention of premature birth. Recently, exogenous mesenchymal stromal cells (MSC) have been shown to prevent and rescue impaired lung development in animal models. Understanding the mechanisms behind the beneficial action of these cells is crucial for a successful, safe, and effective clinical translation of these promising MSC-based cell therapies in neonates. Hypothesis: Endogenous lung-resident MSC contribute to normal lung development and become impaired in conditions resembling premature birth, thus playing a part in the pathogenesis of BPD. Exogenous MSC act by supporting and/or preserving the endogenous mesenchymal cell’s function. Methods and Results: Using lung tissue from aborted fetuses, a novel enzyme/density gradient technique was employed to obtain endogenous human fetal lung mesenchymal cells (FLMSC). The vast majority of the so-isolated cells fulfilled all criteria of MSC, making the herein presented work the first complete description of MSC from human fetal lung tissue. Human umbilical cord-derived (UC)MSC were isolated by enzymatic digestion of the Wharton’s jelly. When cultured in hypoxic atmospheres resembling intrauterine conditions, resident FLMSC exerted a gene expression- and cytokine profile supporting epithelial and endothelial lung development, and secreted extracellular matrix components crucial for normal lung growth. After exposure to hyperoxia – thus mimicking premature birth and subsequent treatment on a neonatal intensive care unit – FLMSC showed signs of transdifferentiation, acquired a pro-inflammatory / anti-angiogeneitic secretory profile, diminished production of crucial extracellular matrix components and send out danger signals to other cells. Conversely, UCMSC secreted various paracrine factors protecting lung cells, and proteins contributing to lung growth and alveolarization. Discussion and Conclusions: The human fetal lung’s mesenchyme at the late canalicular stage of development mainly consists of MSC rather than fibroblasts, thus implying a complex mesenchymal stem-/progenitor cell hierarchy and previously undescribed cellular transdifferentiation processes of human endogenous lung mesenchymal progenitors during late pregnancy. Evidence for a contribution of FLMSC to normal lung development was generated in vitro, suggesting a co-ordination of endothelial and epithelial cell fate by human endogenous lung MSC. When challenged with hyperoxia, FLMSC cells acquire a phenotype contributing to the pathogenesis of the BPD. Conversely, UCMSC harbor the potential to provide the factors that these damaged resident MSC lack to produce. The endogenous MSC may therefore represent a potential target of cell-based therapies of BPD. However, in vivo data obtained from premature animals is inevitable to gain further insights into the contribution of endogenous lung MSC to normal and disrupted lung development and to clinically translate potent and safe MSC-based therapeutics for our most vulnerable patient population - premature infants.

Malgré l’évolution des outils de bio-ingénierie tissulaire et la sophistication des recherches visant à développer de nouveaux substituts cutanés, la prise en charge clinique des brûlures sévères a relativement peu évolué depuis plus de 30 ans. Si la survie est quasiment garantie par l’utilisation de cultures d’épidermes autologues (CEA), le traitement des grands brûlés n’en reste pas moins traumatisant, avec l’émergence de séquelles physiques (cicatrices hypertrophiques, dyschromies, fragilité cutanée), pathophysiologiques (désordres métaboliques et immunitaires), et neuropsychologiques (neuropathies, douleurs chroniques, syndrome de stress post-traumatique). Depuis leur découverte dans les années 1960, les Cellules Stromales Mésenchymateuses (CSM) ont suscité un intérêt grandissant dans le domaine de la thérapie cellulaire, en raison de leurs propriétés trophiques et immunomodulatoires. La découverte de leur haute plasticité face à divers stimuli environnementaux a plus récemment ouvert le champ de nouvelles stratégies de thérapie ciblée appelées « préconditionnement ». Ainsi, cette thèse a pour objet l’évaluation d’une stratégie de préconditionnement de CSM, afin d’en potentialiser l’action thérapeutique, dans le cadre du traitement de brûlures par CEA. Ce travail de thèse a été partagé en trois unités expérimentales. Tout d’abord la stratégie de préconditionnement a été mise au point sur des modèles de cicatrisation in vitro, permettant ainsi de souligner le rôle intéressant de l’interleukine-1β (IL-1β) et de la substance P (SP). Ensuite, l’efficacité et les mécanismes d’action de ces facteurs de préconditionnement ont été évalués in vitro, puis in vivo sur un modèle de plaie excisionnelle, en utilisant les CSM ou leurs produits de sécrétion. Il a ainsi pu être démontré que l’IL-1β améliorait l’efficacité des CSM en promouvant leur activité anti-inflammatoire, pro-migratoire et de remodelage. Il a également été montré que cet effet était en partie lié à un mécanisme impliquant la voie du TGF-β1. Enfin, la plus-value de cette stratégie thérapeutique a fait l’objet d’une étude in vivo sur un modèle de brûlure profonde mimant la prise en charge du patient sévèrement brûlé. Malgré divers problèmes techniques limitant la prise de greffe de CEA dans ce modèle, l’effet anti-inflammatoire et pro-angiogénique des CSM a pu être confirmé. Ces résultats semblent donc montrer l’intérêt d’une thérapie par CSM préconditionnées. Des études précliniques complémentaires sont maintenant requises pour vérifier la plus-value d’une telle thérapie dans le contexte de la brûlure cutanée. / Since the 1980’s, little progress has been made in the management of major burns, in spite of several research advances in the field of skin tissue engineering and regenerative medicine. Developed in 1975, Cultured Epithelial Autografts (CEA) are the last-in-date significant breakthrough, allowing patient survival in most critical cases. However, patients still have to cope with debilitating sequelae including hypertrophic scars, skin fragility, immunometabolic dysfunctions, chronic pain and post-traumatic stress disorder. Mesenchymal Stromal Cells (MSC) have raised an increasing interest during the past 50 years due to their trophic and immunomodulatory properties. Recent findings about their high plasticity to external stimuli have fostered the development of new targeted therapies known as “preconditioning strategies”. This PhD work thus aimed to assess a MSC preconditioning strategy for the treatment of major burns using CEA. The present work was divided into three main experimental parts. First, in vitro experiments were developed in order to set up the preconditioning strategy, and revealed the interesting role of both interleukin-1β (IL-1β) and substance P (SP). Then, both effectiveness and mechanism of action of these preconditioning modalities were assessed in vitro and in vivo, either using MSC or their secretory products. It was thus shown that IL-1β could potentiate MSC effectiveness through the promotion of pro-migratory, anti-inflammatory and pro-remodeling activities. This effect was shown to be partly mediated by the TGF-β1 signaling pathway. At last, this preconditioning strategy was evaluated in a third degree burn rat model mimicking the surgical treatment applied to severe burn patients. Despite technical hurdles limiting CEA engraftment, anti-inflammatory and pro-angiogenic properties of MSCs were confirmed in this model. These preliminary results underline the potential of a preconditioned MSC therapy in wound healing. Additional preclinical studies are now required to corroborate the benefit of such a therapy in major burns.