269659 Designer Collagen Hydrogels to Regulate Satellite Cell Phenotype

Monday, October 29, 2012: 8:48 AM
Cambria West (Westin )
Veronica Rodriguez-Rivera1, Richard L. Goodwin2, Mary O. Morales3, Nikkhoo Mohsen1, Louis Terracio4, John W. Weidner1 and Michael J. Yost5, (1)Chemical Engineering Department, University of South Carolina, Columbia, SC, (2)Department of Cell Biology and Anatomy, University of South Carolina-School of Medicine, Columbia, SC, (3)Department of Surgery, University of South Carolina-School of Medicine, Columbia, SC, (4)New York University-College of Dentistry, New York, NY, (5)Department of Surgery, Medical University of South Carolina, Charleston, SC

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).

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