The mechanism that drives valve calcification has been likened to that of bone formation and/or a dystrophic process which includes the deposition of hydroxyapatite mineral 
; nevertheless the process which mediates the formation of calcified lesions on aortic valve cusps remains uncertain. Investigators have speculated that valve calcification involves a transdifferentiation of VICs into osteoblasts, which then mediate bone-like mineral formation 
. The implication of such an osteoblast-like mineralization process in the aortic valve has prompted the development of in vitro
models to examine the disease process. Easily cultured and fast-growing PAVICs are often used as a simplified model for aortic valve calcification 
, however, their efficacy in representing the disease has yet to be established. This study aimed to characterize the ‘calcified’ OST+TGF-β1 PAVICs nodule composition, compare them to those created by a confirmed mineralizing culture model (MOBs), and report any calcium phosphate deposition within the PAVICs nodules.
We observed that PAVICs grown in OST+TGF-β1 medium formed nodular structures that stained positively for ARS, as has been previously described 
. Such nodules were notable for their distinct three-dimensional morphologies that are reminiscent of nodules formed from MOBs, which also stained positively for ARS. Nevertheless, when we examined the ultrastructure of such nodules by TEM, no electron-dense mineral deposits were observed, as were readily identifiable in our previously published report of nodules formed from MOBs 
. Furthermore, Raman spectroscopy measurements clearly showed an absence of mineralization in the OST+TGF-β1 PAVICs, whilst MOBs nodules demonstrated distinct peaks, indicative of phosphate and type-B carbonate-substituted mineral. These results suggest that ‘calcified’ nodules formed from PAVICs under the conditions examined here do not form mineral deposits and that ARS staining is a poor method to identify mineral deposits in such cultures. Notwithstanding, these data do not preclude the possibility of osteogenic transdifferentiation of PAVICs or exclude the chance that they could form mineral under different conditions.
A number of studies have suggested that PAVICs and human aortic VICs may differentiate to osteoblast-like cells during calcified valve disease progression 
. A study by Chen et al
. demonstrated that both mesenchymal and osteogenic progenitor cells exist within the primary PAVICs mixed cell population. Their results further exposed that PAVICs have the ability to transdifferentiate into myofibrogenic, adipogenic, osteogenic and chodrogenic lineages in vitro
and thus potentially in vivo
. To probe PAVICs nodules’ potential for osteoblastic differentiation when grown in OST+TGF-β1 medium, we examined expression of two genes: type I collagen (COL1A1
) and osteocalcin (BGLAP)
. It has been reported that calcified human aortic valve interstitial cells have an increase in osteocalcin RNA expression (a late marker for bone differentiation) 
. Whilst we noted an up regulation of type I collagen in OST+TGF-β1 grown PAVICs, BGLAP
expression remained stable in PAVICs grown in OST+TGF-β1 and this level of expression was significantly lower than expression levels in CTL PAVICs at the same time points. This suggests that PAVICs grown in OST+TGF-β1 for up to 21 days were not differentiating into osteoblasts. A previous study demonstrated PAVICs grown in mineralization medium for up to eight days did not display the same level of increased alkaline phosphatase (an early mineralization marker) as osteoblasts in culture 
. The lack of osteoblastic differentiation in this study may be attributed to a wide range of factors including their growth on stiff tissue culture plastic/glass substrates 
and/or TGF-β1 supplementation.
In this study the OST media was supplemented with TGF-β1 due to its physiological importance in tissue calcification. Studies have shown qualitatively higher levels of TGF-β1 in the ECM that co-localized with areas of calcification in diseased human aortic valves 
. Additionally, the inflammatory response has been implicated as an important contributing factor in disease onset, which suggests that a local availability of TGF-β1 
may increase during the initial stages of disease progression. Nevertheless, the connection between TGF-β1 use in this in vitro
system and disease progression is still unclear. Osman et al
. showed that supplementation of human VIC cultures with members of the TGF-β family (including TGF-β1) prompted the cells to adopt a more osteoblast-like phenotype by inducing the secretion of proinflammatory cytokines which may play an important role in pathological valvular calcification 
. Our results here show that PAVICs grown in OST+TGF-β1 medium do not show evidence of osteoblastic transdifferentiation and thus these ‘calcified nodules’ have yet to demonstrate their relationship to calcified aortic valve disease progression. The absence of mineral within CTL+TGF-β1 and OST medium PAVICs nodules suggests the lack of mineralization is not due to osteogenic supplementation or the additional TGF-β1 supplementation.
