journal of the mechanical behavior of biomedical materials 134 (2022) 105384

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Journal of the Mechanical Behavior of Biomedical Materials

journal homepage: www.elsevier.com/locate/jmbbm

A methodological framework for nanomechanical characterization of soft

biomaterials and polymers
Sofia E. Arevalo a, *, Donna M. Ebenstein b, Lisa A. Pruitt a
a
Department of Mechanical Engineering, University of California, Berkeley, CA, USA
b
Department of Biomedical Engineering, Bucknell University, Lewisburg, PA, USA

A B S T R A C T

Nanoindentation utilizes a hard indenter probe to deform the sample surface in order to measure local properties, such as indentation modulus and hardness. Initially
intended for characterization of elastic and elastic-plastic materials, nanoindentation has more recently been utilized for viscoelastic solids as well as hydrated and
soft biological materials. An advantage to nanoindentation is the ability to determine the nano- and microscale properties of materials with complex microstructures
as well as those of limited sample dimension. Nanoindentation finds utility in the characterization of structural tissues, hydrogels, polymers and composites.
Nevertheless, testing complexities such as adhesion and surface detection exist in nanoindentation of compliant viscoelastic solids and hydrated materials. These
challenges require appropriate modifications in methodology and use of appropriate contact models to analyze nanoindentation data. A full discussion of protocol
adjustments has yet to be assembled into a robust nanoindentation testing framework of soft biomaterials and polymers. We utilize existing nanoindentation
literature and testing expertise in our laboratories to (1) address challenges and potential errors when performing indentations on soft or hydrated materials, (2)
explore best practices for mitigating experimental error, and (3) develop a nanoindentation framework that serves researchers as a primer for nanoindentation testing
of soft/hydrated biomaterials and polymers.

1. Introduction The lack of standardized guidelines for performing nanoindentation

on soft, hydrated materials is an ongoing roadblock to validating
Nanoindentation techniques were developed primarily for linearly nanoindentation and poses a risk of reporting erroneous nano­
elastic, isotropic and homogenous materials; yet, there is ongoing in­ mechanical measurements. Standardized procedures for characterizing
terest in utilizing this tool for the characterization of soft, hydrated the nanomechanical properties of metallic and stiff materials (i.e.,
materials with hierarchical microstructures. Nanoindentation probes MEMS, semiconductor components and protective coatings) exist
materials at the nano- and micro-length-scales, proving advantageous (ASTM E 2546 and ISO 14577); yet, these lack experimental modifica­
for mechanical characterization of samples with limited dimensions or tions and appropriate models for collecting and analyzing nano­
complex microstructures. An advantage of nanoindentation is its ability indentation data for soft and hydrated biomaterials (Fischer-Cripps,
to map the local mechanical behavior of heterogeneous materials (Oyen 2006). Variability in testing protocols may arise from the presence of
et al., 2012). Nanoindentation is particularly appealing as a mechanical adhesion, difficulty in detecting surfaces, fluid-probe interactions, or
characterization tool of biological tissues with heterogenous micro­ viscoelastic behaviors (Farine, 2013). Further, a lack of standardization
structures, irregular dimensions and compliant anisotropic behaviors leads researchers to use inappropriate data collection and analysis, or
(Carrillo et al., 2005). Soft, hydrated biomaterials such as hydrogels each to develop their own protocol, so it is difficult to compare results
(Galli et al., 2009; Kaufman et al., 2008; Kohn and Ebenstein, 2013) and between studies in different groups or know how the indentation
cartilage (Franke et al., 2011; Simha et al., 2007) have employed modulus should compare to materials properties (such as modulus)
nanoindentation methods to ascertain local mechanical properties such measured thorugh bulk testing methods like tension and compression. In
as modulus and hardness. sum, standard procedures for characterizing soft, hydrated and hetero­
As research in the fields of biomaterials, tissues and nature-inspired geneous systems are needed. In the interim, we review the literature to
materials increases, there is ongoing demand for nanoscale character­ offer a methodological framework for nanomechanical characterization
ization techniques that can elucidate structure-property relationships in of soft, hydrated biomaterials and polymers.
these complex systems (Ebenstein and Pruitt, 2004). A limited number of review papers address the complications

* Corresponding author. Department of Mechanical Engineering, University of California, 2121, Etcheverry Hall, Berkeley, CA, 94720-1740, United States.
E-mail address: [email protected] (S.E. Arevalo).

