See

discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/225927850

A method to determine Youngs modulus of

soft gels for cell adhesion
ARTICLE in ACTA MECHANICA SINICA AUGUST 2009
Impact Factor: 0.89 DOI: 10.1007/s10409-009-0270-6

CITATIONS

READS

11

229

5 AUTHORS, INCLUDING:
Xiaoling Peng

Jianyong Huang

Shanghai Ocean University

32 PUBLICATIONS 259 CITATIONS

16 PUBLICATIONS 168 CITATIONS

SEE PROFILE

SEE PROFILE

Chunyang Xiong
42 PUBLICATIONS 386 CITATIONS
SEE PROFILE

Jing Fang
Peking University
195 PUBLICATIONS 1,018 CITATIONS
SEE PROFILE

Available from: Jianyong Huang

Retrieved on: 24 September 2015

Acta Mech Sin (2009) 25:565570

DOI 10.1007/s10409-009-0270-6

TECHNICAL NOTE

A method to determine Youngs modulus of soft gels

for cell adhesion
Xiaoling Peng Jianyong Huang Lei Qin
Chunyang Xiong Jing Fang

Received: 4 January 2009 / Accepted: 9 April 2009 / Published online: 30 May 2009
The Chinese Society of Theoretical and Applied Mechanics and Springer-Verlag GmbH 2009

Abstract A convenient technique is reported in this note

for measuring elastic modulus of extremely soft material for
cellular adhesion. Specimens of bending cylinder under gravity are used to avoid contact problem between testing device
and sample, and a beam model is presented for evaluating
the curvatures of gel beams with large elastic deformation. A
self-adaptive algorithm is also proposed to search for the best
estimation of gels elastic moduli by comparing the experimental bending curvatures with those computed from the
beam model with preestimated moduli. Application to the
measurement of the property of polyacrylamide gels indicates that the material compliance varies with the concentrations of bis-acrylamide, and the gels become softer after
being immersed in a culture medium for a period of time, no
matter to what extent they are polymerized.
Keywords Youngs modulus Bending measurement
Cell adhesion Polyacrylamide gel
1 Introduction
Interaction between an elastic substrate and adherent cells
has received extensive attention since it plays an important role in regulating cellular functions and behaviors in
contraction, migration, and invasion [14]. With the help
of distortion of flexible substrates or elastic deformation of
micro-columns induced by the adherent cells, the cellular
The project supported by the National Basic Research Program
(2007CB935602), the National Natural Science Foundation of China
(90607004, 10672005).
X. Peng J. Huang L. Qin C. Xiong (B) J. Fang
Laboratory of Cell Mechanics, Department of Biomedical Engineering,
Peking University, 100871 Beijing, China
e-mail: cyxiong@pku.edu.cn

mechanical responses can be investigated by quantitative

analyses [510]. To match the mechanical properties of the
cells, some extremely soft polymeric gels, such as polydimethylsiloxane (PDMS), polyacrylamide (PAA), are used
as the flexible substrates to culture various cells so that their
biological activities can be evaluated. It has been reported that
elastic stiffness of the substrate gels exerts markedly influences on mechanical behaviors and biochemical expressions
of the adherent cells [59]. Therefore, accurate determination of Youngs moduli of these extremely soft gels becomes
essential for obtaining exact responses of the cells to the substrate flexibilities. PAA gel is among the widely used materials for exploring the responses of cells to their substrates
since the hardness of this polymer can be modulated by varying percentages of monomers and its varied modulus is of the
order of that of cells [11].
The gels, such as PDMS and PAA, usually display large
elastic deformation in resisting the applied forces. According to the tension results reported by Pelham et al. [4], when
the specimens with a cross-section area of 3 mm 10 mm
were loaded by a stretching force of 0.103 N, the PAA gels
could undergo a strain to the extent of 3.830%, showing
the fact that the material behaved as linear stressstrain relation in this strain range. Likewise, the indentation tests on
gels with a small steel ball [7], as well as the studies using a
micro-probe in atom force microscopy (AFM) [12] exhibited
similar properties [13]. From the view point of experimental techniques, the method of stretching gel specimens often
fails due to stress concentration at the clamping end of the
specimen with low strength intensity. For the technique using
AFM or nanoindentation [14] to detect the probe force and
the indented depth, interfacial adhesions are usually involved
in the contact surfaces between the materials, and thus the
surface adhesions have strong influence on the deduction of
Youngs modulus of the material, and adhesive work needs

