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Research Paper

Nanomechanical measurements of polyethylene glycolhydrogels using atomic force microscopy

Zouheir Drira, Vamsi K. Yadavallin

Department of Chemical and Life Science Engineering, 601 W. Main Street, Virginia Commonwealth University, Richmond,

VA 23284, United States

a r t i c l e i n f o

Article history:

Received 19 March 2012

Received in revised form

21 September 2012

Accepted 23 September 2012

Available online 7 November 2012

Keywords:

Poly(ethylene glycol)

Hydrogel

Nanoindentation

Young’s modulus

nt matter & 2012 Elsevie.1016/j.jmbbm.2012.09.01

hor. Tel.: þ1 804 828 0587;vyadavalli@vcu.edu (V.K.

a b s t r a c t

Poly(ethylene glycol) (PEG)-based hydrogels are among the most widely used synthetic

polymers for biomedical applications. Critical parameters of importance for PEG hydrogels

are their mechanical properties which can be highly tuned. While properties such as elastic

moduli have been measured at the bulk scale, it is often important to measure them at the

micro and nanoscales. Further, non-destructive measurements of material properties can

enable in situ and high-throughput monitoring for applications including modulating cellular

interactions. In this research, the elastic modulus and the stiffness of polyethylene glycol

diacrylate (PEG-DA) hydrogel matrices at the nanoscale are determined via nanoindentation

using an atomic force microscope (AFM). The effect of varying parameters including monomer

molecular weight, initiator concentration and rates of hydration on the mechanical strength

of photopolymerized hydrogels were investigated. We present the effects of indentation

parameters including loads and indent depths on such measurements. Mechanical char-

acteristics of versatile PEG hydrogels can be adjusted based on polymer chain length and

crosslinking, while completely hydrated hydrogels have mechanical properties similar to

articular cartilage. A better understanding of these properties can enable tailoring hydrogel

based biomaterials for various applications in scaffolds and tissue engineering.

& 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Hydrogels are hydrophilic, crosslinked polymeric networks

capable of uptake of large quantities of water or biological

fluids (Peppas et al., 2000). As multifunctional materials,

they are used in a variety of applications including as

scaffolds for tissue engineering, vehicles for drug delivery,

contact lenses, cosmetic products and biosensors (Jeong

et al., 2002; Khademhosseini and Langer, 2007; Miyata et al.,

1999; Nicolson and Vogt, 2001; Ulijn et al., 2007; Van Tomme

et al., 2008). Poly(ethylene glycol) (PEG) is a water soluble

r Ltd. All rights reserved5

fax: þ1 804 828 3846.Yadavalli).

hydrogel that has wide ranging applications in biomedical

and biological areas due to its high biocompatibility, hydro-

philicity and non-toxicity (Iza et al., 1998; Jang et al., 2009;

Peyton et al., 2006). With a range of viscoelastic character-

istics, the ability to allow transfer of gases and nutrients and

ease of fabrication, these hydrogels are highly suitable for use

as constructs to engineer tissues as well as for cell trans-

plantation. When fully hydrated, the large water content in

PEG hydrogels gives them physical characteristics similar

to soft tissues (Ahearne et al., 2005). Partially hydrated

hydrogels have found applications as wound dressings to

.

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accelerate the healing process (Cai and Gupta, 2000; Yoshii

et al., 1999).

Hydrogels can be engineered to form three-dimensional

scaffolds composed of extracellular matrix molecules, which

provide structural support, adhesive sites and mechanical as

well as biomechanical signals to cells. With the control of

surface properties becoming significantly important in bio-

materials and tissue engineering, characterization of the

mechanical properties of materials at the micro and nanos-

cale is of outstanding interest. Recent work has shown that

cellular interactions are guided by the nanoscale architecture

of the surfaces on which they are tethered. The behavior and

lineage of cells is dictated by the stiffness and mechanical

nature of the surfaces (Discher et al., 2005; Levental et al.,

2007; Solon et al., 2007). Engineering the nanoscale topogra-

phy and mechanical properties of cellular scaffolds can be

used to elicit specific cellular responses, simulate tissue

environments and direct cell fate and behavior (Brandl

et al., 2007; Discher et al., 2005; Stevens and George, 2005;

Tsang and Bhatia, 2004).

While there have been a number of studies on the bulk

scale properties of hydrogel materials, it is vital to measure

properties at the same length scales that cells interact,

specifically at the nano and micro scales. Cell–cell and cell-

extracellular matrix focal adhesions are modulated by nanos-

cale topography and mechanical properties, typically only on

the order of tens of nanometers (Selhuber-Unkel et al., 2010).

