Substrate properties influence calcification in valvular interstitial cell culture.
OBJECTIVE
Valvular calcification is an active, cell-mediated process that results in significant morbidity and mortality. In standard culture, valvular interstitial cells (VICs) elicit significant calcification as a result of myofibroblast activation, and this limits their use in characterization studies. The study aim was to identify culturing substrates that would suppress atypical VIC calcification, and to investigate culture substrates representing a more physiological system.


METHODS
Several culture platforms were selected to compare and contrast the influence of biochemical and mechanical properties on VIC calcification. Substrates investigated included: tissue culture polystyrene (TCPS), TCPS coated with either fibronectin or fibrin, and an elastic poly(ethylene glycol) (PEG) hydrogel, also with fibronectin or fibrin coupled to the surface. Experiments were repeated with profibrotic growth factor transforming growth factor-beta 1 (TGF-beta1). VIC calcification was characterized by calcific nodule formation, alkaline phosphatase activity and calcium accumulation. Gene and protein expression of alpha smooth muscle actin (aSMA) and core binding factor-1 (CBFa-1) were analyzed with qRT-PCR and immunostaining.


RESULTS
Unmodified TCPS substrates had an innate ability to promote the markers of calcification studied. The addition of TGF-beta1 enhanced levels of all osteoblastic markers studied. When TCPS surfaces were modified with fibronectin, all markers for calcification were repressed, but alphaSMA - a marker for myofibroblastic activity was unchanged. Meanwhile, fibrin-modified TCPS surfaces enhanced calcification over unmodified TCPS substrates. On soft PEG hydrogels, all markers for calcification were repressed, regardless of the surface chemistry, while alphaSMA expression remained unaffected.


CONCLUSIONS
Collectively, VIC properties are highly linked to the culture microenvironment. Both, the biochemical and mechanical environment of tissue culture has an effect on the spontaneous calcification of VICs, and may also have a profound effect on their molecular properties, as related to an understanding of the disease process in vivo.
Benton
Julie A
JA
Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80309-0424, USA.
Kern
Hanna B
HB
Anseth
Kristi S
KS
D000199:Actins / Q000378:metabolism; D000818:Animals; D001021:Aortic Valve / Q000473:pathology*; D015415:Biomarkers / Q000378:metabolism; D002114:Calcinosis / Q000473:pathology*; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D046508:Culture Techniques / Q000379:methods; D005337:Fibrin; D005353:Fibronectins; D007150:Immunohistochemistry; D011092:Polyethylene Glycols; D016133:Polymerase Chain Reaction; D011137:Polystyrenes; D013501:Surface-Active Agents; D013552:Swine; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D000199:Actins; D015415:Biomarkers; D050659:Core Binding Factor Alpha 1 Subunit; D005353:Fibronectins; D011137:Polystyrenes; D013501:Surface-Active Agents; D053773:Transforming Growth Factor beta1; D011092:Polyethylene Glycols; D005337:Fibrin