123

566

to be considered in the models in the process of mechanical

property evaluation [15].
This report presents a new technique for measuring the
Youngs modulus of soft gels, which are useful for cell culture
and adhesion. A bending specimen of gel cylinder with natural gravity load is adopted for measuring the beam deflection,
since the manipulation is straightforward and the relation
between the material deformation and the elastic modulus
is simple. Theoretical analysis of large deformation is carried out to provide an analytical model for processing the
measured displacement data, and numerical simulations are
performed to validate the proposed method. The results for
characterizing properties of porous PAA gels are presented
to show the variation of elastic modulus with time after the
specimens are immersed into a cellular culture medium.

X. Peng et al.

Fig. 1 An image of the bent PAA cylinder, which is produced by pushing the gel out of the pinhead of a syringe and bending under gravity

2 Specimen preparation and computational algorithm

The gel material was made from polyacrylamide prepolymer prepared as described by Dembo et al. [6]. A mixture containing acrylamide (10%, Sigma) and bis-acrylamide
was pumped into a syringe (1 ml) for polymerization using
TEMED (N , N , N
, N
-tetramethylethylenediamine) and
ammonium persulfate. Three sets of samples of different
percentages (0.03, 0.13 and 0.26%) of bis-acrylamide were
produced. In the experiments, one set was tested immediately
after polymerization (50 min), while the other two sets were
tested after being immersed in Dulbeccos modified Eagles
medium (DEME), containing 10% fetal bovine serum
(v/v, Hyclone) and antibiotics (50 mg/ml streptomycin,
50 U/ml penicillin), and maintained in an oven at 37 C for
24 and 48 h.
The bending specimens were prepared by simply pushing the polymerized polyacrylamide out of the pinhead of
the syringe, which was placed in a horizontal bench to allow
the polymer to move out and drop downwards by gravity,
as shown in Fig. 1. The length of the polyacrylamide beam
was measured by the moving distance of the piston as the
specimen incision leaves the syringe output. Images of the
bending PAA specimen were collected by a CCD camera to
measure the exact diameter of the polymer cylinder, and to
record the curvature of beam deflection.
Taking into account the interaction between shear strain,
extension strain and geometrically large bending deformation, we can derive an analytical expression for the beam
deflection, as the material keeps linear stressstrain relationship [16]. For a circular cross-section beam with radius R,
the natural deformation under gravity, as illustrated in Fig. 2,
can be described as

s
q(s t) cos[ (t)]dt
q cos
d
= t
+
,
(1)
dt
E I ( )
G A( )

123

Fig. 2 An illustration of the bent beam with large elastic deformation,

used for the finite difference solution of the deflection

where is the beam slope, s the arc length of the bent beam,
t the length from the fixed end to the current point, q the
equivalent load of gravity, E the Youngs modulus, G the
shear modulus and the shear coefficient (equal to 4/3 for
the circular cross-section [16]). The first term in Eq. (1) represents the bending effect, and the second denotes the shear
effect which should be taken into account when the geometric
structure of the specimen can not satisfy the slender condition. For large deflection of the gel beam under gravity load,
the bending rigidity E I ( ) and shear rigidity G A( )/ are
altered along the neutral axis as a result of the change in the
radius of cross-section. The Poissons ratio of PAA gels is
known as 0.48 with approximately incompressible property
[17,18]. Taken together, all these factors are considered in
solving the deflection of the largely deformed beam by means
of a finite difference approach, as given in the following.

