RESUMO
PURPOSE: Ongoing studies demonstrate that the murine lacrimal gland is capable of repair after experimentally induced injury. It was recently reported that repair of the lacrimal gland involved the mobilization of mesenchymal stem cells (MSCs). These cells expressed the type VI intermediate filament protein nestin whose expression was upregulated during the repair phase. The aim of the present study was to investigate the roles of vimentin, a type III intermediate filament protein and a marker of epithelial-mesenchymal transition (EMT) in repair of the lacrimal gland. METHODS: Injury was induced by direct injection of interleukin (IL)-1 into the exorbital lacrimal gland. MSCs were prepared from injured glands using tissue explants. Expression of vimentin and the transcription factor Snai1, a master regulator of EMT, was determined by RT-PCR, Western blotting analysis, and immunofluorescence. RESULTS: These data show that vimentin expression, at both the mRNA and the protein levels, was upregulated during the repair phase (2-3 days postinjury) and returned to the control level when repair ended. Temporal expression of Snai1 mirrored that of vimentin and was localized in cell nuclei. Cultured MSCs isolated from injured lacrimal glands expressed Snai1 and vimentin alongside nestin and alpha smooth muscle actin (another biomarker of EMT). There was a strong positive correlation between Snai1 expression and vimentin expression. CONCLUSIONS: It was found that EMT is induced during repair of the lacrimal gland to generate MSCs to initiate repair, and that mesenchymal-epithelial transition is then activated to form acinar and ductal epithelial cells.
Assuntos
Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/fisiologia , Traumatismos Oculares/fisiopatologia , Aparelho Lacrimal/lesões , Cicatrização/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Separação Celular , Células Cultivadas , Traumatismos Oculares/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/fisiologia , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMO
Silk scaffolds have been successfully used for a variety of tissue engineering applications due to their biocompatibility, diverse physical characteristics, and ability to support cell attachment and proliferation. Our prior characterization of 4-day postnatal rat tooth bud cells grown on hexafluoro-2-propanol (HFIP) silk scaffolds showed that the silk scaffolds not only supported osteodentin formation, but also guided the size and shape of the formed osteodentin. In this study, interactions between human dental pulp cells and HFIP and aqueous based silk scaffolds were studied under both in vitro and in vivo conditions. Silk scaffold porosity and incorporation of RGD and DMP peptides were examined. We found that the degradation of aqueous based silk is much faster than HFIP based silk scaffolds. Also, HFIP based silk scaffolds supported the soft dental pulp formation better than the aqueous based silk scaffolds. No distinct hard tissue regeneration was found in any of the implants, with or without additional cells. We conclude that alternative silk scaffold materials, and hDSC pre-seeding cell treatments or sorting and enrichment methods, need to be considered for successful dental hard tissue regeneration.
Assuntos
Polpa Dentária/citologia , Propanóis/farmacologia , Seda/farmacologia , Células-Tronco/citologia , Alicerces Teciduais/química , Água/farmacologia , Adolescente , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Separação Celular , Células Cultivadas , Feminino , Fluoresceínas/metabolismo , Humanos , Implantes Experimentais , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fosfoproteínas/farmacologia , Implantação de Prótese , Ratos , Ratos Nus , Células Estromais/citologia , Células Estromais/efeitos dos fármacosRESUMO
While it is known that precise dental epithelial-mesenchymal (DE-DM) cell interactions provide critical functions in tooth development, reliable methods to establish proper DE-DM cell interactions for tooth regeneration have yet to be established. To address this challenge, and to generate bioengineered teeth of predetermined size and shape, in this study, we characterize three dimensional (3D) pre-fabricated DE-DM cell constructs. Human dental pulp cell seeded Collagen gel layers were co-cultured with porcine DE cells suspended in Growth Factor Reduced (GFR) Matrigel. The resulting 3D DE-DM cell layers were cultured in vitro, or implanted and grown subcutaneously in vivo in nude rats. Molecular, histological and immunohistochemical (IHC) analyses of harvested implants revealed organized DE-DM cell interactions, the induced expression of dental tissue-specific markers Amelogenin (AM) and Dentin Sialophosphoprotein (DSPP), and basement membrane markers Laminin 5 and collagen IV, and irregular mineralized tissue formation after 4 weeks. We anticipate that these studies will facilitate the eventual establishment of reliable methods to elaborate dental tissues, and full sized teeth of specified sized and shape.