Here, we have begun to explore how substrate modulus serves as a mechanical cue to regulate the fate of activated valvular myofibroblasts. Our studies revealed striking phenotypic changes from activated myofibroblasts to less proliferative, quiescent-like fibroblasts when the culture substrate’s elastic modulus was reduced from 32 kPa to 7 kPa. Apoptosis was minimally associated with this de-differentiation process. Within 6 hours of in situ substrate elasticity reduction, gene signatures of myofibroblasts (α-SMA and CTGF) were down-regulated, while a fibroblast gene (vimentin) stayed at a similar level, confirming myofibroblast de-activation and suggesting potential signaling cascade mechanisms. Mechanically-reprogrammed VICs on stiff-to-soft gels were able to proliferate and re-initiate expression of myofibroblast genes in response to chemical cues. Considering the extensive health effects of tissue fibrosis, our study provides insight into possibly reducing fibrosis through preventing myofibroblastic activation and will assist with strategic in vitro tissue engineering to replace or re-organize severely fibrotic or calcified tissue.
Human tissues have stiffnesses ranging from ~0.1 kPa to ~20 GPa 
. To recapitulate native stiffness in vitro
, it is critical to culture cells on substrata with a physiologically relevant stiffness for understanding their functions. Previous studies indicated that normal valve fibrosa have a bulk elastic modulus from 0.8–8 kPa 
. When healthy valves become stenotic, osteoid, which is crosslinked collagen matrix as precursor to bone, has been detected in the valve 
. While the stiffness of calcified valves has not been measured to our knowledge, Engler et al.
have shown that osteoid matrix secreted by human mesenchymal stem cells has E
10 kPa 4
. To mimic these microenvironments, we synthesized hydrogels with either a normal mesenchyme-like modulus (~7 kPa, soft gels) or a pathological osteoid-like modulus (~32 kPa, stiff gels) to probe the cell fate of VICs. Kloxin et al.
previously demonstrated that valvular myofibroblast differentiation was promoted on stiff gels, but inhibited on soft gels 
. Similarly, fibroblasts isolated from different tissues, including lung 
and liver 
, have been shown to activate with E
>15 kPa and maintain the α-SMA negative fibroblast phenotype when the microenvironment had E
10 kPa. We observed consistent results for VICs cultured on either stiff or soft gels (). In addition, when we irradiated stiff gels with UV light to reduce the substrate modulus, valvular myofibroblasts were de-activated and lost previously formed α-SMA stress fibers (). Similar behavior was observed for rat pulmonary fibroblasts (Fig. S4
and Text S1
), indicating a general role of substrate modulus in regulating the differentiation of myofibroblasts.
In these experiments, VICs were cultured on a 2-dimensional surface. While this is different from the 3-dimensional (3D) valve tissue in which the endogenous cells reside, a 2D culture approach has several advantages in understanding basic biological systems. 2D surfaces of functionalized biomaterials have served as unique tools for understanding how cells collectively migrate and how they differentiate in response to stiffness or shape 
. Additionally, 2D culture enables one to readily monitor and image cells over time using real time microscopy tracking tools and to collect intracellular proteins or RNA more easily compared with 3D cultures. Currently, two types of scaffolds have been used for the 3D culture of VICs, enzymatically degradable synthetic gels 
and natural matrices comprised of collagen 
or hyaluronic acid 
. Both of these materials have the complication that cells are changing their mechano-environment by degrading the matrix, so it becomes difficult to know the mechanical properties of the matrix in the pericellular region. In contrast, if VICs are encapsulated in non-degradable matrices with precisely defined mechanical properties, they remain in a rounded and un-natural morphology. Thus, it is difficult to de-couple the effect of modulus and cell spreading on the cell fate within a 3D highly cross-linked matrix. For these reasons, our studies focus on isolating and understanding the effect of modulus on cell fate in 2D and believe that this knowledge will be helpful in better understanding VIC function in more complex, 3D matrices in future studies.