A method to determine Youngs modulus

567

Fig. 3 Flow chart of the

iteration algorithm for data
processing of deflection
experiments

By assuming that the beam is divided into N segments,

each with length of h, the position of the nth node is determined by
xn = xn1 + h(1 + n ) cos n

(2)

yn = yn1 + h(1 + n ) sin n

(3)

where n is the beam slope at the nth node, n the tensile

strain which can be obtained by integrating Eq. (1) in two
steps. The first step is to calculate (d/dt) in an iterative
form

 N  (k1)
(k1)
 (k)
qh m=n
xm
xn
d
=
,
(4)
(k1)
dt n
E In
where the superscript k is the current iteration number. This
iteration process stops
when the residual error satisfies the

 (k)
(k1) 
limited condition x N x N  e (e is a beforehand
given error). And the second step is to solve by forward
difference, or n = n1 +(d/dt)n h(1+n ). Thus the beam
slope can be expressed as
n+1 =

q(s t) cos n 2
h + 2n n1 .
E In

(5)

Figure 3 displays a flow chart to describe the computation

process. It is worth noting that the accumulation of computational errors in each iterative step, as implied in the flow
chart of Fig. 3, is most likely to induce divergence of the
differential solution and accordingly leads to unsteady computational results. Therefore, a boundary constraint must be
introduced here by limiting
the beam slop within
a range of





,
to
avoid
diverh,
0, 2 , say n = min n1 + d
dt n1
2
gence during the iterative computation.
And then, a self-adaptive algorithm is developed to determinate the soft material Youngs moduli from the beam
deflections. For a real image of the bent beam captured by
the CCD camera, we adopt a four-order polynomial, say
y = f (x), to fit the curve of the deformed cantilever. On
the other hand, by employing the above iteration program,
a series of computational beam curves can be obtained by
varying the value of Youngs modulus within a prescribed
range roughly pre-estimated. The discrepancies between the
real beam shape and the specified calculated curves can be
quantitatively characterized as

r 

yi f (xi ) 2
2
(r 1)
(6)
=
f (xi )
i=1

123

568

X. Peng et al.

Fig. 4 Comparison between the bending curves solved by finite

differential method (Matlab) and by FEM simulation (ANSYS)

where (xi , yi ) represents the ith sampling point selected from

the computational beam curves, whereas (xi , f (xi )) denotes
the corresponding location of the actual deformed cantilever; r is the total number of sampling points. Differentiating
Eq. (6) with respect to the Youngs modulus and setting the
outcome equal to zero, we can thus obtain an optimal estimation of the Youngs modulus E. The self-adapted process
is carried out to search for the minimum value of the error
function, and the entire algorithm is programmed in Matlab
(MathWorks, Natick, MA).

3 Results and discussions

To validate the feasibility of the proposed method for processing experimental data, we firstly computed a soft-gel cantilever by using the finite element method (FEM) (ANSYS,
Inc. Houston, PA) and the present method, respectively. The
beam was chosen with 18 mm of length and 4.5 mm in diameter, and Youngs modulus is 13kPa and Poisson ratio 0.48.
In the computation with ANSYS, the BEAM 189 element
type was selected for modeling the structure; and the keys
of large deformation analysis and linearly elastic material
were switched on. Figure 4 gives a comparison between the
beam curves from FEM (dotted line) simulation and from
our finite difference computation (solid line), with the same
set of geometrical and mechanical parameters. The maximum discrepancy between the two deflection curves is only
0.037 mm, less than one pixel (0.05 mm) in the corresponding digital images, indicating that both results are in close
agreement.
Based on the curvature pictures of the bent cylinders,
the Youngs moduli of the PAA material were obtained via the
fitting algorithm of the beam images, as showed in Fig. 5. The
moduli of the non-immersed PAA gels were 13.1 2.1 kPa,
34.74.1 kPa and 59.9 4.7 kPa, respectively, as the percentages of bis-acrylamide in the soft gels were 0.03, 0.13 and
0.26%. With experimental uncertainties taken into account,
these moduli are of the same order as the results obtained by
using other methods, as previously reported in Refs. [4,6,17].
More specifically, the modulus of non-immersed PAA with

123

Fig. 5 Youngs moduli of PAA gels with different concentrations of

cross-linker, showing their changes before and after being immersed in
DMEM medium for one and two days