To date, most mechanical measurements have been primarily

measured at the macro and microscopic level using tensile,

compressive and dynamic mechanical methods to capture

elastic and viscoelastic behaviors (Anseth et al., 1996; Lee

et al., 2009). On the other hand, current strategies to modify

cellular function and morphology involve the engineering of

surfaces by altering their mechanical properties at the

nanoscale. There is therefore a need to develop techniques

that extend experimentation from the macro to the

nanometer range. The recent ability to investigate small

loads on the order of nanonewtons and displacement of a

few nanometers has greatly encouraged the study of such

nanomechanical properties of materials (Li and Bhushan,

2002). In particular, the strategy of nanoindentation has

emerged as a valuable tool to measure and analyze the

material properties of different materials including tissues,

bone, cartilage, cell-membranes as well as metal composites

and polymers (Constantinides et al., 2008; Ebenstein and

Pruitt, 2006; Franke et al., 2007; Hengsberger et al., 2002;

Kim et al., 2002; Klapperich et al., 2001). However, despite this

utility, there have been limited reports on using nanoinden-

tation as a strategy to measure the nanomechanical proper-

ties of polymers, particularly hydrogels (Hu et al., 2012;

McConney et al., 2010).

In this manuscript, we present systematic nanoscale mea-

surements of mechanical properties such as the elastic

modulus (Ec) and the stiffness (S) of the widely used PEG

hydrogel. Specifically, we form these matrices via photopoly-

merization of polyethylene glycol diacrylate (PEG-DA).

A strategy of atomic force microscopy (AFM) based nanoin-

dentation is used to probe the effect of varying parameters

in the fabrication of PEG-DA hydrogels including monomer

molecular weight, initiator concentration and rates of hydration.

In addition to reporting such important nanoscale measure-

ments, we address some key challenges: Traditional nanoin-

dentation experiments are not well adapted for soft materials

(moduluso5 MPa), particularly without damaging the

samples or having large tip–surface adhesion forces (Ebenstein

and Pruitt, 2004, 2006). This has made measurements of

biologically relevant materials difficult. Secondly, current

experimental tools have consisted of applying a vertical

indent to a surface and estimating the mechanical properties

from the deformation caused by the indent. The samples are

therefore unusable following the measurement process. Here,

the AFM is used as a non-destructive indenter on PEG

hydrogel samples in both the dry and hydrated states. By

controlling the depth of the indent, the loading force and rate,

it is possible to investigate soft samples repeatedly and in a

potentially high-throughput and non-destructive fashion.

Understanding the mechanical properties can enable the

design of tissue engineering substrates where the elastic

moduli can be better tuned to modulate cellular responses

along desired routes.

2. Experimental section

2.1. Materials and methods

Poly(ethylene glycol) diacrylates (PEG-DA) with reported

molecular weights of 258, 575 and 700 Da were purchased

from Sigma-Aldrich Co. (St Louis, MO). Photoinitiator

Darocur 1173 (Hydroxy-2-methyl-1-phenyl-1-propanone)

was obtained from Ciba Specialty Chemicals Corporation

(Tarrytown, NY). Deionized water (resistivity 18 mO cm) was

obtained from a Milli Q water purification system (Millipore

Corporation, Danvers MA) and Ethanol (200 proof, absolute,

anhydrous, Shelbyville, KY) were used for experiments.

Photopolymerization of the PEG polymer was performed in

a UV chamber—wavelength 360 nm (Loctite Zeta 7401, Loctite

Corporation, Rocky Hill, CT). The Atomic Force Microscope

(AFM) (MFP-3D, Asylum Research, Santa Barbara, CA) was

used to obtain images and perform nanoindentation on the

PEG-DA hydrogel samples. To confirm tip morphology and

radius, scanning electron microscopy (SEM) images were

obtained before and after nanoindentation experiments

using a JEOL JSM-5610LV instrument (Tokyo Japan).

2.2. Fabrication of PEG-DA hydrogels

PEG-DA hydrogel samples were prepared by a two step

process as reported earlier (Revzin et al., 2001). 2 ml of the

polymer precursor was mixed with 10 ml of Darocur 1173

(0.5% photoinitiator) and vortexed for 20 s to obtain a well

mixed solution. The same procedure was repeated at 1.0%

and 1.5% initiator. Following UV radiation (1–5 s exposure)

and photopolymerization of the solution in mold, clear,

uniform, and rectangular PEG-DA samples were obtained

reproducibly in the form of hydrogel slabs of �1.5 mm

thickness. Hydrated hydrogel slabs were obtained by incuba-

tion in water to study the effect on the mechanical proper-

ties. Water content was estimated by weighing the samples in

the hydrated state and in a completely dry state following

Fig. 1 – (A) AFM scan of a 20 lm area of the PEG-DA hydrogel

sample formed by photopolymerization of the monomer

(inset). (B) Schematic of the indentation process and the

resulting force–displacement curve used to calculate

mechanical properties.