TEM and histological staining demonstrated that PAVICs nodules grown in OST+TGF-β1 were marked by an abundant proteinaceous ECM, which contained collagen, but without a specific arrangement or orientation. Collagen production is mediated by VICs in vivo
as part of normal valve maintenance, however, disruption of this process has also been associated with calcified aortic valve disease progression 
. Valvular fibrosis and over-activated VICs have been implicated in the early stages of calcified valve pathobiology 
, including a recent suggestion of calcified aortic valve stenosis being more appropriately viewed as a fibrocalcific disease 
. Our current study confirms TGF-β1 supplementation likely promotes and/or maintains an activated myofibroblastic phenotype in PAVICs and production of ECM in vitro
. The relationship, if one exists, between in vitro
PAVIC-mediated ECM production and the fibrotic stage of aortic valve calcification, however, has yet to be established.
PAVICs-mediated production of fibrous ECM was further explored using Raman spectroscopy. Like the histological analyses, Raman spectroscopy further identified the abundant proteinaceous content of PAVICs nodules cultured in OST+TGF-β1 medium. Our PLS-DA model successfully distinguished between the two experimental PAVICs systems based on the ECM produced by PAVICs grown in OST+TGF-β1 medium. The model also clearly indicated the collagen content within the nodules was a heavy contributor to the model variables, and thus collagen is a distinguishing element between the groups. Taken together, these results suggest Raman spectroscopy may be an effective means to successfully and non-invasively monitor ECM production in live PAVIC systems in vitro.
Cross sections of OST+TGF-β1 PAVICs nodules showed positive expression of αSMA, as did monolayers of PAVICs grown in CTL medium, implicating a myofibroblastic phenotype 
. Our results show that the cells at the centre of these nodules are not αSMA positive and thus may have a different phenotype or be undergoing apoptosis, as has been previously suggested 
. TGF-β1 has been suggested to promote myofibroblastic expression particularly when incorporated on stiff substrates 
and calcification via an apoptosis pathway 
, thus suggesting a non-osteoblastic state 
. The results presented in this study show that PAVICs cultured in OST+TGF-β1 medium for 21 days, a relatively late time point for in vitro culture 
, do not further transdifferentiate from the activated myofibroblastic phenotype into osteoblast-like cells or contain calcium phosphate within the ‘calcified nodules’. It remains uncertain as to whether VICs must pass through an intermediate stage of activated VICs to become osteoblast-like VICs 
. Further investigations are needed to establish if the OST+TGF-β1 PAVICs model has any relationship to early (preosteoblastic) stages of calcified aortic valve disease.
The inhibition of nodule formation in cultured VICs has been explored through the addition of statins, pravastatin 
, nitric oxide donors, as well as other cell permeate superoxide scavengers 
. The response and transformations of VICs grown in vitro
to various treatments raise interesting questions regarding the relationship between these cells and the complex in vivo
environment. As investigations into the pathobiology of aortic valve calcification progress, characterization of both systems using a variety of techniques offers promise of bridging this gap.
This study combines gold standard biological techniques as well as advanced material characterization techniques including rapid, non-invasive Raman spectroscopy. The results show PAVICs grown in OST+TGF-β1 media for up to 21 days express an activated myofibroblastic phenotype and produce a predominantly collagen ECM, however, demonstrate no evidence of further transdifferentiation into an osteoblastic phenotype and/or calcium phosphate deposition. Additionally these PAVICs nodules did not contain any calcium phosphate materials as seen in human aortic valve calcification. We have thus established a clear limitation of cultured PAVICs grown in CTL and OST+TGF-β1 media as they do not appear to transdifferentiate into osteoblastic-like cells nor form mineral deposits indicative of calcified aortic valve disease 
. This study also provides further information on the collagen-rich ECM produced in PAVICs nodules grown in OST+TGF-β1 medium and the heterogeneous nature of these nodules. This characterisation of in vitro
PAVICs systems is critical in further understanding PAVICs behavior in culture and for comparison to aortic valve calcification.