0.03% bis-acrylamide is about twice as large as that reported

in Ref. [4], whereas the other sets show no distinct differences. More important, our results show that immersion of
the specimens in DMEM brings a sharp decrease of the
Youngs modulus, which has not been paid attention in all
previous reports. As shown in Fig. 5, the moduli of the samples immersed for 24 h are nearly one half of those without
immersion. The question why the polymer moduli decline
during this period is still open and needs further physicalchemistry analysis. Anyhow, there is no evident discrepancy
between the immersed samples for 24h and 48 h, suggesting that for the PAA gel used to culture adherent cells, the
elastic modulus of the soft substrate becomes stable after
the material has been immersed in DMEM for a period of
time, possibly more than 24 h. Actually, for most experiments dedicated to the study of interaction between adherent cells and polymeric substrates, the substrates are usually
immersed in culture medium for at least several hours or
even more than one day, in order to guarantee steady cell
adhesions. During this process, the immersion might well
have altered the mechanical properties of the gel substrates,
especially for those hydrated materials like PAA. This feature seems neglected in previous measurements, in which the
Youngs moduli of the elastic substrates were normally surveyed just after gel polymerization without any immersion
process [4,6,17], and thus might induce big errors in evaluating the response of adherent cells to substrate flexibilities.
It is therefore recommended that the Youngs moduli of the
soft gels be measured under the same condition as the real
one these materials will experience.
Note that the above theoretical analysis and experimental
data processing is based on a premise that the gel material lies
within linear elasticity range even when the bent specimens

A method to determine Youngs modulus

569

Table 1 The suggested lengths for the PAA gel-cantilever specimens

to keep the material within linear stressstrain relationship
Duration (h)

Concentration of bis-acrylamide (%)

0.03

0.13

0.26

19 mm

35 mm

45 mm

24

14 mm

25 mm

35 mm

48

14 mm

25 mm

35 mm

adhesive effect of contact in using AFM probing to measure force and deformation. We present analytical expression
to describe large elastic deflection of the flexible gel beam
and develop the algorithm to fit the beam curvature so as
to obtain optimal estimation of the Youngs modulus. The
comparison of our results with those from FEM simulation
validates the proposed method in data processing, and the
application to property measurement of PAA material indicates that the elastic moduli of the gel vary with the percentages of the cross-linker involved. The present results show
that the PAA gel becomes more compliant after immersed
in DMEM for a period of time, which is important in estimating the mechanical properties of the soft matter and in
evaluating the responses of the cultured cells to the stiffness
change of the flexible substrates [5,10].

References

Fig. 6 Comparison of the real curvature shape of a specimen containing 0.03% bis-acrylamide with the best-fitted bent beam calculated
by the iterative computation with the optimal estimation of Youngs
modulus

undergo large geometrical deformation. In actual tests, therefore, the PAA specimen bending must be constrained within
a limited strain range so that such an assumption is satisfied.
As mentioned above, for this kind of soft materials including
PDMS and PAA, a number of tensile (or compressive) tests
[4,6,19] confirmed that they responded to stress in a linearly elastic manner if the applied strain was less than 30%,
and thus these requirements can be achieved in our specimen
design by properly controlling the original lengths of the PAA
cylinders, as given in Table 1, which are determined from
FEM analysis (ANSYS) with preestimated Youngs moduli.
Moreover, the best-fitted bent curve derived by our processing algorithm indeed agrees well with the corresponding real
cantilever shape, as shown in Fig. 6, with a mean error less
than 0.05 mm, which not only further verifies that the PAA
behaves as a linearly elastic material in our experiment, but
also implies that it is feasible to use the current data processing to precisely estimate the elastic modulus.
In conclusion, we propose an efficient way to measure the
Youngs modulus of soft gels with the aid of beam bending under gravity. The specimen preparation is very simple,
with neither fracture of sample clamping in tensile tests, nor

