title: Surface chemistry regulates valvular interstitial cell differentiation in vitro.
abstract: The primary driver for valvular calcification is the differentiation of valvular interstitial cells (VICs) into a diseased phenotype. However, the factors leading to the onset of osteoblastic-like VICs (obVICs) and resulting calcification are not fully understood. This study isolates the effect of substrate surface chemistry on in vitro VIC differentiation and calcified tissue formation. Using ω-functionalized alkanethiol self-assembled monolayers (SAMs) on gold [CH3 (hydrophobic), OH (hydrophilic), COOH (COO(-), negative at physiological pH), and NH2 (NH3(+), positive at physiological pH)], we have demonstrated that surface chemistry modulates VIC phenotype and calcified tissue deposition independent of osteoblastic-inducing media additives. Over seven days VICs exhibited surface-dependent differences in cell proliferation (COO(-)=NH3(+)>OH>CH3), morphology, and osteoblastic potential. Both NH3(+)and CH3-terminated SAMs promoted calcified tissue formation while COO(-)-terminated SAMs showed no calcification. VICs on NH3(+)-SAMs exhibited the most osteoblastic phenotypic markers through robust nodule formation, up-regulated osteocalcin and α-smooth muscle actin expression, and adoption of a round/rhomboid morphology indicative of osteoblastic differentiation. With the slowest proliferation, VICs on CH3-SAMs promoted calcified aggregate formation through cell detachment and increased cell death indicative of dystrophic calcification. Furthermore, induction of calcified tissue deposition on NH3(+) and CH3-SAMs was distinctly different than that of media induced osteoblastic VICs. These results demonstrate that substrate surface chemistry alters VIC behavior and plays an important role in calcified tissue formation. In addition, we have identified two novel methods of calcified VIC induction in vitro. Further study of these environments may yield new models for in vitro testing of therapeutics for calcified valve stenosis, although additional studies need to be conducted to correlate results to in vivo models.
  
  
  CONCLUSIONS
  Valvular interstitial cell (VIC) differentiation and aortic valve calcification is associated with increased risk of mortality and onset of other cardiovascular disorders. This research examines effects of in vitro substrate surface chemistry on VIC differentiation and has led to the identification of two materials-based initiation mechanisms of osteoblastic-like calcified tissue formation independent of soluble signaling methods. Such findings are important for their potential to study signaling cascades responsible for valvular heart disease initiation and progression as well providing in vitro disease models for drug development. We have also identified a VIC activating in vitro environment that does not exhibit confluence induced nodule formation with promise for the development of tissue regenerating scaffolds.
authors.affiliation: Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM, USA; Nanoscience and Microsystems Engineering, University of New Mexico, Albuquerque, NM, USA. Electronic address: mrush@unm.edu.
authors.forename: Matthew N
authors.initials: MN
authors.lastname: Rush
authors.affiliation: Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM, USA; Biomedical Science Graduate Program, University of New Mexico, Albuquerque, NM, USA. Electronic address: kcoombs@salud.unm.edu.
authors.forename: Kent E
authors.initials: KE
authors.lastname: Coombs
authors.affiliation: Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM, USA; Nanoscience and Microsystems Engineering, University of New Mexico, Albuquerque, NM, USA; Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM, USA. Electronic address: edirk@unm.edu.
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Hedberg-Dirk
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D015870:Gene Expression; D006351:Heart Valves / Q000166:cytology* / Q000378:metabolism; D066298:In Vitro Techniques; D013499:Surface Properties; D013552:Swine
chemical_list: D015415:Biomarkers
keywords: Calcification; Differentiation; Heart valve; Self-assembled monolayers; Valvular interstitial cells