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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 ?-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 ?-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.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.

SUBMITTER: Rush MN

PROVIDER: S-EPMC4648670 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Surface chemistry regulates valvular interstitial cell differentiation in vitro.

Rush Matthew N MN Coombs Kent E KE Hedberg-Dirk Elizabeth L EL

Acta biomaterialia 20150930

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 (hydrophil ...[more]

PMID: 26428193

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Project description:The development of a substance or inhibitor-based treatment strategy for the prevention of aortic valve stenosis is a challenge and a main focus of medical research in this area. One strategy may be to use the tankyrase inhibitor XAV-939, which leads to Axin stabilisation and subsequent destruction of the ?-catenin complex and dephosphorylation of ?-catenin. The dephosphorylated active form of ?-catenin (non-phospho-?-catenin) then promotes nuclear transcription that leads to osteogenesis. The aims of the present study were to develop an experimental system for inducing in vitro calcification of human aortic valvular interstitial cells (VICs) to investigate the potential anti-calcific effect of XAV-939 and to analyse expression of the Wnt signalling proteins and Sox9, a chondrogenesis regulator, in this model. Calcification of human VIC cultures was induced by cultivation in an osteogenic medium and the effect of co-incubation with 1?M XAV-939 was monitored. Calcification was quantified when mineral deposits were visible in culture and was histologically verified by von Kossa or Alizarin red staining and by IR-spectroscopy. Protein expression of alkaline phosphatase, Axin, ?-catenin and Sox9 were quantified by western blotting. In 58% of the VIC preparations, calcification was induced in an osteogenic culture medium and was accompanied by upregulation of alkaline phosphatase. The calcification induction was prevented by the XAV-939 co-treatment and the alkaline phosphatase upregulation was suppressed. As expected, Axin was upregulated, but the levels of active non-phospho-?-catenin were also enhanced. Sox9 was induced during XAV-939 treatment but apparently not as a result of downregulation of ?-catenin signalling. XAV-939 was therefore able to prevent calcification of human VIC cultures, and XAV-939 treatment was accompanied by upregulation of active non-phospho-?-catenin. Although XAV-939 does not downregulate active ?-catenin, treatment with XAV-939 results in Sox9 upregulation that may prevent the calcification process.

Project description:A quantitative understanding of the complex interactions between cells, soluble factors, and the biological and mechanical properties of biomaterials is required to guide cell remodeling toward regeneration of healthy tissue rather than fibrocontractive tissue. In the present study, we characterized the combined effects of boundary stiffness and transforming growth factor-?1 (TGF-?1) on cell-generated forces and collagen accumulation. We first generated a quantitative map of cell-generated tension in response to these factors by culturing valvular interstitial cells (VICs) within micro-scale fibrin gels between compliant posts (0.15-1.05 nN/nm) in chemically-defined media with TGF-?1 (0-5 ng/mL). The VICs generated 100-3000 nN/cell after one week of culture, and multiple regression modeling demonstrated, for the first time, quantitative interaction (synergy) between these factors in a three-dimensional culture system. We then isolated passive and active components of tension within the micro-tissues and found that cells cultured with high levels of stiffness and TGF-?1 expressed myofibroblast markers and generated substantial residual tension in the matrix yet, surprisingly, were not able to generate additional tension in response to membrane depolarization signifying a state of continual maximal contraction. In contrast, negligible residual tension was stored in the low stiffness and TGF-?1 groups indicating a lower potential for shrinkage upon release. We then studied if ECM could be generated under the low tension environment and found that TGF-?1, but not EGF, increased de novo collagen accumulation in both low and high tension environments roughly equally. Combined, these findings suggest that isometric cell force, passive retraction, and collagen production can be tuned by independently altering boundary stiffness and TGF-?1 concentration. The ability to stimulate matrix production without inducing high active tension will aid in the development of robust tissue engineered heart valves and other connective tissue replacements where minimizing tissue shrinkage upon implantation is critical.

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Surface chemistry regulates valvular interstitial cell differentiation in vitro.

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Surface chemistry regulates valvular interstitial cell differentiation in vitro.

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