1. Galbraith, C.G., Sheetz, M.P.: Forces on adhesive contacts affect

cell function. Curr. Opin. Cell Biol. 10, 566571 (1998)
2. Vogel, V., Sheetz, M.P.: Local force and geometry sensing regulate
cell functions. Nat. Rev. Mol. Cell Biol. 7, 265275 (2006)
3. Yeung, T., Georges, P.C., Flanagan, L.A., Marg, B., Ortiz, M.,
Funaki, M., Zahir, N., Ming, W.Y., Weaver, V., Janmey,
P.A.: Effects of substrate stiffness on cell morphology, cytoskeletal
structure, and adhesion. Cell Motil. Cytoskelet. 60, 2434 (2005)
4. Pelham, R.J., Wang, Y.L.: Cell locomotion and focal adhesions
are regulated by substrate flexibility. Proc. Natl. Acad. Sci.
USA. 94, 1366113665 (1997)
5. Qin, L., Huang, J.Y., Xiong, C.Y., Zhang, Y.Y., Fang, J.: Dynamical stress characterization and energy evaluation of single cardiac
myocyte actuating on flexible substrate. Biochem. Biophys. Res.
Commun. 360, 352356 (2007)
6. Dembo, M., Wang, Y.L.: Stresses at the cell-to-substrate interface
during locomotion of fibroblasts. Biophys. J. 76, 23072316 (1999)
7. Munevar, S., Wang, Y.L., Dembo, M.: Traction force microscopy
of migrating normal and H-Ras transformed 3T3 fibroblasts. Biophys. J. 80, 17441757 (2001)
8. Wang, N., Tolic-Norrelykke, I.M., Chen, J.X., Mijailovich, S.M.,
Butler, J.P., Fredberg, J.J., Stamenovic, D.: Cell prestress. I. stiffness and prestress are closely associated in adherent contractile
cells. Am. J. Physiol. Cell Physiol. 282, C606616 (2002)
9. Lee, J., Leonard, M., Oliver, T., Ishihara, A., Jacobson, K.: Traction
force generated by locomoting keratocytes. J. Cell Biol. 127, 1957
1964 (1994)
10. Huang, J.Y., Peng, X.L., Qin, L., Zhu, T., Xiong, C.Y., Zhang, Y.Y.,
Fang, J.: Determination of cellular tractions on elastic substrate
based on an integral Boussinesq solution. J. Biomech. Eng.-Trans.
ASME. 131, 061009 (2009). doi:10.1115/1.3118767
11. Li, Y., Wen, C., Xie, H., Ye, A., Yin, Y.: Mechanical property analysis of stored red blood cell using optical tweezers. Colloids Surf.
B. 70, 169173 (2009)
12. Dimitriadis, E.K., Horkay, F., Maresca, J., Kachar, B., Chadwick,
R.S.: Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82, 2798
2810 (2002)
13. Zhang, M., Zheng, Y.P., Mak, A.F.T.: Estimating the effective
Youngs modulus of tissue from indentation testsnonlinear finite
element analysis of effects of friction and large deformation. Med.
Eng. Phys. 19, 512517 (1997)

123

570
14. Chen, S., Liu, L., Wang, T.: Nanoindentation of thin-film-substrate system: determination of film hardness and Youngs modulus.
Acta Mech. Sin. 20(4), 383392 (2004)
15. Wu, K.C., You, H.: Determination of solid material elastic modulus and surface energy based on JKR contact model. Appl. Surf.
Sci. 253, 85308537 (2007)
16. Timoshenko, S., Gere, J.M.: Mechanics of Materials. Van Nostrand
Reinhold, New York (1972)
17. Boudou, T., Ohayon, J., Arntz, Y., Finet, G., Picart, C., Tracqui, P.:
An extended modeling of the micropipette aspiration experiment

123

X. Peng et al.
for the characterization of the Youngs modulus and Poissons ratio
of adherent thin biological samples: Numerical and experimental
studies. J. Biomech. 39, 16771685 (2006)
18. Tracqui, P., Ohayon, J., Boudou, T.: Theoretical analysis of the
adaptive contractile behaviour of a single cardiomyocyte cultured
on elastic substrates with varying stiffness. J. Theor. Biol. 255,
92105 (2008)
19. Xiang, Y., LaVan, D.A.: Analysis of soft cantilevers as force transducers. Appl. Phys. Lett. 90, 133901 (2007)