Estudo in vitro da produção de quimiocinas e pró-colágeno I por fibroblastos de gengiva, ligamento periodontal e polpa dental humanos

Fibroblastos sao as celulas mais numerosas encontradas nos tecidos orais como gengiva, ligamento periodontal e polpa dental. Alem de exercerem funcao estrutural, estas celulas tambem desempenham papel importante na resposta imune destes tecidos atraves do reconhecimento de antigenos e producao de mediadores inflamatorios e citocinas. Evidencias apontam ainda para o fato de que fibroblastos nao constituem um grupo unico de celulas. Sendo assim, os objetivos deste estudo foram: (I) avaliar a producao diferencial de fibroblastos de gengiva, ligamento periodontal e polpa dental de dentes permanentes e deciduos quanto a producao das quimiocinas CCL3 e CXCL12; (II) avaliar a producao de pro-colageno I pelas celulas de polpa e (III) avaliar a expressao diferencial dos fibroblastos quanto a microRNAs. Dentes recentemente extraidos (terceiros molares higidos) e fragmentos de gengivas saudaveis de tres pacientes adultos foram obtidos no Laboratorio de Farmacologia e Fisiologia Clinica da Faculdade de Odontologia de Bauru. Caninos deciduos de dois pacientes com indicacao para extracao por motivos ortodonticos foram obtidos na Clinica de Odontopediatria da mesma unidade. Culturas primarias de fibroblastos de gengiva (n=3), ligamento periodontal (n=3) e polpa de dente permanente (n=3) e polpa dental de dente deciduo (n=2) foram estabelecidas a partir de tecidos humanos por meio de tecnica de explant. Apos a quarta passagem, a producao de CCL3 e de CXCL12 foi avaliada apos estimulo com concentracoes crescentes (0 10 µg/mL) de acido lipoteicoico de Enterococcus faecalis (EfLTA), lipopolissacarideo de Porphyromonas gingivalis (PgLPS) ou LPS de Escherichia coli (EcLPS) por ELISA apos 1, 6 e 24 h. O RNAm para as quimiocinas no grupo estimulado com EcLPS por 24 h foi avaliado por transcricao reversa seguida de reacao em cadeia da polimerase quantitativa. A producao de pro-colageno I por celulas de polpa estimuladas com EfLTA e PgLPS foi avaliada por imunofluorescencia. O perfil... Fibroblasts are the dominant cells within oral tissues such as gingiva, periodontal ligament and dental pulp. Besides the architectural maintenance of the connective tissues, fibroblasts are also involved in connective tissue immune response through antigen recognition and production of inflammatory mediators and cytokines. Recent studies also demonstrated that fibroblasts do not constitute a unique group of cells. Taken this togeter, the objectives of the present study were: (I) to evaluate the production of the chemokines CCL3 and CXCL12 by human gingival, periodontal ligament as well as permanent and deciduous dental pulp fibroblasts; (II) to evaluate the production of procollagen I by dental pulp fibroblasts and (III) to evaluate the differential pattern of expression of microRNAs by the oral fibroblasts. Recently extracted teeth (non-carious third molars) and fragments of healthy gingiva from three adults were obtained at the Laboratory for Clinical Pharmacology and Physiology at Dental School of Bauru. Deciduous canines from two patients with orthodontic indication for extraction were obtained at Pediatrics Clinics of Dental School of Bauru. Primary cultures of fibroblasts from gingiva (n=3), periodontal ligament (n=3) as well as permanent pulp (n=3) and deciduous pulp (n=2) were established through an explant technique. After the fourth passage, fibroblasts were challenged with increasing concentrations (0 10 µg/mL) of Enterococcus faecalis lipoteichoic acid (EfLTA), Porphyromonas gingivalis lipopolysaccharide (PgLPS) or Escherichia coli LPS (EcLPS) for 1, 6 and 24 h. The chemokines were assessed through ELISA while the mRNA for CCL3 and CXCL12 (EcLPS at 24 h) were assessed through reverse transcription followed by quantitative polymerase chain reaction. The expression of microRNAs was screened through a microarray assay. The production of CCL3 on cell supernatants was detected in all cellular groups, with higher amounts at gingival fibroblasts...