Low intermittent flow promotes rat mesenchymal stem cell differentiation in logarithmic fluid shear device

Abstract

Bone marrow mesenchymal stem cells are an ideal candidate for bone tissue engineering due to their osteogenic potential. Along with chemical, mechanical signals such as fluid shear stress have been found to influence their differentiation characteristics. But the range of fluid shear experienced in vivo is too wide and difficult to generate in a single device. We have designed a microfluidic device that could generate four orders of shear stresses on adherent cells. This was achieved using a unique hydraulic resistance combination and linear optimization to the lesser total length of the circuit, making the device compact and yet generating four logarithmically increasing shear stresses. Numerical simulation depicts that, at an inlet velocity of 160 μl/min, our device generated shear stresses from 1.03 Pa to 1.09 mPa. In this condition, we successfully cultured primary rat bone marrow mesenchymal stem cells (rBMSCs) in the device for a prolonged period of time in the incubator environment (four days). Higher cell proliferation rate was observed in the intermittent flow at 1.09 mPa. At 10 mPa, both upregulation of osteogenic genes and higher alkaline phosphatase activity were observed. These results suggest that the intermittent shear of the order of 10 mPa can competently enhance osteogenic differentiation of rBMSCs compared to static culture.

Induction of osteogenic differentiation of bone marrow stromal cells on 3D polyester-based scaffolds solely by subphysiological fluidic stimulation in a laminar flow bioreactor

“…Studies using human bone marrow-derived MSC reported that shear stress at 0.4–2.2 Pa increased the expression of BMP-2, bone sialoprotein (BSP), osteopontin (OPN) and ALP together with enhanced calcium deposition within 7 days. 16 – 18 Further comparable results were reported in MSC isolated from rodents at 1.09 mPa–1.03 Pa. 19 Compared with 2D models, however, there is only limited evidence on fluid flow-induced osteogenesis in the 3D environment. Human fetal osteoblasts, hFOB 1.19, subjected to cyclic fluid shear stress at 3.93 mPa for 28 days on functionalised polycaprolactone/hydroxyapatite scaffolds, exhibited increased ALP activity, extracellular matrix (ECM) formation and mineralisation.…”

“…24,46,47 Hence, the results provide evidence that osteogenesis of BMSC on 3D non-osteoinductive scaffolds can be induced solely by fluid flow in the absence of osteogenic supplements as reported in 2D systems where fluid effect is purely represented as shear stress. 14,15,19,20 Various types of bioreactors have been developed for bone tissue engineering. Each of the systems has distinctive features, and therefore, the identification of experimental parameters is the first step in reconciling the experimental data.…”

“… 24 , 46 , 47 Hence, the results provide evidence that osteogenesis of BMSC on 3D non-osteoinductive scaffolds can be induced solely by fluid flow in the absence of osteogenic supplements as reported in 2D systems where fluid effect is purely represented as shear stress. 14 , 15 , 19 , 20 …”

“…Previous studies using a 2D microfluidic system showed that sub-physiological shear stress was sufficient to upregulate osteogenic markers. 17 , 19 Therefore, we further evaluated fluid-induced osteogenesis in a 3D environment where fluid effects were exerted not only as shear stress but also as pressure. In both FL-L and FL-H, the mRNA expression of early-to-mid phase osteogenic markers, RUNX2, SP7, IBSP, ALPL and SPP1 was consistently upregulated over the period of 21 days.…”

Biophysical Stimuli as the Fourth Pillar of Bone Tissue Engineering

“…Load-induced FFSS could change cell shape. One classic 2D model to explore the effect of load-induced FFSS is parallel-plate flow chamber ( Yourek et al, 2010 ; Dash et al, 2020 ). Using this model, it was confirmed that short-term continuous FFSS (9 dynes/cm 2 ) for 24 h could promote the osteogenic differentiation of MSCs without chemically osteoinductive molecules ( Yourek et al, 2010 ).…”