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.

Isolated effect of material stiffness on valvular interstitial cell differentiation

“…Cellular functions such as adhesion, spreading, proliferation and gene expression are affected by the chemical and physical factors of the extracellular environment . Cells respond to these extracellular cues through mechanosensing proteins, which activate cell signaling cascades .…”

“…Given the importance of substrate stiffness in controlling mechanosensing behavior, investigations into the effects of matrix mechanics on cell signaling have become more prevalent. However, in vitro studies examining substrate's mechanical properties effects on cells have been limited due to a lack of material platforms capable of isolating stiffness over a significant range without changing the material's surface chemistry …”

“…During aortic valve disease, changes in protein deposition and overall stiffness of the valve are known to increase over time . The primary cells of the aortic valve, valvular interstitial cells (VICs), are a heterogeneous and dynamic population that are particularly sensitive to the substrate on which they are grown . In vitro , VICs expanded on tissue culture polystyrene (TCPS) exhibit myofibroblastic characteristics with an elongated spindle shaped morphology, rapid proliferation, and expression of alpha smooth muscle actin (αSMA) .…”

“…In vitro , VICs expanded on tissue culture polystyrene (TCPS) exhibit myofibroblastic characteristics with an elongated spindle shaped morphology, rapid proliferation, and expression of alpha smooth muscle actin (αSMA) . Over time, VICs on TCPS adopt an osteoblastic‐like (OB) phenotype attributed to cell confluence . It has also been suggested that stiffening of the aortic valve's extracellular matrix (ECM) may be able to signal VICs to differentiate to a disease inducing OB phenotype .…”

“…Classically, αSMA has been used as a marker for myofibroblastic activation of VICs. More recently, however, αSMA expression has also been shown to increase in OB VICs . While studies have begun to look at the role of αSMA as a marker of osteoblastic potential, at this point seminal studies of stiffness have not included OB markers such as osteocalcin …”