title: A small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes.
abstract: Mechanical forces are translated into biochemical signals and contribute to cell differentiation and phenotype maintenance. Mesenchymal stem cells and their tissue-specific offspring, as osteoblasts and chondrocytes, cells of cardiovascular tissues and lung cells are sensitive to mechanical loading but molecules and mechanisms involved have to be unraveled. It is well established that cellular mechanotransduction is mediated e.g. by activation of the transcription factor SP1 and by kinase signaling cascades resulting in the activation of the AP1 complex. To investigate cellular mechanisms involved in mechanotransduction and to analyze substances, which modulate cellular mechanosensitivity reporter gene constructs, which can be transfected into cells of interest might be helpful. Suitable small-scale bioreactor systems and mechanosensitive reporter gene constructs are lacking. To analyze the molecular mechanisms of mechanotransduction and its crosstalk with biochemically induced signal transduction, AP1 and SP1 luciferase reporter gene constructs were cloned and transfected into various cell lines and primary cells. A newly developed bioreactor and small-scale 24-well polyurethane dishes were used to apply cyclic stretching to the transfected cells. 1 Hz cyclic stretching for 30 min in this system resulted in a significant stimulation of AP1 and SP1 mediated luciferase activity compared to unstimulated cells. In summary we describe a small-scale cell culture/bioreactor system capable of analyzing subcellular crosstalk mechanisms in mechanotransduction, mechanosensitivity of primary cells and of screening the activity of putative mechanosensitizers as new targets, e.g. for the treatment of bone loss caused by both disuse and signal transduction related alterations of mechanotransduction.
authors.affiliation: Orthopedic Center for Musculoskeletal Research, University of Würzburg, Brettreichstrasse 11, D-97074 Würzburg, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Seefried
authors.forename: Sigrid
authors.initials: S
authors.lastname: Mueller-Deubert
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwarz
authors.forename: Thomas
authors.initials: T
authors.lastname: Lind
authors.forename: Birgit
authors.initials: B
authors.lastname: Mentrup
authors.forename: Melanie
authors.initials: M
authors.lastname: Kober
authors.forename: Denitsa
authors.initials: D
authors.lastname: Docheva
authors.forename: Astrid
authors.initials: A
authors.lastname: Liedert
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
authors.forename: Anita
authors.initials: A
authors.lastname: Ignatius
authors.forename: Matthias
authors.initials: M
authors.lastname: Schieker
authors.forename: Lutz
authors.initials: L
authors.lastname: Claes
authors.forename: Winfried
authors.initials: W
authors.lastname: Wilke
authors.forename: Franz
authors.initials: F
authors.lastname: Jakob
authors.forename: Regina
authors.initials: R
authors.lastname: Ebert
mesh_terms: D019149:Bioreactors; D002352:Carrier Proteins / Q000096:biosynthesis; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000295:instrumentation; D002460:Cell Line; D049109:Cell Proliferation; D016207:Cytokines / Q000096:biosynthesis; D020342:Finite Element Analysis; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D040542:Mechanotransduction, Cellular; D059630:Mesenchymal Stem Cells / Q000502:physiology; D011140:Polyurethanes; D011994:Recombinant Proteins / Q000096:biosynthesis; D020218:Response Elements; D016329:Sp1 Transcription Factor / Q000235:genetics; D013312:Stress, Physiological / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D016207:Cytokines; D011140:Polyurethanes; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; C066845:pleiotrophin; D008156:Luciferases