https://doi.org/10.1016/j.jmbbm.2022.105384
Received 25 March 2022; Received in revised form 8 July 2022; Accepted 15 July 2022
Available online 1 August 2022
1751-6161/© 2022 Published by Elsevier Ltd.
S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

associated with testing soft, hydrated or viscoelastic biomaterials As the use of nanoindentation expands to other fields, there is a
(Table 1). In the seminal nanoindentation review paper on nano­ pressing need to provide an educational paper that is accessible to those
indentation in biomaterials, Ebenstein and Pruitt emphasize the sus­ not fully versed in the field of nanomechanical characterization. This
ceptibility of adhesive interaction between the probe and sample work aims to serve as a comprehensive guide for edifying novices to
(Ebenstein and Pruitt, 2006). A practical guide for analyzing nano­ nanoindentation on proper testing practices, thereby minimizing
indentation data with an emphasis on biological materials was later experimental errors and ensuring efficacy in measurements. Through
published by Oyen and Cook (2009) and serves as an introduction to this review, we develop a framework that encompasses a broad range of
various deformation modes found during indentation - elastic, plastic, nanoindentation users who seek to employ nanoindentation in diverse
viscous and fracture; however, it does not focus on the intricacy of research fields. We aim to encourage accessibility while ensuring effi­
studying the nanomechanical properties of soft biomaterials. Oyen later cacy and reliability in the nanomechanical measurements. As such, the
highlights the future of nanoindentation in her review of nano­ framework provides guidance on all aspects of the nanoindentation
indentation of hydrated materials and tissues (Oyen, 2015). Oyen rec­ experiment, including probe selection, experimental set-up, and data
ognizes that soft-biological materials are prone to adhesion effects and analysis methods, highlighting common data collection challenges and
may have different failure modalities that pose experimental challenges how to address them.
where analysis of data needs further elaboration for obtaining reliable
results. More recently, Chang and co-workers explore the potential er­ 2. Nanoindentation: materials research and other small-scale
rors and parameters that may influence nanomechanical measurements testing techniques
and provide a brief discussion on the contact mechanics within the ap­
plications of nanoindentation (Chang et al., 2016). These review articles 2.1. Nanoindentation research development from 1988 to present
serve as a foundation to introduce the complex multi-faceted approach
of performing a nanomechanical analysis of biological materials. How­ Since its inception in the 1980s, nanoindentation research has
ever, they provide few practical pointers on how to select the best expanded from thin films to ceramics, alloys, polymers and biomaterials.
methods for data collection and analysis when studying a new material. We performed a systematic literature review focused on nano­
indentation research for the following materials – hydrogels, soft poly­
mers, soft biological materials, soft cartilage and thin-films. Each result
Table 1 was carefully reviewed to ensure that depth sensing nanoindentation
Review papers on static nanoindentation of soft materials and biological mate­ instrumentation was used in the research. As illustrated in Fig. 1, in the
rials (hydrated and non-hydrated) spanning from early 2003 to 2020. 1990s, nanoindentation emerged as a tool for measuring and mapping
Reference Material Aims the mechanical properties of thin films, as at the time it was the only
standardized technique that measured properties of small volumes of
Perepelkin et al., Soft Materials • Discussion of various depth sensing
2020 (Perepelkin indentation approaches stiff materials.
et al., 2020) • Exploration of factors contributing Nanoindentation was primarily designed for testing elastic and
to erroneous measurements elastic-plastic materials and this trend is reflected in the extensive
• Provide an overview of methods
number of early research materials (Fig. 1). An uptick of nano­
that account for adhesion
Qian et al., 2018 ( Soft Biological • Discussion of constitutive behavior
indentation research for other materials ensued in the late 1990’s with
Qian and Zhao, Materials of soft biomaterials the advancement of nanoindentation analytical models for analyzing
2018) • Discussion of experimental load-displacement curves, namely after Oliver and Pharr published their
methodology and challenges from seminal paper on nanoindentation (Oliver and Pharr, 1992a). Further,
nanoindentation of soft
an increase in nanoindentation research may also be a consequence of
biomaterials
• Exploration of the applications of the technology becoming more affordable and accessible and techno­
nanoindentation logical advancements leading to improved capability of capturing local
Chang et al., 2016 ( Biological • Discussion of other small-length
Chang et al., 2016) Materials scale testing techniques
• Examination of the potential errors
and parameters that may influence
nano-mechanical measurements
• Provide an overview of contact
mechanisms
• Provide a guideline for
experimental settings
Oyen, 2015 (Oyen, Hydrated • Discussion of the challenges
2015) Biological tissues associated with handling and
and hydrogels testing hydrated materials
• Discussion on the models used to
extract mechanical properties
• Provide a review of key results
from recent nanoindentation
experiments
• Discussion on the outlook for the
future of nanoindentation
Ebenstein et al., 2006 Biological • Provide a review of the research
(Ebenstein and materials impact of nanoindentation
Pruitt, 2006) • Discussion of the mechanics of
nanoindentation
• Discussion of probe selection,
adhesion, surface preparation and
hydration
Haque 2003 (Haque, Biological • Exploration of the applications of Fig. 1. Progression of nanoindentation research in the field of thin-films (yel­
2003a) materials, nanoindentation low), alloys (green), ceramics (green-blue), polymers (blue), and biomaterials
biomaterials • Discussion of the materials already
(dark-blue). The materials encompassed in the biomaterials category include
explored by nanoindentation
hydrogels and biological materials, for example cartilage and tissues.

