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

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.

Fluid flow‐induced cell stimulation in bone tissue engineering changes due to interstitial tissue formation in vitro

“…Computational models (e.g., computational fluid dynamics [CFD], multiphasic finite element [FE]) have been used for calculating the local WSS for a variety of applied flow rates 4‐8 . In most previous CFD analyses, the models merely included the empty scaffold geometries which were obtained either from computer aided design (CAD) or micro‐computer tomography (μCT) scanning images 7‐11 . It was assumed that cells were initially attached flatly onto the scaffold surface prior to ECM deposition with the underlying assumption that the solid volume of cells was negligible and would not affect the fluid flow comparing to that in the empty scaffold 7,9‐11 .…”

Modeling Host-Pathogen Interactions in the Context of the Microenvironment: Three-Dimensional Cell Culture Comes of Age

“…1B, panel a) (8,127). Fluid shear influences cell proliferation, differentiation, morphology, and function (30,114,(128)(129)(130)(131)(132)(133)(134)(135)(136)(137)(138)(139)(140). Models developed within the dynamic RWV environment experience excellent mass transfer of nutrients/wastes and exhibit enhanced structure, differentiation, function, and multicellular complexity relative to 2-D monolayers (11,80,(141)(142)(143)(144)(145)(146)(147)(148)(149)(150)(151)(152)(153)(154).…”

Strategy for achieving standardized bone models

“…The homogeneous porous media flow (HPMF) model, which considers the scaffold as a homogeneous permeable solid, has been used in recent studies (Kleinhans et al, ; Vetsch, Betts, Muller, & Hofmann, ) to encompass scaffold hydraulic properties (e.g., porosity and permeability) while reducing the complexity of the simulation. Figure a shows the shear stress distribution at the median transverse cut plane predicted by this model for a flowrate of 12 ml/min (Vetsch et al, ). Figure b shows the shear stress distribution in a similar scaffold generated by finite element analysis (FEA) based on the actual scaffold geometry for a lower flowrate of 0.3 ml/min.…”

“…Limit of the homogeneous porous media flow (HPMF) model. (a) Wall shear stress map at the transverse cut plane (50%) in an 8‐mm diameter salt‐leached scaffold (pore size 315–400 µm, porosity 55%) for a 12 ml/min flowrate, calculated with HPMF (from Vetsch et al, ). (b) Wall shear stress map at the transverse cut plane (50%) in an 8 mm diameter salt‐leached scaffold (pore size 250–425 µm, porosity 80%) for a 0.3 ml/min flowrate, calculated with finite element analysis based on CFD using the actual scaffold geometry (from Liu et al, )…”

“…In addition, depending on the study, different viscosity values are declared for similar culture media, from 0.78(Bacabac et al, 2005) to 1.45Liu et al, 2018;Olivares, Marshal, Planell, & Lacroix, 2009; Zhao, Vaughan, & F I G U R E 1 1 Limit of the homogeneous porous media flow (HPMF) model. (a) Wall shear stress map at the transverse cut plane (50%) in an 8-mm diameter salt-leached scaffold (pore size 315-400 µm, porosity 55%) for a 12 ml/min flowrate, calculated with HPMF (fromVetsch et al, 2017). (b) Wall shear stress map at the transverse cut plane (50%) in an 8 mm diameter salt-leached scaffold (pore size 250-425 µm, porosity 80%) for a 0.3 ml/min flowrate, calculated with finite element analysis based on CFD using the actual scaffold geometry (fromLiu et al, 2018) HADIDA AND MARCHAT | 261McNamara, 2016), creating a strong bias in calculated shear stress values for both CFD analysis and the simplified mathematical approach.As the Goldstein approximation seemed to be the most prevalent source of the seldom declared shear stress values in the literature until a few years ago, the current literature relies on sparse and possibly unreliable data.…”