VALIDATED CYCLIC SHEAR STRESS SIGNIFICANTLY UP-REGULATESN BY HUMAN TENOCYTES

Russell Tucker (1,2), Sarah Franklin (2), Duanduan Chen (1), Yiannis Ventikos (1), Per Henningsson (3), Richard Bomphrey (3), Mark Thompson (1,2)

1. Department of Engineering Science, University of Oxford, UK; 2. Institute of Musculoskeletal Sciences, University of Oxford, UK; 3 Department of Zoology,

University of Oxford, UK.

Introduction

Stimulating cells is vital for understanding cell

behaviour, providing a foundation for developing

tissue engineering applications. In vitro stimulation

devices are regularly used to study the effect of

mechanical forces on cell cultures; however these

devices are inherently limited as the method and

type of stimulation can vary between research

institutions and results can’t be attributed to a

validated force. Work by Thompson et al.

highlighted the presence of fluid flow in a device

thought to be primarily concerned with the

application of a substrate strain. This work presents

a new, simple, validated stimulation device that has

a high throughput and can be accurately reproduced

across research groups for varying phenotypes.

Methods

Cyclic fluid flow in a 6-well plate, induced with a

seesaw rocker (Stuart SSL4) allowed for accuracy

and repeatability at a consistent angle of tilt of 7 °.

2 ml of 0.5 % FCS/DMEM added to each well

drove shear stresses at the well base where human

tenocytes (up to passage 3) were cultured to

confluency. Digital particle image velocimetry

(PIV) captured velocity vectors (in a 60 x 60 mm

flow field) of a thin suspension of 48 �m

fluorescent microspheres seeded within a plate well

moving along a 10 mJ laser sheet in the central

plane. PIV vectors were compared to

computational fluid dynamics (CFD, ESI-ACE)

simulations (where Volume of Fluids was

employed to track the free surface), to validate

model assumptions and verify shear stresses at the

cell layer. Interrupted (8 hours with a break of 20

hours at the half-way point) and continuous (7

days) cyclic fluid stress was applied at 5 RPM.

ECM synthesis was investigated using biochemical

assays (SircolTM and DMB) and normalised with

dsDNA content (PicoGreen) and viability checked

with live/dead stain.

Results

Stimulation under both conditions resulted in a

significant increase in collagen and GAG secretion

(Fig 1A). With an increased duration of

stimulation, protein secretion increases non-

linearly. Flow patterns were similar in PIV (Fig 1B)

and CFD and maximal velocities matched to an

order of magnitude. CFD showed maximal shear

stresses at the cell layer for 5 RPM to be 0.13 Pa.

Figure 1: A) Collagen at the cell layer is

significantly up-regulated with the application of 8

hours of cyclic fluid shear stress (P <0.001). B)

PIV velocity vector transect of a 6 well plate

chamber at an angle of tilt of 7°. Velocity peaks at

±75mms-1 (orange and dark blue).

Discussion

It is essential that in vitro stimulation devices are

validated for biological results to be comparable

and meaningful. Fluid shear stress of 0.13 Pa at 5

RPM, significantly up-regulates the secretion of

proteins that are crucial in the formation of tendon

structure. Previous authors [Archambault, 2002]

have shown tenocytes to be sensitive to similar

shear stress levels. Tenocytes are known to possess

primary cilia which may be involved in the up-

regulated ECM response to fluid flow. Future work

will consider the effect of fluid flow on alignment

of cilia, cytoskeleton and cell across the culture

well and investigate the role of shear stress

magnitude on cellular response. A deeper

understanding of mechanotransduction is essential

for engineering replacement tissue to treat

musculoskeletal disease.

References

Thompson et al, J Biomech Model Mechanobiol,

10 559-564 (2011)

Archambault et al, J Biomech 35 303-309 (2002)

A

B

ECM SECRETIO

Presentation 1494 − Topic 31. Mechanobiology and cell biomechanics S437

ESB2012: 18th Congress of the European Society of Biomechanics Journal of Biomechanics 45(S1)