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drying in a convection oven. Clear homogeneous samples

without any cracking or discoloration were used for the

indentation experiments. PEG-DA hydrogels with the shortest

chains (MW 258 Da) tended to crack unless polymerized for a

short (o1 s) time. On the other hand, PEG-DA 575 and 700

hydrogel samples were easy to fabricate and formed uniform

and well shaped slabs even at longer exposures.

2.3. AFM-nanoindentation

In this work, the AFM ‘‘tip’’ refers to the part of the probe that

actually interacts with the surface. The tips are typically

pyramidal or spherical and the area of contact with the

surface is used in calculating the modulus. The AFM tip is

positioned at the end of a thin, flexible beam called the

‘‘cantilever’’. These cantilevers are typically triangular (V) or

rectangular (diving board or I-shaped). The tip and cantilever

comprise the AFM probe. The nanoindentation experiments

were conducted on PEG-DA samples using two different

probes: AC160 TS (Olympus Research (Tokyo, Japan)) and

PPP-ZEIHR (NanoSensors (Neuchatel, Switzerland)) with

nominal spring constants varying from 30–40 N/m and

15–27 N/m, respectively. These were used to image the topo-

graphy of the hydrogel surfaces, as well as for nanoindenta-

tion experiments. Experiments were performed on dry

hydrogel samples with three different molecular weights

(PEG-DA MW 258, 575 and 700) and different initiator

compositions. The widely used PEG-DA of MW 575 with 1%

initiator was used to estimate mechanical properties in the

hydrated state.

2.4. Spring constant calculation

Mechanical applications involving the AFM depend on the

accurate knowledge of the properties of the AFM probe. Spring

constant values (k) are typically provided by the manufac-

turer, but it is important to measure them prior to every

nanoindentation experiment to quantify the forces measured

(Butt et al., 2005). To minimize the effect of tip–surface

adhesion, (AC160 TS, nominal k¼30–40 N/m and PPP-ZEIHR,

nominal k¼10–60 N/m), spring constants were measured on a

hard and clean mica surface using the thermal fluctuation

method. The same procedure was repeated 6 times on

different points of a mica surface to obtain the average

calculated spring constant as the value used for analysis. It

is important to note that most methods used to calibrate the

AFM probe may have an associated error that can propagate

in calculation of the mechanical properties of the material of

interest (Butt et al., 2005).

2.5. Imaging and nanoindentation experiments

Hydrogel surfaces were imaged in non-contact mode in order

to measure the topography of the soft biomaterials and

polymers with minimal damage to both the sample and the

tip (Yang et al., 2007). During a typical nanoindentation test,

force and displacement are recorded as a three-dimensional

indenter tip is pressed into the surface with a prescribed

loading and unloading profile (Fig. 1B). The response of

interest is where the force F and penetration depth h are

varied and measured by analyzing the loading—unloading

curves generated (Haque, 2003). The primary difference

between the nanoindentation used in this work and to that

reported earlier is that the polymer sample is not damaged

during the indentation procedure. Different loads and dis-

placements were investigated, taking care that the surface

was not damaged during the experiments, as verified by AFM

imaging.

3. Results and discussion

Poly(ethylene glycol) (PEG) is a widely used hydrogel in

biomedical engineering both as coatings and as tissue

engineering scaffolds (Harris, 1992). The diacrylate form of

PEG (PEG-DA) is obtained by substituting the hydroxyl end

groups with acrylate functionalities (Fig. 1A). This enables

crosslinking the polymer efficiently via photopolymerization

(Hennink and van Nostrum, 2002; Hu et al., 2009). Photopoly-

merization allows for a high degree of spatial and temporal

control, fast curing rates and importantly, polymerization

in situ from aqueous precursors in a minimally invasive

manner (Nguyen and West, 2002). The formation of cross-

linked PEG hydrogel networks is based on the UV-initiated

free-radical polymerization of the pendant acrylate end group

of PEG monomers. When exposed to UV light in the presence

of a photoinitiator, acrylate groups form reactive free radical

sites resulting in the formation of highly crosslinked net-

works. This allows the creation of hydrogel matrices of

varying geometries and shapes at various length scales.

