Effect of inflammation on mesenchymal stem cell from periodontal tissues

Orientador: Karina Gonzales Silvério RuizTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O presente estudo avaliou o efeito do processo inflamatório sobre as propriedades das células mesenquimais indiferenciadas do ligamento periodontal (PDLSCs) e sobre a expressão dos marcadores mesenquimais nos tecidos periodontais. Inicialmente as PDLSCs purificadas para o marcador CD105 (hPDL-CD105+) foram expostas ao extrato proteico total da espécie bacteriana Porphyromonas gingivalis (PgPE), e avaliadas quanto ao metabolismo celular, atividade inflamatória, capacidade proliferativa e diferenciação osteogênica. Na sequência, foi analisada a alteração dos marcadores de perfil mesenquimal indiferenciado após a indução da doença periodontal in vivo, e confirmada in vitro. Para isso, hPDL-CD105+ foram expostas à diferentes concentrações de PgPE e avaliadas quanto ao metabolismo celular (ensaio de MTT), a fim de determinar a concentração de PgPE não-citotóxica. Apoptose (marcadores Annexin V e 7-AAD) foi avaliada pela técnica da citometria de fluxo. A atividade pró-inflamatória foi investigada por 1) expressão gênica de interleucina 1-beta (IL-1ß), interleucina 6 (IL-6) e (fator de necrose tumoral alfa (TNF-a), 2) a presença da proteína IL-6 por imunofluorescência 3) qPCR array da via de sinalização IL6/STAT3. Para a avaliação da capacidade de diferenciação osteogênica, foi realizado o ensaio de formação de nódulos minerais, com coloração de vermelho de alizarina (AR) e expressão dos genes para Runt-related transcription factor 2 (RUNX2), fosfatase alcalina (ALP) e osteocalcina (OCN) por RT-qPCR. Doença periodontal experimental por ligadura foi induzida em ratos Wistar durante 7 dias, após este período o grupo somente com indução da doença sofreu eutanásia (n=8) e outro grupo recebeu raspagem da superfície radicular após a remoção da ligadura (n=8), e junto dos animais controle (n=8), sofreram a eutanásia aos 14 dias. Dos tecidos periodontais coletados de ratos foram avaliados a expressão dos marcadores relacionados ao fenótipo mesenquimal indiferenciado (STRO-1, CD105, CD166, CD146), por meio de ensaio de imunohistoquímica e expressão gênica por RT-qPCR. Os mesmos marcadores foram avaliados por citometria de fluxo nas hPDL-CD105+ expostas à PgPE. Os resultados mostraram que doses menores que 3 ?g/ml se mostraram não-citotóxicas, e não foram capazes de induzir apoptose celular. hPDL-CD105+ apresentaram aumento na expressão de citocinas pró-inflamatórias expostas à 2ug/ml de PgPE, e confirmados por um aumento na expressão de 28 genes inflamatórios envolvidos com a via IL6/STAT3. Adicionalmente, essas células apresentaram manutenção do potencial osteogênico com deposição significativa de nódulos minerais e expressão dos genes osteogênicos após exposição à PgPE. Nos tecidos periodontais, a doença periodontal inflamatória induziu um aumento na expressão gênica dos marcadores de superfície CD105 e CD166, e este resultado foi confirmado em nível proteico in situ. Nas hPDL-CD105+ estimuladas com PgPE, houve aumento do marcador de indiferenciação celular STRO-1. E este aumento está correlacionado positivamente ao aumento de expressão gênica da IL-6 nas hPDL-CD105+. Esses dados sugerem que houve uma clara modulação dos marcadores de superfície celulares relacionado ao fenótipo mesenquimal indiferenciado in vitro e in vivo, e que a presença da PgPE não impactou na viabilidade celular e a capacidade de diferenciação osteogênica das hPDL-CD105+, preservando suas propriedades biológicasAbstract: The present study evaluated the effect of the inflammatory process on mesenchymal stem cell properties from the periodontal ligament (PDLSCs) and on the expression of the mesenchymal cell surface markers in the periodontal tissues. Initially PDLSCs purified with CD105 marker (hPDL-CD105+) were exposed to Porphyromonas gingivalis total protein extract (PgPE), and cells metabolism, inflammatory activity, proliferative capacity and osteogenic differentiation were evaluated. Subsequently, it was analyzed the expression of the mesenchymal cell surface markers after the induction of periodontal disease in vivo, and confirmed in vitro. For this, hPDL-CD105+ were exposed to different concentrations of PgPE and evaluated for cellular metabolism (MTT assay) to determine the non-cytotoxic PgPE concentration. Apoptosis (Annexin V and 7-AAD markers) was assessed by flow cytometry technique. Proinflammatory activity was investigated by 1) gene expression of interleukin 1-beta (IL-1?), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-?) by RT-qPCR, 2) presence of IL-6 with immunofluorescence assay 3) qPCR array of IL6/STAT3 signaling pathway. To evaluate osteogenic differentiation capacity, mineral nodules formation was assessed with alizarin red staining (AR) and gene expression for Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) by RT-qPCR. Experimental periodontal disease by ligature was induced in Wistar rats for 7 days, after this period one group with disease induction only was euthanized (n=8) and one group was received scaling and root planning after removal of the ligature (n=8), and together with the control group (n=8), were euthanized at day 14. From the periodontal tissues collected from rats, the expression of mesenchymal cell surface markers (STRO-1, CD105, CD166, CD146) was evaluated by immunohistochemistry and RT-qPCR gene expression. The same cell surface markers were evaluated by flow cytometry in hPDL-CD105+ exposed to PgPE. Results showed that doses lower than 3 ?g/ml were non-cytotoxic, and were not able to induce apoptosis. hPDL-CD105+ showed increased expression of proinflammatory cytokines exposed to 2ug/ml PgPE, and this was confirmed by an increased expression of 28 inflammatory genes related to the IL6/STAT3 pathway. In addition, osteogenic potential was maintained in the presence of PgPE, with significant deposition of mineral nodules and expression of the osteogenic genes. In rat periodontal tissues, inflammatory disease induced an increase in CD105 and CD166 surface markers gene expression and this result was confirmed in situ at protein level. hPDL-CD105+ stimulated with PgPE showed an increase in STRO-1 stem cell marker. And this increase is positively correlated to increased IL-6 gene expression in hPDL-CD105+. These data suggest that there was a clear modulation of the cell surface markers related to the mesenchymal stem cell phenotype in vitro and in vivo, and that the presence of PgPE did not affect cell viability and osteogenic differentiation capacity of hPDL-CD105+, preserving its biological propertiesDoutoradoPeriodontiaDoutora em Clínica Odontológica159202/2013-22014/01827-9CNPQFAPES