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

nano-length scale mechanical properties. characterization of articular cartilage (Korhonen et al., 2002). Buffinton
The proportion of publications each year for nanoindentation of et al. compared the mechanical behavior of polyacrylamide gels
biomaterials is small compared to thin films and alloys (Fig. 1). Nano­ measured using pipette aspiration and bulk uniaxial compression to
indentation research for hydrogels, soft biological and soft cartilage nanoindentation. In the same study, the researchers compared the
materials is fairly recent; the earliest publications appeared in the early properties of silicone elastomers (<1 MPa) measured using nano­
2000s but research in this realm is expanding (Fig. 1). Similarly, an indentation to properties measured via pipette aspiration, compression,
increasing trend is observed for nanoindentation research of soft poly­ and tension (Buffinton et al., 2015a). Through comparison to bulk
mers. The abundance of literature that exists for non-biomaterial sys­ compression and tension results, Buffinton and co-workers concluded
tems is likely owed to the established experimental testing protocols that that pipette aspiration and nanoindentation were both suitable me­
promote confidence in and reliability of data. Since biomaterials are a chanical characterization techniques for soft biomaterials, with the
newly developing field for nanoindentation, there is a need to address additional benefit, relative to bulk testing, of allowing localized testing
the challenges and errors in order to advance nanoindentation research in heterogeneous or irregularly shaped samples (Buffinton et al., 2015a).
of soft, hydrated biomaterials and polymers. Numerous review publi­ Comparisons of nanoindentation and atomic force microscopy
cations highlight the utility of nanoindentation as a method that can (AFM) are more prevalent in the biomaterials literature, since both have
assist in the physical understanding of materials and the local properties been used extensively to indent soft materials. For this reason, we
of surfaces, interfaces and highly hierarchical structures (Ebenstein and compare AFM with nanoindentation (Table 2). AFM utilizes a nanoscale-
Pruitt, 2006; Oyen, 2015; Qian and Zhao, 2018; Haque, 2003b; Oral sized pyramidal probe attached to a small cantilever; as the cantilever
et al., 2004), so nanoindentation is likely to be applied to diverse ma­ bends, a laser diode and a split photodetector detects the bending. This
terials in the future. As nanoindentation becomes more prolific in other bending is indicative of the probe-sample interaction force. In AFM, the
fields, it is imperative to develop a framework that can assist researchers indenter penetrates the sample at a slight angle to the surface; by
to make the appropriate choices for obtaining reliable nanomechanical contrast, the indenter travels to the surface vertically in nano­
measurements. indentation. Similar to AFM, nanoindentation can measure the stiffness
of soft and hydrated materials, however, it is more limited in load and
displacement range. Furthermore, AFM was developed for scanning
2.2. Nanoscale mechanical characterization techniques (When is it (topographic characterization) and adapted to mechanical analysis,
appropriate?) while nanoindentation was developed for mechanical testing and
adapted to scanning. In summary, while AFM has better spatial resolu­
Many biological materials and biomaterials have a structural hier­ tion it is more challenging to get accurate mechanical properties due to
archy that influences their bulk mechanical behavior. There is growing the reasons mentioned above. Conversely, nanoindentation has less
interest in mechanical characterization of structure-property relation­ spatial resolution for scanning and testing, but there exists more accu­
ships, in particular across multi-scaled or hierarchal length scales, for rate mechanical models.
synthesis of tailored nature-inspired or hierarchical materials for spe­ The choice for using nanoindentation versus AFM is strongly influ­
cific use in load-bearing applications (Lakes, 1993). This need motivates enced by the best working range for the instruments and the sample. For
the urgency for robust testing across nano- and micro-scale domains. example, nanoindentation is better suited for stiffer biological materials
Nanoindentation provides an appealing option for understanding the like bone, while AFM is best for characterizing extremely soft materials
mechanical properties across multiple length scales of biological systems like cells and proteins (Chang et al., 2016).
and biomaterials. A noteworthy observation is that the location on the load vs. nano­
Aside from nanoindentation, other methods for mechanical testing of indentation plot may differ for the same sample material. This is due, in
small and soft materials include pipette aspiration and atomic force part, to the variety of indentation parameters used when indenting the
microscopy. A handful of researchers have compared the mechanical materials showcased in Fig. 2, such as maximum load, maximum
behavior of soft, hydrated biological and biomaterials across various displacement, loading rate, probe geometry, and probe size. For
testing modalities. For example, nanoindentation has been coupled with example, for the same stiffness material and maximum indent load, the
traditional methods of confined and unconfined compression in the

Table 2
Advantages and disadvantages for using atomic force microscopy (AFM) and nanoindentation as a mechanical characterization technique. As for the instrumentation,
in AFM, the approach is angled, while nanoindentation is a vertical approach.
Atomic Force Microscopy Nanoindentation

Advantages • Higher load and depth resolution • Larger load and displacement range (max-displacement 5–10 μm)
• Suitable for ultra-soft materials • Suitable for stiff materials
• Suitable for very thin materials • More accurate mechanical models and mechanical properties
• Superior image and spatial resolution • Suitable image resolution on stiff materials
• Easier to test in fluids

Disadvantages • Limited load and displacement range (max-displacement 100 nm) • Lower load and depth resolutions
• Challenging to extract accurate mechanical properties • Not optimized for ultra-soft materials nor testing in fluids
• Sample dimension constraints
• Intensive sample preparation
• Slow scan time

Instrumentation

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

Fig. 2. Load (uN) and indentation depth (um) ranges for AFM, nanoindentation and micro-indentation, including load-depth nanoindentation data points of
representative materials to illustrate the range of properties being measured. Materials include polymers, biological materials, thin films, and ceramics/glasses. The
load-indentation depth values were obtained from the literature (Ebenstein and Pruitt, 2004; Arevalo and Pruitt, 2020; Cuy et al., 2002; il Jang and Pharr, 2008;
Hodzic et al., 1999; Dey et al., 2011; Zysset et al., 1999; Grunlan et al., 2001; Ebenstein et al., 2002; Tong and Ebenstein, 2015; Kaufman and Klapperich, 2009;
Klapperich et al., 2001; Katti et al., 2006; Mao et al., 2011; Barnoush et al., 2010).

maximum indent depth is expected to increase as the diameter of a Further, Fig. 2 shows how the nanoindentation load and displace­
conospherical probe decreases. Similarly, in viscoelastic materials for ment ranges compare with AFM and microindentation ranges. It is
which the stiffness increases with indentation loading rate, even if the interesting to note that although the displacement range of nano­
maximum indent depth is held constant, the maximum load is expected indentation spans all three testing methods, the load range (~10 μN –
to increase as the loading rate increases. These examples show that 10 mN) is unique. It is also notable that all the nanoindentation data
careful attention is necessary when studying complex materials’ nano­ collected with blunt, larger diameter probes (e.g., conospherical, flat
mechanical properties and comparing results across studies with punch) fall within the microindentation displacement range. In com­
different experimental parameters. parison, the data predominantly collected using sharper probes (e.g.,

Fig. 3. General guideline to help determine whether nanoindentation is within range to characterize the sample (Oyen, 2011). In this an analysis, Pmax is the
maximum load, R is the radius of conospherical probe, and hmax is the estimated maximum depth. Er is the unknown reduced elastic modulus (in this condition it is
the plane strain modulus) that is determined from an indentation test and is limited by the macroscale elastic modulus of the material.

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

Berkovich) fall in the AFM range. These results suggest that nano­ for a number of reasons including the variation in contact stress beneath
indentation, AFM, or microindentation are suitable for characterizing the indenter relative to the length scales of underlying material con­
some materials. However, the testing parameters need to be different so stituents. The most common probe geometries used for nanoindentation
that the loads fall into the optimal ranges for those systems. include Berkovich, conospherical, conical, flat-end conical and flat
When deciding on the best small-scale characterization technique, punch (see Fig. 4). Berkovich (3-sided pyramidal probes) and conical
knowledge of the macroscale elastic modulus of the material helps probes are commonly considered sharp probes. Flat punch, flat-end
identify whether the indentation depth or indentation load is within the conical and conospherical indenters are examples of blunt probes;
resolution limits of the testing system. Oyen (2011) provides a general however, the acuity of conospherical probes may be increased by
back-of-the-envelope calculation to aid in determining whether a ma­ decreasing the probe diameter.
terial is suitable for nanoindentation; the process is illustrated in Fig. 3. Geometry of probes is an important consideration for nano­
This method makes use of the maximum-load (Pmax) equation that is indentation. The advantages and disadvantages of flat punch, spherical
based on the Hertz relationship between load and displacement for a and pyramidal indentation probes is summarized in Table 3. Spherical
spherical indenter. By using this method, we can get a quick assessment probes (Fig. 4) are advantageous in that there is a delayed onset of
of the material suitability for nanoindentation based on the knowledge plastic deformation while the sharper conical or pyramidal probes
of the macroscale properties and the radius of the indenter probe. Use of induce elastic-plastic deformation even at small displacements. Flat
this equation requires the following assumptions: (1) the material ex­ punch probes are advantageous in that they provide a constant, known
hibits minimal adhesion to the probe; (2) the material deforms within its contact area, while contact area changes with depth for all other probe
linear elastic regime under the applied stress and strain rate produced by geometries. However, the issues with a flat punch are the presence of
spherical probe of radius, R; (3) any effects of finite thickness are stress concentration at edges and the challenge with achieving full
neglected for the indent depth, h; and (4) the probe is made from a contact, as it is very difficult to achieve a perfect parallel surface on
comparably rigid material, such that the elastic properties of the probe samples. Another probe geometry utilized in characterizing soft mate­
can be neglected. The calculation serves as a rough estimate of suit­ rials, the flat-ended cone punch (Fig. 4), offers the benefits of using a
ability for using nanoindentation (Fig. 3). Once the approximate flat-punch while reducing the high stress concentration at the corners. In
maximum load or maximum displacement has been calculated, the contrast to flat punches, flat-ended cones are easier to manufacture, and
values can be compared against the range of the nanoindenter instru­ can offer a constant contact area for small displacements, simplifying the
ment. While the load and displacement range may vary across different analysis (Tong and Ebenstein, 2015). The flat-ended cone punch has
manufacturers, customarily the displacement may fall within 10− 2 − been successfully used in the characterization of cartilage and poly­
103 μm and the load within 10− 1 − 107 μN, dependent on urethane (Simha et al., 2007).
instrumentation. A compendium of literature related to nanoindentation of bio­
Lastly, careful selection of the probe geometry and size is imperative materials is provided in Table 4, which offers relevant information on
to the overall gathering of data, and guidance on selecting a suitable the probe utilized including its geometry, material, and probe-size
probe geometry and size will be discussed in subsequent sections. (radius), followed by the biomaterial and the measured elastic moduli.
As demonstrated in Table 3, the conospherical probe is widely employed
3. Nanoindentation: probe selection, load function and data for a variety of softer biomaterials. In contrast, the Berkovich is preva­
analysis lently used for characterizing bone-tissue (Edward Hoffler et al., 2005)
and stiff PDMS formulations (Wang et al., 2015).
In depth-sensing nanoindentation, the load and displacement are One factor to consider when deciding on an indentation probe is the
monitored continuously as a small probe is pressed into a sample stiffness of the material to be tested – is the sample a soft material or a stiff
following a user-prescribed profile. The load-displacement data can later material? As an example, we consider indentation of gels using a flat
be analyzed using an appropriate contact mechanics model to extract punch. For stiff gels whose modulus is on the order of 100 kPa and
mechanical properties such as modulus and hardness. Before an indenter above, the general recommendation is to use smaller diameter punches
can perform an indent, the probe needs to know when it is in contact whose diameter is on the order of 500 μm (Liu et al., 2009). In contrast,
with the surface. A common method for surface detection is the use of a as the gel stiffness decreases toward the 1 kPa range, a larger diameter
set-point force. Using this method, the indenter probe is slowly stepped punch of 1.75–2 mm is recommended (Liu et al., 2009). The challenge
towards the surface until a predefined force, the set-point force, is sensed with using a diameter that is too small on a soft material is that the low
by the instrument. Once that force is detected, the instrument assumes contact stiffness makes it difficult to properly identify the contact
the probe is in contact with the sample and will begin the indent and interface and reach a stable equilibrium during surface detection,
data collection process. thereby, preventing the initiation of the indentation test. Similarly,
The first crucial step in the design of a nanoindentation protocol is using a diameter that is too large for a material whose surface is not
identifying an indenter geometry and size that is most appropriate for nominally flat hinders achieving a complete contact (Liu et al., 2009). In
the scope of the research and the material in question. The next step is general, the softer the material, the blunter the probe recommended.
setting up the time profile, which provides the instructions (testing pa­ A second factor to consider when deciding on an indentation probe is
rameters) to the nanoindenter – rate, maximum load/displacement, the size scale of the features of interest in the sample material – are you
holding times and whether the test is in load or displacement control. interested in tissue level/bulk properties, or the properties of individual con­
Lastly, the data analysis of nanoindentation involves selecting a suitable stituents? For heterogenous or porous materials, nanomechanical mea­
model to analyze the load-displacement curves and extracting the me­ surements can vary significantly depending on whether a sharp or blunt
chanical properties. This review will explain in detail the importance of probe is used, and can also be affected by the radius of the blunt probe
each step of the indentation process and provide suggestions to ensure (Oyen et al., 2012). Varying the dimension of the probe enables the
collection of reliable mechanical properties. mechanical characterization across macro-, micro-, and nano-length
scales; however, increasing the dimension of probes decreases spatial
resolution (Ebenstein and Pruitt, 2006). The selection of probe geometry
3.1. Probe geometry and size and size are parameters that depend on the length scale of the micro­
structure or constituents present in the material and the goal of the
Judicious choice of probe geometry and size is essential for acquiring research. For instance, a large diameter spherical probe (>50 μm) is
reproducible and accurate measurements of the desired material. The better suited for measuring tissue level properties in soft tissues because
probe geometry and size influences the nanomechanical measurement the diameter is larger than typical dimensions of cells and structural

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

Fig. 4. Schematic illustration of the various probe geometries used in nanoindentation – Berkovich, Conospherical, Conical, Cone punch, and Flat punch,. In this
diagram, P is the applied load, d or h is the distance from the surface of the sample to maximum penetration depth, a is the radius of the projected contact between
the tip and the sample, and b is the radius of the flattened end of the cone punch.

using a smaller (90 μm diameter) probe overestimated the reported

Table 3
values. Hence, measured modulus may have a dependence on indenter
Presentation of the advantages and disadvantages of commonly used nano­
probe size, especially when probe sizes span constituent length scales.
indentation probes in tissues and polymers and a summary of the materials that
are best suited for characterization with given probe geometries.
Many tissue studies seeking to characterize constituent behavior choose
an indenter size that is less than 10% of the expected length-scale of the
Advantages Disadvantages Materials
modulus variation, as this enables accurate prediction of the mechanical
Flat + Data interpretation - Most limited in • Large diameter variations in tissues with significant modulus heterogeneity (Armitage
Punch is straightforward spatial resolution flat punches are and Oyen, 2017).
+ Detecting and (compared to preferred for
measuring the sharp indenters) testing very soft
In summary, researchers have the choice between different geome­
contact area is - Not suitable for materials tries, and within some of these geometries there is control over size. The
simpler than other characterizing fine stiffness of the material and the size of the target features influence the
tip geometries features choice of your probe – geometry and size. When testing soft materials, a
+ Contact area is less - Alignment of tip
blunt or large diameter probe is preferred in order to avoid puncturing
likely to get affected parallel to sample
by thermal drift or surface is the material and to achieve sufficient contact stiffness to detect when the
creep important and probe is in contact with the sample. The diameter of the blunt probe
challenging should be chosen based on the size scale of the constituents of interest or
- Stress singularities the desired spatial resolution of testing (there is a trade-off between
present at the
edges
increasing resolution and decreasing contact stiffness).
Spherical + Can use analytical - Reduced spatial • Large diameter Indenter probes are made from stiff, hard materials to prevent sub­
models (e.g. JKR) resolution spherical stantial probe deformation and wear during indentation. Reducing wear
+ Delay onset of (compared to probes are ensures that the probe geometry will not change rapidly with testing,
plastic deformation sharp indenters) preferred for
increasing the durability and consistency of the probe (AFM probes are
+ Geometric similarity - May not be ideal testing very soft
+ Offers increased for probing really materials less expensive but are less durable and need to be replaced frequently).
contact stiffness fine features • Smaller Using a stiff material ensures that the deformation observed in the
without high stress diameter indent is dominated by the deformation of the sample rather than the
concentrations spherical probe. As such, the material recommendations for fabricating probes
+ Wide range of probes are
diameters available preferred for
varies based on the stiffness and hardness of the material being indented.
(for multiscale testing stiffer Typically, stiff, hard materials such as diamond or sapphire are used to
analysis or to probe materials fabricate commercial indenter probes, under the assumption that probes
constituents or • Preferred for are used to test hard, stiff materials. However, lower stiffness materials
tissue level testing depth-
such as glass and tungsten can be equally suitable when nanoindentation
properties) dependent
materials is applied to soft biomaterials with moduli more than three orders of
Pyramidal + Yields much smaller - Sharp edges may • Most suitable magnitude lower than traditional indentation substrates. Glass has the
contact area damage soft for fine features advantage of being able to fabricate a truly spherical probe at a much
+ Geometric similarity materials • Preferred for lower cost than sapphire (which can also be perfectly spherical) or
+ Suitable to probe - Harder to detect stiffer materials
diamond (which is difficult to fabricate as a perfect sphere at the nano/
fine features surface of soft • Most
materials commonly used micro scale due to its faceted crystal structure). Glass is also easy to
probe geometry functionalize.
Common indenter probe materials are diamond, sapphire, glass,
tungsten or alumina. Commercial indenter probes are made from dia­
fibers (Ebenstein and Pruitt, 2004). On the other hand, if the mechanical
mond and sapphire, while custom-made indenters include glass, tung­
properties of the individual constituents are needed then the dimension
sten or alumina. Custom-made probes are often tailored for specific
of the probe should be adjusted to maximize the spatial resolution and
researcher needs. In particular, there is ongoing research into the use of
allow measurement at precise locations (Ebenstein and Pruitt, 2006).
alternative probe materials to mitigate probe-sample adhesion (Simha
A small body of work has explored the role of indenter size on the
et al., 2007). For example, Grunlan and co-workers prepared tungsten
measured elastic modulus of soft materials. For example, Oyen and co-
probes of various probe diameters to compare the mechanical property
workers investigated the size effects in indentation of hydrated biolog­
measurements against those obtained using diamond (Grunlan et al.,
ical tissues by utilizing micro- and nanoindentation techniques. They
2001). They attributed observed differences to the probe-adhesion and
discovered that elastic properties were consistent across length-scale but
suggested that tungsten probes may prevent adhesion effects during
the time-dependent mechanical response were substantially different
indentation. Others have utilized indenter probe functionalization to
between large and small contact radii for the same tissue specimen
mitigate adhesion between the probe and sample (adhesion effects are
(Oyen et al., 2012). Additionally, Simha and co-workers explored the
described in subsequent sections). Slaboch and colleagues pioneered the
effects of probe size on the mechanical properties of bovine cartilage and
use of keratose-functionalized probes to characterize blood clots after
polyurethane (Simha et al., 2007); they observed that the modulus
prior research showed a reduction of blood/tissue adhesion in medical
measurements obtained using larger (2 mm and 4 mm) indenter probes
devices (Slaboch et al., 2012). While probe functionalization in the
agreed with the literature values. By contrast, the modulus measured

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Table 4
Summary of literature pertaining to nanoindentation of biomaterials. Table includes probe geometry, probe material, probe radius, biomaterial characterized and
indentation elastic modulus.
Probe Material Probe Radius Material Characterized (Reference) E (MPa)
Probe Geometry

Berkovich Diamond PDMS (Deuschle et al., 2007) /

Bone tissue (Ebenstein et al., 2002) /
Flat Punch Sapphire 25 μm poly-2-hydroxyethylmethacrylate (pHEMA) /
Sapphire 25 μm PuraMatrix-collagen hydrogels (Kaufman and Klapperich, 2009) /
Flat ended cone Diamond 97.72 μm Polyacrylamide gel (Tong and Ebenstein, 2015) /
Diamond 5 μm Urethane (Simha et al., 2007) 4.73
Diamond 5 μm Cartilage (Simha et al., 2007) 2.56
Diamond 90 μm Urethane (Simha et al., 2007) 4.41
Diamond 90 μm Cartilage (Simha et al., 2007) 2.32
Diamond 90 μm Cartilage ii (Simha et al., 2007) 3.43–4.73
Diamond 190 μm Urethane (Simha et al., 2007) 4.34
Diamond 310 μm Urethane (Simha et al., 2007) 4.62
Diamond 310 μm Cartilage (Simha et al., 2007) 1.26
Diamond 1000 μm Cartilage ii (Simha et al., 2007) 1.21–1.62
Conospherical Diamond 100 μm PEG (Kohn and Ebenstein, 2013) 0.6–8.5
Diamond 100 μm PDMS (Carrillo et al., 2005) 0.7–2.6
Diamond 100 μm Hematoma, Fibrous Tissue, Calcified Tissue 0.02–2.6, 0.05–21.5, 0.04–21300
Diamond 100 μm Ebenstein et al. (2009)
Diamond 100 μm Porcine Costal Cartilage (Gupta et al., 2009) 1–2.4
Diamond 1 μm Plaque (Ebenstein et al., 2002) 0.06–9
Diamond 50 μm PDMS (Cao et al., 2005) 1.9 ± 2.3

Diamond 55 μm pHEMA (Kaufman et al., 2008) \

Diamond 55 μm PS-4 Polymer (Oyen et al., 2012) \
Borosilicate G 100 μm PDMS (Kohn and Ebenstein, 2013) \
Borosilicate G 75–150 μm PDMS (Ebenstein, 2011) 0.2–2.2
Sapphire 283 μm Cartilage, Bone (Oyen et al., 2012) \
Sapphire 400 μm PS-4 Polymer (Oyen et al., 2012) \
Sapphire 200 μm Silicon (Buffinton et al., 2015b) \
Sapphire 500 μm Polyacrylamide Gels (Buffinton et al., 2015b) \
Glass Sphere 500 μm Polyacrylamide gel (Tong and Ebenstein, 2015) \
Alumina 400 μm Poly(acrylamide) gels (Galli et al., 2009) \

nanoindentation community is not widely employed, it is a commonly nanoindentation; they offer high strength and hardness as well as ease of
employed practice in the AFM community to mitigate probe-and-sample manufacturability across various probe diameters. It is worth noting that
adhesion. when using diamond-based probes, conospherical probes will not be
In summary, diamond-based probes are the gold standard of perfectly spherical due to diamond’s faceted crystal structure, and some

