(14b) Designer Collagen Hydrogels to Regulate Satellite Cell Phenotype

Introduction: Tissue engineering strategies
that employ cells to repair and replace damaged tissue is a rapidly growing and
evolving field. The regenerative capabilities of muscle stem cells, satellite
cells, are lost once they are cultured in a rigid environment such as
polystyrene plastic, which has an elastic modulus of 3 GPa.
In order for the satellite cells to remain undifferentiated and proliferative,
it is imperative to recreate the biophysical properties of the stem cells niche
that includes an appropriate extracellular matrix and basement membrane. Using a
tunable collagen/laminin substrate we determined that
an elastic modulus of 12 KPa maintained satellite cells in a quiescent/undifferentiated state
which corresponds to the in vivo
satellite cell niche moduli. These data indicate that
the fate and activation of skeletal muscle stem cells can be modulated by its
mechanical environment.

Materials
and Methods: The
shear modulus of collagen/laminin hydrogels
were measured using a Rheometrics Fluid Spectrometer
II.
Satellite cells were
isolated and purified using BD Biosciences FACS Aria II cell sorter. Positive
fractions were collected, counted and cultured on hydrogels
with different moduli. Hydrogels
were stained for integrin α-7, MyoD, Pax7, and F-actin subjected
to confocal microscopic analysis to determine the
progression of skeletal muscle differentiation.

Results
and Discussion: The
shear modulus was obtained for each collagen/laminin hydrogel concentration and converted into Young's moduli. A linear regression fit of the elastic modulus and
the collagen concentration allows the fabrication of tunable collagen/laminin hydrogels to mimic in vivo niches stiffness to control a specific
phenotype.

A significant reduction in the expression of the integrin α7 was observed in the confocal
microscopy images of 14 to 20 mg/mL hydrogels, which corresponds to the elasticity seen in vivo compared to a stiffer niche as
seen in figure 1. Furthermore, the specific stage of the satellite cells in the myogenesis pathway was determined. Pax7 is a transcription
factor expressed in quiescent satellite cells. MyoD is
a transcription factor for myogenic differentiation. Morphological changes in the cellular cytoskeleton
as determined by the F-actin staining corresponds to the degree of myogenic differentiation, as well as reduced Pax7 and MyoD expression at concentrations of 14 and 20 mg/mL. 14
mg/mL hydrogel have a
modulus of 12 KPa.

Conclusions:
Previous studies
and our data using natural ECM components suggest that the mechanical environment
plays a significant role in the regulation of the skeletal muscle precursor cell
niche. Further testing is required to confirm these findings. By maintaining
the satellite cell population in the appropriate niche, we will be able to
maintain their abilities and employ them for regenerative therapies.

Acknowledgements: This work was supported by
NIH-NIDCR IROIDE019355 (MJ Yost, PI).