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.
Previous methods for investigating material stiffness on cell behavior have focused on the use of substrates with limited ranges of stiffness and/or fluctuating surface chemistries. Using the co-polymer system of n-octyl methacrylate crosslinked with diethylene glycol dimethacrylate (DEGDMA/nOM), we developed a new cell culture platform to analyze the isolated effects of stiffness independent from changes in surface chemistry. Materials ranging from 25 kPa to 4,700 kPa were fabricated. Surface analysis including goiniometry and X-ray photoelectron spectroscopy (XPS) confirmed consistent surface chemistry across all formulations examined. The mechanosensitive cell type valvular interstitial cell (VIC) was cultured DEGDMA/nOM substrates of differing stiffness. Results indicate that order of magnitude changes in stiffness do not increase gene expression of VIC alpha-smooth muscle actin (αSMA). However, structural organization of αSMA is altered on stiffer substrates, corresponding with the appearance of the osteoblastic marker osteocalcin and nodule formation. This research presents the co-polymer DEGDMA/nOM as ideal substrate to investigate the influence of stiffness on VIC differentiation without the confounding effects of changing material surface chemistry. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 51-61, 2017.
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