The fate of myofibroblasts after normal tissue repair has been an ongoing debate. In granulation and scar tissue, massive apoptosis has been observed 
. Induction of apoptosis has been associated with matrix tension. For example, sudden release of collagen gels from their anchor causes programmed cell death in human dermal fibroblasts 
. Additionally, compliant substrata with E <1 kPa have been shown to induce significantly higher caspase 3 activity than stiff substrata in lung fibroblasts 
. Consistently, we observed a small but significant increase of apoptosis on stiff-to-soft gels in comparison to stiff gels on day 5 (), indicating that some valvular myofibroblasts underwent apoptosis in response to reduction in modulus. However, this level of apoptosis on stiff-to-soft gels was similar to that observed for cells cultured on statically soft substrates. Additionally, the average level of apoptosis in VICs was ~5% on stiff-to-soft gels, which was too small to account for a nearly 35% decrease in the myofibroblast population. Therefore, most myofibroblasts did not undergo apoptosis in response to substrate modulus reduction. These findings within the context of the literature suggest that there may be different thresholds of substrate modulus for regulating myofibroblast activation and apoptosis. While E
~7 kPa is sufficient to de-activate valvular myofibroblasts without inducing significant apoptosis, we speculate that further reduction of E
below or around 1 kPa would induce most cells to undergo apoptosis. Additionally, softening the substrate did not select for specific populations of cells, as the cell number counted as described in the Text S1
was not changed significantly from day 3 to day 5 across all gel moduli (Fig. S1
), and cells did not proliferate or undergo apoptosis significantly over time. There were slightly fewer cells attached on soft gels at day 1 than stiff gels, so we observed fewer cells on soft gels than on stiff gels from day 3 to day 5 (Fig. S1
Since valvular myofibroblasts did not undergo significant programmed cell death, we hypothesized that these cells de-differentiated into a dormant, or quiescent-like, fibroblast state. Myofibroblasts are differentiated from fibroblasts through increased α-SMA expression and its organization into stress fibers, which is regulated by mechanical stress 
. When cells adhere to surfaces, traction forces are generated based on the resistance of the matrix to cellular adhesion and movement 
. Cells on substrates with higher moduli have been shown to exert higher traction forces as measured by deformation of embedded fluorescent beads 
. Mechanical strain generated on higher substrate moduli activate intracellular signaling through p38 MAPK, Rho kinase and focal adhesion kinase to up-regulate transcription of α-SMA and subsequently incorporation of α-SMA into stress fibers 
. Our results confirmed that α-SMA stress fibers in VICs are dependent on substrate modulus. Based on , α-SMA stress fibers in VICs were disassembled after 2 days of lowering substrate elasticity. Myofibroblast activation on substrates with varying moduli was independent of their time in culture (), indicating minimal influence from soluble factors in the medium. On stiff-to-soft gels, we observed a higher percentage of activated myofibroblasts (~25%) than that on soft gels (~10%). This indicates that not every myofibroblast can be efficiently de-activated by modulus reduction on stiff-to-soft gels.
Myofibroblasts not only differ from fibroblasts in the formation of α-SMA stress fibers, but also have a distinct gene expression profile 
. Through previous research, gene signatures to distinguish myofibroblasts from fibroblasts have been revealed, such as α-SMA and CTGF. α-SMA is highly regulated at the transcriptional level with multiple serum response elements and CArG motifs in the promoter region of the gene 
. CTGF expression is involved in the pathogenesis of fibrosis for various tissues and is tightly associated with the myofibroblast phenotype 
. Both genes are more highly expressed by myofibroblasts than fibroblasts. As shown in , these myofibroblast genes, α-SMA and CTGF, were significantly down-regulated with substrate modulus reduction, and the expression level of these genes was similar on stiff-to-soft gels compared with soft gels, suggesting reversion of activated VICs to a fibroblast-like phenotype. The reduction of these mRNAs was observed 6 hours after irradiating stiff gels to make them soft, indicating that cells change their molecular phenotype quickly in response to the mechanical cues. Uniquely, in comparison to substrates fabricated with discrete stiffness, changing substrate modulus in situ
using PD-PEG gels enabled us to track dynamic transcriptional changes during myofibroblast de-activation and further reveal the molecular mechanisms regulating this process. As CTGF has been shown to be down-regulated through the YAP/TAZ pathway on soft substrata 
, it is possible that this signaling is involved in the early phase of myofibroblast deactivation on stiff-to-soft gels. Hinz et al.
have discovered that latent TGF-β1 from the ECM is activated by contraction of α-SMA stress fibers in myofibroblasts 
. Given that VICs rarely form α-SMA stress fibers on soft or stiff-to-soft substrata, this result indicates a limited ability to activate TGF-β1 from their microenvironment. This mechanism may act at a later phase to reinforce the un-activated fibroblast phenotype on stiff-to-soft gels.
Another functionally significant characteristic of myofibroblasts is their high rate of proliferation. Lung fibroblasts cultured on substrates with high modulus (E
~100 kPa) exhibited increased myofibroblast activation and more proliferation 
. In fibrotic lesions, a large number of myofibroblasts, generated through cell proliferation, exacerbates the inflammatory response and collagen deposition 
. In contrast, VICs residing in healthy compliant valve matrices are mostly quiescent 
. We found that the number of proliferating VICs was decreased by ~30% on stiff-to-soft gels in comparison to stiff gels. This result indicates that lowering substrate modulus inhibits cell cycle progression and directs cells to a more quiescent-like phenotype. In particular, a higher fraction of the cell population stalled in the G2 or mitosis (M) phase of the cell cycle on soft or stiff-to-soft gels than on stiff gels (Fig. S2
), suggesting that mechanical tension conferred by substrate modulus is an important regulator for the G2/M phase of the cell cycle. From both , reducing substrate modulus not only down-regulated myofibroblast differentiation, but also controlled the proliferative response of these cells.
The myofibroblast phenotype has been suggested to be plastic, where myofibroblasts can be inhibited through different means including TGF-β1 antagonist treatment 
and low substrate modulus 
. If the valvular myofibroblasts were reprogrammed to quiescent fibroblasts on stiff-to-soft gels, then these cells should maintain the fibroblast gene expression and the potential to proliferate and differentiate into myofibroblasts. Vimentin is an intermediate filament protein expressed in mesenchymal cells, including fibroblasts 
. VICs expanded on plastic plates are all positive for vimentin staining (). This fibroblast property was preserved when substrate modulus was decreased. Based on , mRNA and protein expression of vimentin was present at a similar level in the de-activated cells on stiff-to-soft gels, compared with cells on either stiff or soft gels, indicating that the de-activated cells were still fibroblasts. Our results also suggest that these deactivated cells are in a reversible state and respond to FGF2 and increased serum by entering the cell cycle and respond to TGF-β1 by expressing myofibroblast gene markers (). Cell plasticity has become a blooming field of research with the paradigm-shifting discovery of reprogramming adult somatic fibroblasts into pluripotent stem cells by activating four transcription factors 
. A culture substratum with appropriate elastic modulus and binding epitopes shows promise as a complementary approach to reprogram the cells into a developmental stage of interest and to dynamically dictate cell phenotype and fate in a non-invasive manner.
The timing and duration of matrix signaling events are emerging as important factors in myofibroblastic differentiation plasticity and ultimate cell fate. We observe myofibroblastic de-activation of VICs with substrate modulus changes at short culture times. In complementary studies, Balestrini et al.
have observed that lung myofibroblasts “memorized” the stiff or soft substrates on which they were propagated for 3 weeks and stayed activated or un-activated even after they had been transferred to substrates with opposite stiffness 
. To compare, our cells have been cultured 7 days on stiff plastic plates before seeding on soft hydrogels for subsequent modulus tuning of 6 days in culture. Further, we observed similar levels of activation for freshly isolated VICs on stiff gels and stiff-to-soft gels as VICs at passage 3 (Fig. S5
). This indicates that our culture of VICs on plastic plates for about a week did not change the cellular response to substrate modulus. While there could be inherent differences between valvular fibroblasts and lung fibroblasts, our results and Balestrini et al. collectively indicate that as the myofibrobalsts mature over time, there may be a time limit on their ability to revert back to fibroblasts.
Differentiation of myofibroblasts is regulated by multiple factors, including cell-cell contact 
, adhesive epitopes 
, TGF-β1 
, and substrate elasticity 
. However, cells in vivo
encounter numerous signals and integrate different types and magnitudes of signals in choosing their fate. For example, cell-cell contact prevents TGF-β1 from inducing epithelial-to-myofibroblast differentiation 
. The ECM protein fibronectin with ED-A domain is required for TGF-β1 mediated myofibroblast differentiation 
. The Wells group has found that portal fibroblasts need both a stiff substrate and TGF-β1 to become myofibroblasts 
. Similarly, we observed that the myofibroblastic differentiation of VICs is regulated by both substrate stiffness and TGF-β1. When VICs were cultured on stiff-to-soft substrata (E
, from ~32 kPa to ~7 kPa), they can still activate fibrogenic genes (CTGF, FN1 and Col1A1) in response to TGF-β1 (). However, these cells fail to develop α-SMA stress fibers on the soft substrata even with TGF-β1 treatment ( and Fig. S3
). We speculate that a stiffer substrate is required for mature valvular myofibroblast formation and the de-activated VICs on stiff-to-soft gels were likely in a proto-myofibroblast state when treated with TGF-β1 
. The PD-PEG gel system provides a powerful tool in studying cellular responses to competing signals in vitro
, for example reduced substrate modulus while simultaneously increasing pro-fibrotic cytokines. This may provide insight into how microenvironment modulus in combination with other chemical or biological cues directs cell fate.
In summary, valvular myofibroblasts were reprogrammed to fibroblast-like cells when substrate modulus was reduced with light in situ from E ~32 kPa to E ~7 kPa. This de-differentiation process is characterized by low occurrences of apoptosis, dissolution of α-SMA stress fibers, down-regulation of differentiation associated genes (α-SMA and CTGF), and a decline in cell proliferation. The de-activated fibroblasts on stiff-to-soft gels can be re-activated by FGF2 and serum to enter the cell cycle and by TGF-β1 to express fibrogenic genes, such as CTGF, Col1A1 and FN1. Our data suggest that the fate of valvular myofibroblasts is regulated by substrate elasticity independent of soluble factors. This can potentially be applied to equivalent myofibroblasts from other tissues and presents a promising approach in tempering tissue fibrosis by de-differentiating activated myofibroblasts. Our study also provides an example of dynamically reprogramming differentiated cells through substrate modulus reduction and shapes the conception of designing user-defined 2-dimensional, or even 3-dimensional, platforms for controlling the developmental stage of cells.