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.
Highlights
Mechanical loading plays an important role in the remodeling of mineralized bone matrix and fracture healing
Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS; order number 10270– 06), penicillin-streptomycin-fungizone (P/S/F), nonessential amino acids (NEAA), basic fibroblast growth factor, β-glycerolphosphate, ascorbic acid (AA), dexamethasone (Dex), alamarBlue1 solution and Quant-iTTM PicoGreen1 double stranded DNA reagent kit were from Gibco (Zug, Switzerland). 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) was from abcr GmbH & Co. (Karlsruhe, Germany)
The two different flow velocities applied in the study presented have been chosen to aim at causing biological responses similar to the biological responses occurring during early fracture healing and bone remodeling in healthy bone tissue, respectively
Summary
Mechanical loading plays an important role in the remodeling of mineralized bone matrix and fracture healing. The mineralized bone matrix is continuously remodeled, as a function of local mechanical stimuli [1]. Osteocytes are considered to be the mechanosensitive cell. They are thought to sense shear stresses (SS) caused by load-induced movement of the interstitial fluid within the lacuno-canalicular system [2]. Stimulation of osteocytes by very high SS leads to the recruitment of osteoblasts to the bone surface and subsequent mineralized bone matrix formation [3]. The precise role of mechanical stimulation on cells in fracture healing is not clearly understood [4]
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