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide fromEscherichia coli (EcLPS). Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR.PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities232FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPNão te

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. Objective : This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities

Transcriptome profile of highly osteoblastic/cementoblastic periodontal ligament cell clones

Heterogeneous cell populations of osteo/cementoblastic (O/C) or fibroblastic phenotypes constitute the periodontal dental ligament (PDL). A better understanding of these PDL cell subpopulations is essential to propose regenerative approaches based on a sound biological rationale. Objective: Our study aimed to clarify the differential transcriptome profile of PDL cells poised to differentiate into the O/C cell lineage. Methodology: To characterize periodontal-derived cells with distinct differentiation capacities, single-cell-derived clones were isolated from adult human PDL progenitor cells and their potential to differentiate into osteo/cementoblastic (O/C) phenotype (C-O clones) or fibroblastic phenotype (C-F clones) was assessed in vitro. The transcriptome profile of the clonal cell lines in standard medium cultivation was evaluated using next-generation sequencing technology (RNA-seq). Over 230 differentially expressed genes (DEG) were identified, in which C-O clones showed a higher number of upregulated genes (193) and 42 downregulated genes. Results: The upregulated genes were associated with the Cadherin and Wnt signaling pathways as well as annotated biological processes, including “anatomical structure development” and “cell adhesion.” Both transcriptome and RT-qPCR showed up-regulation of WNT2, WNT16, and WIF1 in C-O clones. Conclusions: This comprehensive transcriptomic assessment of human PDL progenitor cells revealed that expression of transcripts related to the biological process “anatomical structure development,” Cadherin signaling, and Wnt signaling can identify PDL cells with a higher potential to commit to the O/C phenotype. A better understanding of these pathways and their function in O/C differentiation will help to improve protocols for periodontal regenerative therapies