The AFM was used earlier to study the mechanical properties

of photo-cross-linked, temperature-responsive hydrogel layers

in water (Harmon et al., 2003). It was demonstrated that the

elastic modulus differs as a function of the polymer volume

fraction, in addition to the effects of the cross-linking, density

and degree of ionization. The physical properties of the hydro-

gels thus formed can be controlled by varying the initiator

concentration, the molecular weight of the monomer and its

Fig. 2 – Overlay of several different indentation curves taken

on dry PEG 575 hydrogels at high and low loads.

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concentration. For instance, the initiator concentration can affect

the degree of crosslinking of the polymeric chains and thereby

alter its physical and chemical properties. The ability of these

highly tunable hydrogels to provide a wide range of applications

is due to specific mechanical properties and responsive behavior

to external factors and environmental conditions. In the experi-

ments described, we probe the effect of these parameters by

varying parameters such as loads applied and indent depths for

different hydrogel samples. Different AFM probes are used to

study the effect of varying loading rates on the mechanical

properties—modulus of elasticity, Ec and the stiffness, S. Impor-

tantly, for the first time, the AFM is used as a non-destructive

indenter to determine the mechanical properties of the PEG-DA

hydrogels in a variety of states, including hydrated states.

3.1. AFM imaging and nanoindentation

The Young’s modulus, also known as the tensile modulus or

modulus of elasticity, is a measure of the ratio of the uniaxial

stress to the uniaxial strain of a material. The elastic

modulus can be experimentally determined from the slope

of linear fits to the stress–strain curves (Harmon et al., 2003).

The stiffness, defined as the measure of the resistance of an

elastic body to the deformation under an applied load, is

calculated by measuring the slope of the upper portion of the

unloading curve (Liu et al., 2009). Both these parameters are

important in the measurement, analysis and characterization

of the mechanical properties of these polymers. In particular,

the measurement of stiffness allows continuous measure-

ment of properties as a function of depth and also facilitates

a more accurate identification of the point of contact of the

tip with the surface (Oliver and Pharr, 2004).

AFM imaging of the samples revealed uniform, flat surfaces

with a roughness of approximately 74 nm over a large area of

20–50 mm (Fig. 1A). The nanoindentation experiments con-

sisted of obtaining force–displacement curves by indenting

the surface of the PEG-DA hydrogel slabs. Following the

imaging of the surface, 25 different points were selected on

the surface and the cantilever was moved to each point to

obtain measurements under four different indent modes

(high and low load, high and low displacement). Under high

and low load (150 nN and 20 nN, respectively), the force

pushing the tip into the surface thereby deforming the

material is controlled. The corresponding depth or displace-

ment is a function of the material being indented. Under high

and low displacement indent modes, the depth is controlled,

and the tip travels a fixed depth into the surface (100 nm or

10 nm, respectively). Care was taken to observe that the

surface was not damaged post-indentation.

Nanoindentation can discriminate between similar, low-

modulus, hydrated samples (Kaufman et al., 2008). The most

well-known and applied theory to describe the elastic defor-

mation and analyze the mechanical properties of materials

via nanoindentation is the Hertz model. An important

approximation in this model is the absence of adhesion or

surface forces. Therefore, while using AFM, the Hertz model

applies under high loads and low surface forces (Butt et al.,

2005; Cappella and Dietler, 1999). A better approximation for

low-modulus (softer) materials is obtained using the

Oliver–Pharr model (Pharr and Oliver, 1992). Here it is

assumed that during the unloading from the surface, only

the elastic displacement is recovered (Oliver and Pharr, 2004).

Details on the model and its use in fitting nanoindentation

curves have been covered in excellent papers on the subject

and the equations have been skipped in this work (Lin and

Horkay, 2008; Oliver and Pharr, 2004).

Identical procedures were repeated with the four indent

modes for PEG-DA hydrogels with MW of 258, 575 and 700.

The high load indent mode resulted in the highest signal to

noise ratio and was commonly used. At lower load indents, the

tip interaction with the PEG-DA surface increases (the force

applied is around 50 nN and depth traveled is r100 nm)

whereby softer cantilevers are not well suited to perform

indentation tests. Fig. 2 shows an example of data collected

in the overlay of several different indentation curves. At high

load and displacement modes, the curves have a high signal to

noise ratio and were easier to fit via the Oliver–Pharr model to

determine the elastic modulus and the stiffness. On the other

hand, at a low load and especially for the low displacement

modes, the force curves reveal expected higher noise levels and

significant tip–surface adhesion resulting in difficulty in reliably

fitting data.

3.2. Variation of molecular weight

Polyethylene glycol hydrogels with three different monomer

weights of 258, 575 and 700 Da were initially studied. These

correspond to commonly used PEGs for fabricating scaffolds

for tissue engineering (Drury and Mooney, 2003). These

hydrogels were initially investigated in the dry condition.

