Flow velocity-driven differentiation of human mesenchymal stromal cells in silk fibroin scaffolds: A combined experimental and computational approach

PLoS One. 2017 Jul 7;12(7):e0180781. doi: 10.1371/journal.pone.0180781. eCollection 2017.

Abstract

Mechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (vlow = 0.001m/s and vhigh = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at vlow compared to vhigh and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at vhigh whereas the amount of DNA was increased at vlow. The biological response at vlow might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (vlow) or bone remodeling (vhigh). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading.

MeSH terms

  • Biomechanical Phenomena
  • Bioreactors
  • Bone Regeneration / physiology
  • Bone and Bones / cytology
  • Bone and Bones / physiology
  • Calcification, Physiologic*
  • Cell Culture Techniques
  • Cell Differentiation / drug effects
  • Cell Proliferation
  • Extracellular Matrix / metabolism
  • Fibroins / chemistry
  • Fibroins / pharmacology*
  • Humans
  • Intercellular Signaling Peptides and Proteins / pharmacology
  • Mesenchymal Stem Cells / cytology*
  • Mesenchymal Stem Cells / drug effects
  • Mesenchymal Stem Cells / physiology
  • Osteogenesis*
  • Primary Cell Culture
  • Rheology
  • Stress, Mechanical
  • Time-Lapse Imaging
  • Tissue Engineering / methods*
  • Tissue Scaffolds
  • X-Ray Microtomography

Substances

  • Intercellular Signaling Peptides and Proteins
  • Fibroins

Grants and funding

The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP/2007-2013): FP7-NMP-2010-LARGE-4: BIODESIGN - Rational Bioactive Materials Design for Tissue Regeneration (JRV, DCB, RM, SH) and ERC-2013-StG: REMOTE - Real-time monitoring of load induced remodeling in tissue-engineered bone (SH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.