O efeito do processo inflamatório sobre as propriedades biológicas das células mesenquimais indiferenciadas do ligamento periodontal de humanos

A doença periodontal inflamatória caracteriza-se pela destruição dos tecidos que suportam os dentes, os quais incluem ligamento periodontal, cemento dental e osso alveolar. A forma de terapia mais comumente empregada no tratamento da doença periodontal é a raspagem e alisamento da superfície radicular (terapia mecânica), a qual tem como efeito clínico a diminuição da profundidade de sondagem das bolsas periodontais e a estabilização das medidas do nível clínico de inserção quando combinadas com um bom controle de biofilme. Porém, após este tratamento, ocorre o padrão de cura do tipo reparação (epitélio juncional longo) devido à alta taxa proliferativa do tecido epitelial quando comparado aos tecidos mesenquimais envolvidos (ligamento periodontal, cemento dental e osso alveolar), não ocorrendo a regeneração. Atualmente, sabe-se que o sucesso da regeneração dos tecidos periodontais depende da migração e proliferação das células indiferenciadas do ligamento periodontal remanescente, acompanhada pela subsequente diferenciação em osteoblastos, cementoblastos e fibroblastos. Sendo a periodontite uma doença inflamatória crônica, tem sido sugerido que as estruturas periodontais remanescentes, teriam a sua habilidade para promover a regeneração tecidual comprometida. A doença periodontal possui caráter multifatorial, porém, seu fator etiológico primário é o biofilme, sendo que a Porphyromonas gingivalis tem sido considerada um dos principais patógenos associados ao desenvolvimento e progressão da doença periodontal inflamatória crônica (periodontite), e atualmente sabe-se que seu lipopolissacarídeo é reconhecido pelas células por receptores Toll-like, já identificados em células mesenquimais do ligamento periodontal. Assim, propõe-se que as células mesenquimais indiferenciadas sofreriam alterações em suas propriedades quando expostas a um ambiente rico em toxinas bacterianas, comprometendo a sua capacidade regenerativa.Periodontal disease is characterized by inflammatory destruction of tissues which support the teeth including periodontal ligament, dental cementum and alveolar bone. The most common form of therapy used in the treatment of periodontal disease is the scaling and root planning (mechanical therapy), which decreases clinical probing depth of periodontal pockets and stabilizes the measures of clinical attachment level when combined with good biofilm control. However, after this treatment, the result is periodontal repair (long junctional epithelium) due to the high proliferative rate of epithelial tissue as compared to mesenchymal tissues involved (periodontal ligament, dental cementum and alveolar bone), not occurring regeneration. Currently, it is known that successful periodontal tissue regeneration depends on the migration and proliferation of stem cells from the remaining periodontal ligament, followed by subsequent differentiation into osteoblasts, cementoblasts and fibroblasts. Being periodontitis a chronic inflammatory disease it has been suggested that periodontal remaining structures would have compromised ability to promote tissue regeneration. Periodontal disease is a multifactorial disease, however, its primary etiological factor is biofilm, and Porphyromonas gingivalis has been considered one of the major pathogens associated with the development and progression of chronic inflammatory periodontal disease, and currently it is known that its lipopolysaccharides are recognized by the Toll-like receptors, which have been identified in mesenchymal stem cells of the periodontal ligament. Therefore, it is proposed that mesenchymal stem cells from periodontal ligament would change their properties when exposed to a rich environment of bacterial toxins, compromising its regenerative capacity