Fig. 3 shows the elastic modulus and stiffness of the samples

as a function of the molecular weight. This shows that the

mechanical properties of the polymers increase with the

molecular weight although they tend to level off at higher

molecular weights. This is consistent with measurements

made of pure hydrogel samples at the bulk level, wherein

the longer chains also have reduced crosslinking densities

resulting in a leveling off of elastic moduli (Iza et al., 1998).

PEG-DA 575, which is more typically used in biomedical

applications, was subsequently used for analysis in which

the initiator composition and water content were varied.

3.3. Variation of the initiator composition

Three different hydrogel samples were fabricated with vary-

ing amounts of initiator, and then tested. The objective was

Fig. 3 – Variation in the elastic modulus and stiffness of

PEG-DA as a function of molecular weight. All measurements

of the pure hydrogels were taken in a dry condition.

Fig. 4 – Effect of changing the amount of photoinitiator on

the elastic moduli of PEG-DA 575.

Fig. 5 – Elastic modulus and stiffness of PEG-DA 575 with

differing water content.

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to determine the effect of the initiator in determining the

final crosslinking of the diacrylate monomer. Hydrogel sam-

ples were tested using 0.5%, 1% and 1.5% initiator v/v. Fig. 4

shows the elastic modulus and stiffness average values

obtained after fitting the unloading part of the curves with

the Oliver–Pharr model. The elastic modulus and the stiffness

of the hydrogel matrices increased from 0.5% initiator to

1.5%. This reflects that the initiator concentration affects the

crosslinking degree of each sample with a higher initiator

resulting in a stiffer polymer. The 1% initiator concentration

was adopted for subsequently investigating the hydrogels in

varying hydration states.

3.4. Variation of hydration condition of the hydrogels

PEG hydrogels have been used in varying states of hydration

for different applications—from dry coatings to partially

hydrated sheets as wound dressings (Cai and Gupta, 2000)

to fully hydrated constructs in tissue engineering

(Khademhosseini and Langer, 2007). Performing nanoinden-

tation experiments on hydrated hydrogel surfaces is of

great interest in enabling the design of suitable cellular

matrices with controlled mechanical properties. Fig. 5 shows

the change in mechanical properties as a function of hydra-

tion. Initially, partially hydrated PEG-DA 575 samples were

investigated with 63% and 86% water content, respectively.

Wet samples were assessed on the basis of the final water

content of the material. Following photopolymerization, each

sample was washed with water for 8–10 min and dried. Wet

hydrogel samples were prepared by rehydrating the sample

in water over 24 h and allowing them to swell to an equili-

brated state. The size and weight of the hydrogel increase

confirming the swelling of the sample. Once the sample was

removed from the water and weighed, the surface was blown

in a gentle stream of air prior to nanoindentation experi-

ments. This step was essential to prevent hydrostatic attrac-

tion between the tip and surface. The humidity in the AFM

chamber is controlled to keep the moisture level constant.

After the nanoindentation, the sample was weighed again

and the percent of water content in the hydrogel

was calculated. Under high load indent, the PEG-DA 575

sample with 63% water content had an elastic modulus

4.3370.28 MPa and a stiffness of 2.4870.27 N/m. Since the

values for elastic modulus and stiffness were close to that of

the dry PEG-DA 575 (1% initiator), it is likely that the

indentation was performed on a dry surface. This was further

observed with hydrogels at a lower water content which

indicated that while the bulk water content is �63%, a dry

layer of the hyrogel sample several hundred nanometers

deep changes the surface properties of such samples. How-

ever, with 86% water, the PEG-DA 575 elastic modulus

dropped to 2.8570.35 MPa, while the stiffness increased to

4.3270.12 N/m, which shows that the water has an important

effect in enabling such hydrogel materials to approach

properties of tissue.

Since a fully hydrated hydrogel surrounded by a liquid

environment (such as serum or water) mimics applications

involving cellular scaffolds, it is important to measure the

nanomechanical properties of the hydrogel in a fully

hydrated state. To achieve this, the hydrogel with a water

content close to 100% was indented directly under water. This

experiment is particularly challenging given the limited

control of the experimental parameters under water. It must

be noted that indenting soft polymeric samples can result in

a high degree of adhesion to the surface, rendering errors in

recording nanomechanical measurements. Consequently,

data was obtained only for high displacement and high

loading for this condition using a stiff cantilever.

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Complex viscoelastic–elastic responses induced during the

indentation of soft polymers result in several challenges

typical for polymers having strain and strain-rate dependent

properties. The response of these polymeric materials affects

the measurement of the mechanical properties such as

elastic modulus and hardness which are a function of the

surface contact-indenter geometry, depth as well as the

loading rate (Briscoe et al., 1998). Various theoretical and

empirical models have been developed to analyze the

force–displacement curves generated to account for some of

these parameters. The mechanical properties of the hydrogel

increase as the amount of the initiator increase in the

precursor. The degree of hydration dramatically affects the

mechanical behavior of the PEG-DA. The presence of water

within the hydrogel network weakens the internal as well the

external mechanical properties, leading to smaller values of

elastic modulus and stiffness compared with dry polymers.

3.5. Effect of cantilever stiffness

Experiments were conducted using a stiff cantilever (AC 160

with k �40 N/m). Therefore, it was essential to use another

cantilever is important for comparison. Nanoindentation

experiments were therefore conducted using a softer canti-

lever (PPP-ZEIHR with a nominal force constant �20 N/m)

keeping the loading rate the same. Each cantilever was

calibrated to obtain the precise values of the spring constants.

Using a softer cantilever, the elastic modulus values of the

PEG-DA (1.8370.03 MPa) as well as the stiffness (1.1870.01 N/m)

were comparable to the stiffer cantilever—1.4670.07 MPa for

the elastic modulus and 0.8270.04 N/m for the stiffness.

These numbers are well within the range of the experimental

uncertainty that may be expected from even measurements

of the spring constant (see below), thereby confirming the

results. It must be noted that the loading rates in both

experiments were the same. However it is important to note

that in the case of the soft cantilever, a high degree of

tip–surface sticking is observed particularly in the low load

and low displacement modes, often precluding measure-

ment. For example, the use of the PPP-ZEIHR cantilever under

water was not possible since the force interaction and the

thermal fluctuation within the liquid was significant. On

the other hand, this cantilever was useful for experiments

in the dry condition and under high displacement indent

mode. The choice of suitable cantilever is therefore extremely

important in accurate measurement of such properties,

particularly for soft polymeric samples.

3.6. Discussion on elastic modulus and stiffnessmeasurements

The accuracy of hardness and modulus measurement

depends inherently on how well parameters including the

maximum load, the maximum displacement, and the elastic

unloading stiffness can be measured experimentally (Oliver

and Pharr, 2004). At higher molecular weight, the PEG-DA

hydrogel showed an increase in elastic modulus. Under high

load the PEG-DA 575 shows higher elastic modulus as well as

under low load indent mode. The standard deviation was a

marginally higher for the high load. The variation in the

values of the modulus at different points on the hydrogel

surface was low indicating that the surface was uniform. The

PEG-DA 700 elastic modulus did not show a difference in

either mode. The PEG-DA 258 hydrogel has the lowest elastic

modulus with only 1.33 and 1.58 MPa under high and low

modes, respectively. Here, the molecular weight difference is

observed to affect the performance of the hydrogel. This is

reasonable, because in the case of PEG-DA 258 for example,

by applying a load of 150 nN, the tip traveled �60 nm more

than for a 20 nN force. The same situation was observed for

the stiffness values. PEG-DA 575 and 700 hydrogels had a

higher stiffness value in comparison to PEG-DA 258, which

may represent a weaker material.

The load applied has an impact on the response of the

polymer. Under higher loads, the PEG-DA 700 hydrogel has a

higher stiffness value. When a load of 200 nN is applied, the

tip travels �300 nm into the surface. On the other hand, at a

low load of 20 nN, the tip travels �200 nm into the surface.

This implies that a tenfold increase of load does not result in

a proportional increase in penetration. Controlling the

displacement while indenting on the PEG-DA surfaces, can

be more challenging than controlling the load, especially at

low displacements. An identical trend of the elastic modulus

to molecular weight relation was observed. The higher mole-

cular weight corresponds to a higher elastic modulus at both

high load and displacements. However, the low displacement

measurements of the elastic modulus were observed to have

extremely high noise levels. This was expected because the

penetration into the sample was only �10 nm.

By increasing the amount of the crosslinking agent, the

elastic modulus as well as stiffness increased in value. It was

observed that the higher the molecular weight of the hydro-

gel, the higher is the value of the elastic modulus. These

experimental and analysis parts were studied under high

load and displacement indent modes. At lower displacement

indent mode (�10 nm) and load indents, the tip–surface

interaction forces were predominant, which made analysis

difficult. Stiffer cantilevers with a higher spring constant

were found to work better to obtain force–displacement

curves that could be easily analyzed. The PEG-DA hydrogels,

as soft materials required the Oliver–Pharr model as best

suited for our experiments.

3.7. SEM imaging of the AFM probes

Since one of the key parameters in determining the accuracy

of fit is the tip geometry, the morphology was examined

using scanning electron microscopy (SEM) prior to and after

the indentation experiments. This was to determine the

effect of the indentation on the geometry of the tip as a

result of these experiments. Fig. 6 shows a clean tip with no

surface impurities or imperfections, with sharp edges and a

tetrahedral geometry. Following the nanoindentation experi-

ments, another set of SEM images were used to determine if

the experiments resulted in any tip damage. Most typically,

the cantilevers showed no significant change in morphology

after nanoindentation, confirming the suitability of the para-

meters adopted. In contrast, Fig. 6A shows an AFM image of a

hydrogel surface that was intentionally indented (red spots)

using a force on the order of micronewtons (�1–5 mN).

Fig. 6 – (A) AFM images of a PEG-DA surface before and after

a nanoindentation experiment to damage the surface.

The figure on the right shows distinct areas of damage

(red circles) where the nanoindentation was applied. (B) Tip

contamination as visualized by SEM. (For interpretation of

the references to color in this figure legend, the reader is

referred to the web version of this article.)

j o u r n a l o f t h e m e c h a n i c a l b e h a v i o r o f b i o m e d i c a l m a t e r i a l s 1 8 ( 2 0 1 3 ) 2 0 – 2 826

Following such destructive indentation, the SEM showed that

the tip picked up some of the hydrogel debris. In Fig. 6B, the

probe appears to have lost its sharpness resulting in a slightly

curved edge. From SEM images, it was observed that

contamination as well as a change in geometry of the contact

area may occur as a result of such destructive indentation

experiments. In addition to confirming that the tip geometry

must be accurately quantified prior to analysis of the

force–displacement curves measured, this also showed that

the non-destructive indentation via AFM is the best method

to preserve tip integrity and accurately determining the

mechanical properties of such polymeric samples.

3.8. Hydrogel mechanical properties in comparison toother materials of biological significance

The elastic modulus is one of the most common properties

used to characterize the mechanical behavior and describe

the material. The widely used PEG-DA 575 hydrogel was

chosen to be compared with other materials. The elastic

modulus of the pure hydrogel in the dry state varied between

2 and 5 MPa under different conditions. This corresponds well

with earlier reported moduli reported by Gabler et al. within

the range of 3.5 MPa (Stefan Gabler et al., 2009). The depen-

dence on the degree of crosslinking is similar to that

observed in polyhydroxyl ethyl methacrylate (poly-HEMA)

hydrogels where the elastic modulus increased from

0.5670.06 MPa (0.5% initiator) to 2.670.51 MPa (8% initiator)

(Wilder et al., 2006). The modulus in a fully hydrated condi-

tion was �1 MPa, which also compared well with poly-HEMA

copolymer gels (Oishi et al., 2004). In a review of elastic

moduli of several soft biological materials by Levental et al.

(2007) the elastic modulus measurements of a variety of

tissues such as animal and human tissue were reported.

For example, using a tensile method, the elastic modulus of

an Achilles’ tendon of a rat was around 310 MPa, which is

much higher than these hydrogels. Conversely, the modulus

for bovine articular cartilage as measured by compression is

950 kPa, which approaches the value of the fully hydrated

PEG-DA hydrogel. However, typical values for soft mammalian

tissues ranged from �260 to 490 Pa for brain tissue to several

thousand Pa for muscle tissue (Levental et al., 2007). These

much lower values show that pure PEG-DA is probably not

suitable for applications to mimic the natural mechanical

properties of such tissue samples and softer biomaterials are

needed. In order to apply these hydrogels for tissue engineer-

ing and scaffolding, it is necessary to control the mechanical

properties hydrogel matrix. We show that by varying para-

meters such as molecular weight, initiator concentrations and

hydration rates, it is possible to control properties such as the

elastic modulus at the nanoscale. This is particularly signifi-

cant, to achieve properties similar to biological tissues and

mimic the mechanical behavior of real tissues in vivo, thereby

controlling cell behavior and fate.

3.9. Sources of error in nanoindentation measurements

It must be noted that the values reported in this manuscript

as with virtually all nanoindentation experiments are subject

to a lot of variation and sources of error. These errors can

propagate from various sources including:

1.

Lack of accuracy in measuring the spring constant and tip

geometry: Nanoindentation experiments require accurate

knowledge of probe parameters in order to accurately

measure both force–distance and indentation curves.

Due to the challenges faced while controlling the thick-

ness, the structural defects and deviations in geometry,

the manufacturers of AFM probes use wide tolerances in

their specified values of the spring constant, sometimes

up to 50% of the calibration values (Levy and Maaloum,

2002). These variations may result in wide ranges for the

forces and therefore the elastic moduli and stiffness

values calculated. Therefore, re-calibration is mandatory

before usage. There are several methods designed to

measure the spring constant, including using the actual

geometry of the tip (Butt et al., 2005). However, these

include approximations owing to the difficulty of obtain-

ing exact dimensions of the indenter. Thermal fluctuation

is another method used in this research, which consists of

calibrating the cantilever with thermal noise which can be

affected by factors including the laser intensity and spot

position on the cantilever, in addition to its size (Butt et al.,

2005). That is why it is important to repeat the spring

constant calibration several times to minimize the

experimental error.

Similarly, the geometry of the indenter plays a big role in

measuring the mechanical properties of the material of

interest. A change in the tip radius of curvature, for

instance, can significantly affect the experimental results

(Calabri et al., 2008). It can also affect the indentation and

penetration process as well as the analysis since the tip

can deform the sample with different geometry. In the case

of the AC160 tip used in the nanoindentation experiments,

a tetrahedral geometry was assumed as discussed. Our

j o u r n a l o f t h e m e c h a n i c a l b e h a v i o r o f b i o m e d i c a l m a t e r i a l s 1 8 ( 2 0 1 3 ) 2 0 – 2 8 27

calculations implicitly assume that the tip geometry is not

altered as a result of the indentation experiments. In

several cases, the tip can lose its initial sharpness as

expected, becoming a little blunt or contaminated with

the sample (hydrogel) (McConney et al., 2010). These factors

may result in a further loss of accuracy in experiments.

2.

Tip surface interaction: Tip surface interactions and espe-

cially the non-specific interactions provide another impor-

tant source of error. At the nanoscale, the Van der Waals

forces become more significant, resulting in adhesion

between the tip and the surface investigated. In investigating

partially hydrated samples, this can be significant particu-

larly at lower displacements and loads. This is manifested in

the retraction trace of the force–displacement curve. Even

though the tip–surface interactions were significant in some

cases, the tip did not stick to the sample during the

nanoindentation step, resulting in reasonable curves that

were well fit with the Oliver–Pharr model, especially at high

load and displacement. As has been reported earlier, this can

also cause surface detection errors that can cause an error in

the estimation of modulus (Kaufman and Klapperich, 2009).

However, to reduce any possible errors, it is important

to investigate strategies to try and minimize non-specific

adhesion between the tip and the surface including chemical

modification of the tip material.

3.

Inherent errors in the models assumed: Finally, an impor-

tant cause of error stems from the weaknesses of the

associated models themselves. Despite the fact that it is

best used for rigid materials, the Hertz model was initially

used to fit the nanoindentation curves. However, the

unsuitability of the model for softer hydrogels is com-

pounded by challenges in correctly estimating the geome-

try of the indenter. Primarily, choices of geometries and

half-angles for optimal fit rendered this model as unsui-

table. The Oliver–Pharr model, which is primarily suited

for softer materials was used as the primary model to fit

the nanoindentation curves of the PEG-DA hydrogel in

these experiments (Oliver and Pharr, 2004; Pharr and

Oliver, 1992).

4. Conclusions

Calculating and controlling the mechanical properties of

hydrogel polymers is of great interest particularly for a

variety of biomedical applications. While the mechanical

behavior of hydrogels has been estimated at the bulk scale,

there have been limited studies at the micro and nanoscales.

In this research, the mechanical properties of PEG-DA hydro-

gels at the nanoscale were reported by measuring the elastic

modulus as well as stiffness via nanoindentation using an

AFM. This strategy also provides the capability to separate

the mechanical behavior of different material constituents,

using depth sensing to detect phase transformation and

investigate the plasticization of polymers (Klapperich et al.,

2001). The mechanical behavior of PEG-DA hydrogels was

observed to depend on many parameters including the water

content, monomer molecular weight and the photoinitiator

concentration, with the hydrated polymer having mechanical

properties similar to articular cartilage. Further studies are

needed to elucidate the effects of determinants including the

indenter structural shape and physical properties in the

characterization of soft polymeric materials of interest. Better

models and methods are needed to accurately determine the

mechanical properties of soft material via nanoindentation

experiments, particularly in hydrated systems. These include

developments in studying and confronting the challenges

faced with indenter properties and geometry as well as

minimizing the error obtained from the non-specific force

interaction, in addition to determine the indentation material

parameters for contact modeling, stress/strain analysis and

load bearing.

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