A DYNAMIC TUNABLE PLATFORM TO MANIPULATE FIBROBLAST ACTIVATION
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Abstract
Adverse remodeling of the heart after myocardial infarction (MI), also known
as cardiac fibrosis, will lead to the stiffening and anisotropic conversion of
myocardium tissue and stoppage of blood throughout the body, which will eventually
cause death. The annual cost for treatment of MI causes a heavy burden to patients.
However, most current treatments are limited to the implantation of cardiac patches,
cardiac stents, and bypass surgery, which are non-targeted therapies. Moderate
myocardial fibrosis contributes to the positive regeneration of the heart after MI,
whereas excessive fibrosis can lead to irreversible remodeling and impair the function
of the heart. Thus, a systematic investigation of cardiac fibrosis development and
myocardium remodeling after MI is in urgent need in order to unveil the mechanism
of cardiac fibrosis development and translate it into targeted therapeutic strategies.
In vitro substrates have been broadly applied in the biomedical field as disease
model platforms for physiological and pathological processes. Creating an in vitro
model for post-MI tissue study that can mimic the physical properties of an infarcted
heart with spatial-temporal variation is critical as the building block, yet there has not
been an effective device used to recapitulate the dynamic nature of the infarcted heart
tissue. The recent emergence of novel material with stiffness-tunability in response to
an external magnetic field, magnetorheological elastomers (MREs), reveal an
excellent in-situ modulation of stiffness with a wide range which provides a promising
tool for biomedical applications. However, the widening of the hysteresis loop
observed in MREs is not identical to the characteristic hysteresis loop of the soft ferromagnetic material, which was previously attributed to particle motion. Thus,
unraveling particle motion within the matrix is an urgent need and a critical step to
explain the magnetic hysteresis loop widening of MREs, which will facilitate and
expand its biomedical applications.
Ultrasoft MREs made by incorporating carbonyl iron particles into soft
Sylgard 527 polydimethylsiloxane (PDMS) showed large magnetic-field-dependent
changes in their stiffness changes. Our recent studies exhibited the widening of the
magnetic hysteresis loop in these soft MREs compared to the relatively pinched loop
observed for the stiffer MREs. This interesting behavior made us hypothesize that iron
particle displacement in response to the external magnetic field within the soft PDMS
matrix contributes to the magnetic hysteresis loop widening. We experimentally
validated our hypothesis by tracking fluorescently labeled carbonyl iron particle
motion in the MREs responding to various magnetic field strengths. Particle coating
and bioconjugation of fluorophores were also characterized to validate the success of
modification and retention of the magnetic properties of modified carbonyl iron
particles. Our findings for the first time demonstrate the successful modification of
carbonyl iron particles and observe that particles primarily move along the external
magnetic field direction within the MREs material and such particle displacement
results in the magnetic hysteresis loop widening.
One of the physical cues after MI is cardiac tissue stiffness with spatialtemporal
variation. The infarct border zone, a tissue region that is situated between
infarcted tissue and adjacent healthy tissue, exhibits gradient stiffness change. The
border region is of great importance and interest in the inflammatory and remodeling
after MI involving infarction expansion, fibrosis formation, and reparative process.
The efforts researchers have been building have advanced the development of a
variety of in vitro platforms to mimic the microenvironment of infarct border region
allowing us to have a more controllable and cost-effective approach to characterize the
microenvironment features. Whereas the dynamic nature of gradient stiffness of
infarct border zone has not yet been achieved. In this work, we use MREs as our base
material to fabricate an innovative 2D in vitro platform with the capability of in-situ
modulation on the stiffness gradient mimicking the continuously changing microenvironment
of infarcted myocardium. This allows us to examine the inflammatory
response of primary cardiac fibroblasts (NHCF-V) in a spatiotemporal manner
regarding the different biomarkers’ expression, including αSMA, TGF-β, ACTA2,
COLI/III, and MMP1. We found that cardiac fibroblasts exhibit spatially different
activation and cytokine secretion in the infarcted and remote regions within the same
model and presented a phenotypic conversion as time proceeded. COL1A1/3A1 and
MMP1 exhibit a more robust regulation at 24 h. Additionally, the tunability of such a
gradient stiffness model allows us to modulate the stiffness gradient on demand, either
increasing or decreasing the stiffness gradient, to mimic different stages during
remodeling process. Recognizing that remodeling is guided by many different types of
cues that co-exist in vivo, it is also important to recognize that these cues may
cooperate or compete to ultimately direct remodeling. Specifically, we examine the
competition and cooperation of a chemical cue (anti-fibrotic drug, A 83-01) and
mechanical cue (substrate softening) in tandem to attenuate fibrotic biomarker
responses of cardiac fibroblasts. Individual intervention by drug and softening
exhibited a competitive and/or cooperative effect on the combined intervention in
order to regulate fibrosis. This work reveals the spatiotemporal variation of fibrotic
response in cardiac fibroblasts as well as the complexity of anti-fibrotic drug dosing
and stiffness-varying interactions on cardiac fibroblasts. This platform with a more
realistic microenvironment will not only provide us with a unique in vitro tool to study
disease progression mechanisms, such as cardiac fibrosis, but also serve as a cost -
effective, pathophysiological relevant model for potential therapeutics screening.
Topography, particularly the anisotropic structure of heart tissue, is another
important physical cue in the in vivo cellular microenvironment after MI. Integrating
anisotropic features with stiffness-tunable materials not only allow us to construct both
physiological and pathological relevant building block but also enable us to probe
interactions between topography and stiffness cues. In this study, we introduced an
alternative MRE as our in vitro substrate, EcoGel MREs, which retains higher
anisotropic pattern fidelity than conventional MREs. Our results indicate that the
combination of dynamic changes in stiffness with and without topographic cues
induces different effects on the alignment and activation or deactivation of
myofibroblasts. Specifically, cells cultured on patterned substrates exhibited a more
aligned morphology than cells cultured on flat material; moreover, cell alignment was
not dependent on substrate stiffness. On a patterned substrate, there was no significant
change in the number of activated myofibroblasts when the material was temporally
stiffened, but temporal softening caused a significant decrease in myofibroblast
activation, indicating a competing interaction of these characteristics on cell behavior.