Efeito da nicotina sobre as células do ligamento periodontal de humanos

Introdução: A regeneração dos tecidos periodontais de suporte depende da migração e proliferação das células do ligamento periodontal remanescente. In vitro, essas células têm apresentado alterações em relação à sua capacidade prolilerativa e redução na atividade da fosfatase alcalina, quando cultivadas na presença de diferentes concentrações de nicotina. Objetivo: O objetivo do presente estudo foi avaliar o efeito da nicotina nas concentrações de 50ng a 5ug/ml sobre a proliferação e viabilidade celular e formação de nódulos minerais. Métodos:Cultura primária de células do ligamento periodontal foram obtidas a partir de molares inclusos de 5 doadores do sexo masculino e cultivadas na presença ou ausência das concentrações de nicotina e avaliadas quanto a proliferação e viabilidade celular por meio do reagente MTS.e formação de nódulos minerais pelo ensaio de von Kossa. Resultados: A análise dos dados mostrou que todas as concentrações de nicotina testadas estimularam a proliferação celular, não havendo diferença estatisticamente significante comparado ao grupo controle (p=1,00).Adicionalmente,as 5 populações celulares mantiveram o mesmo padrão de formação de nódulo mineral comparadas ao grupo controle. Conclusão: Os resultados preliminares sugerem que as concentrações de nicotina testadas não influenciaram na capacidade proliferativa e de mineralização das células.Introduction:The periodontal support tissue regeneration depends on the migration and proliferation of the remanescent periodontal ligament cells.In vitro, these cells has shown alterations in their capacity of proliferation and alkaline phosphatase activity reduction if cultivated in the presence of different nicotine concentrations.Objectives:The aim of this work was to evaluate the nicotine effect in concentrations from 50ng to 5u,g/ml upon cells proliferation and viability and mineral nodules formation. Methods:Primary culture of the periodontal ligament cells were obtained from impacted molars of 5 male donors and cultivated in the presence or absence of nicotine concentrations and evaluated the cells proliferation and viability with MTS reagent, and the formation of mineral nodules through Von Kossa essay. Results: The data analysis showed that all of the tested nicotine concentrations promoted cell proliferation, not showing statistically significant difference when compared to control group (p=1,00).Additionally, the 5 populations maintained the same patterns of mineral nodules formation when compared to control group. Conclusion: The preliminary results suggest that the tested nicotine concentrations didn\u27t influence the proliferation and mineralization cells capacity

The effect of bacterial lipopolysaccharide on the biological properties of mesenchymal stem cells from human periodontal ligament

Orientador: Karina Gonzales Silvério RuizDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: 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 indiferenciadaAbstract: 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 cellsMestradoPeriodontiaMestra em Clínica Odontológic

Osteogenic potential of periodontal ligament stem cells are unaffected after exposure to lipopolysaccharides

Abstract Periodontitis develops as a result of a continuous interaction between host cells and subgingival pathogenic bacteria. The periodontium has a limited capacity for regeneration, probably due to changes in periodontal ligament stem cells (PDLSCs) phenotype. The aim of this study was to evaluate the effects of lipopolysaccharides from Porphyromonas gingivalis (PgLPS) on mesenchymal phenotype and osteoblast/cementoblast (O/C) potential of PDLSCs. PDLSCs were assessed for Toll-like receptor 2 (TLR2) expression by immunostaining technique. After, cells were exposed to PgLPS, and the following assays were carried out: (i) cell metabolic activity using MTS; (ii) gene expression for IL-1β, TNF-α and OCT-4 by real-time polymerase chain reaction (RT-qPCR); (iii) flow cytometry for STRO-1 and CD105, and (iv) osteogenic differentiation. PDLSCs were positive for TLR2. PgLPS promoted cell proliferation, produced IL-1β and TNF-α, and did not affect the expression of stem cell markers, STRO-1, CD105 and OCT-4. Under osteogenic condition, PDLSCs exposed to PgLPS showed a similar potential to differentiate toward osteoblast/cementoblast phenotype compared to control group as revealed by mineralized matrix deposition and levels of transcripts for RUNX2, ALP and OCN. These results provide evidence that PgLPS induces pro-inflammatory cytokines, but does not change the mesenchymal phenotype and osteoblast/cementoblast differentiation potential of PDLSCs

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. Objective : This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities