title: Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.
abstract: Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and β-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms.
  
  
  CONCLUSIONS
  This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor.
authors.affiliation: Department of Mechanical Engineering, University of Colorado Boulder, Boulder, CO 80309, USA; Department of Industrial Engineering and Biotech Research Center, University of Trento, via Sommarive 9, 38123 Trento, Italy.
authors.forename: Michael
authors.initials: M
authors.lastname: Floren
authors.affiliation: Department of Industrial Engineering and Biotech Research Center, University of Trento, via Sommarive 9, 38123 Trento, Italy.
authors.forename: Walter
authors.initials: W
authors.lastname: Bonani
authors.affiliation: Department of Mechanical Engineering, University of Colorado Boulder, Boulder, CO 80309, USA.
authors.forename: Anirudh
authors.initials: A
authors.lastname: Dharmarajan
authors.affiliation: Department of Industrial Engineering and Biotech Research Center, University of Trento, via Sommarive 9, 38123 Trento, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Motta
authors.affiliation: Department of Industrial Engineering and Biotech Research Center, University of Trento, via Sommarive 9, 38123 Trento, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Migliaresi
authors.affiliation: Department of Mechanical Engineering, University of Colorado Boulder, Boulder, CO 80309, USA. Electronic address: wtan@colorado.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Tan
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D012831:Bombyx; D002152:Calorimetry, Differential Scanning; D002245:Carbon Dioxide / Q000737:chemistry; D002448:Cell Adhesion; D002454:Cell Differentiation; D005349:Fibroins / Q000737:chemistry*; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D010641:Phenotype; D011312:Pressure; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry; D053773:Transforming Growth Factor beta1 / Q000737:chemistry
chemical_list: D001672:Biocompatible Materials; D020100:Hydrogels; D036341:Intercellular Signaling Peptides and Proteins; D053773:Transforming Growth Factor beta1; D002245:Carbon Dioxide; D005349:Fibroins
keywords: Growth factor; Silk fibroin; Stem cell differentiation; Tunable hydrogel