Fig. 5. Calculating the impact of diamond probe, glass probe and neglecting indenter properties on the determination of the reduced elastic modulus of soft polymers
(1 MPa, 50 MPa). In Eq. A, Er is the reduced modulus (in GPa), Ei is the indenter modulus, vi is the Poisson’s ratio of the indenter, Es is the sample modulus, and vs is
the Poisson’s ratio of the sample.

7
S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

geometries such as flat punches are more challenging to manufacture in data collection after surfaced detection.
the tens of microns size range. Therefore, the use of other lower-cost and A related aspect of consideration when developing a time profile is
higher-fidelity probe materials, such as glass, may be preferred when deciding upon the magnitude of the setpoint force used to find the initial
fabricating blunt probes for testing soft materials. The impact of probe surface contact; once the surface is found by the probe sensing the set­
stiffness on modulus measurements in soft materials is demonstrated in point force, the zero displacement is established. A setpoint force, usu­
Fig. 5. As shown in Fig. 5, using different material probes (diamond vs. ally between 0.1 and 10 μN, is specified in the process of developing the
glass) to characterize soft polymers (between 1 MPa and 50 MPa) time profile (Ebenstein, 2011). The higher the setpoint force, the deeper
resulted in negligible differences in reduced modulus calculations, and the probe will sink into the sample when detecting the surface. The
the moduli were comparable to those computed when neglecting displacement resulting from the preload can be quite significant for
indenter properties entirely. This demonstrates that glass is a sufficiently compliant materials (Cao et al., 2005). Hence, the lowest value of set­
stiff probe material to prevent substantial probe deformation during point force that you can use that avoids false engagement with the
indentation of soft polymers. This provides researchers with the flexi­ surface is recommended. Typical values are 0.1–2 μN; using values as
bility to utilize a variety of probe materials when testing soft materials. high as 10 mN is only recommended when necessary for testing in fluids
(to avoid false surface detection due to changing capillary forces on the
shaft, as will be discussed in a later section) and should only be used in
3.2. Load functions conjunction with the time profile with lift-off segments (Fig. 6c) to allow
for proper identification of the true zero of displacement. In summary,
The applied load or displacement profile is another critical experi­ including the lift-off segment (Segment A) at the start of an indent aids in
mental parameter in indentation testing. Triangular and trapezoidal establishing the true “zero point” of contact between the probe and the
profiles are most commonly utilized in nanoindentation (Fig. 6). The sample to correct for sink-in, while the lift-off at the end of the indent
triangular profile consists of load and unload segments (Fig. 6a), while a (Segment D) can be used to ensure that you capture the full indent until
trapezoidal profile includes a holding period at peak load or displace­ the probe fully separates from the sample when there is significant
ment (Fig. 6b). The use of the trapezoidal time profile elucidated in adhesion between the probe and the sample. The load-displacement
(Fig. 6b) is commonly employed for viscoelastic materials because creep response of a viscoelastic material using a Trapezoidal II profile
related artifacts manifest in nanoindentation as a “nose” on the (Fig. 6c) is illustrated in Fig. 6f; the load-displacement profile in the red
unloading curve (Fig. 6d), as reported by Briscoe and colleagues (Briscoe box is the data that would have been captured using the standard
et al., 1998a). This nose is observed on an indentation load-displacement trapezoidal profile (Fig. 6b) without the lift-offs. Including the lift-offs
curve when a material continues to increase in displacement after the allows determination of the zero-displacement point to correct for
applied load is reduced, posing a challenge for accurately determining sink-in.
the unloading curve slope. A trapezoidal loading curve has been shown
to mitigate this creep artifact in polymeric materials (Fig. 6e) (Klap­
perich et al., 2001). 3.3. Load control vs. Displacement control
An alternative profile is shown in Fig. 6c, and it incorporates an
initial lift-off from the sample (Segment A), a loading segment (Segment During a load-controlled experiment, the load applied during
B), a hold period at peak displacement (Segment C), and an unloading indentation is preselected and as the load changes incrementally, the
segment that also lifts-off from the sample (Segment D). This displace­ displacement results depend on the stiffness of the material. The elec­
ment profile is commonly used for testing soft materials to correct for trostatic force compensates for the stiffness of the sample and maintains
probe sink-in (when the probe is already several hundred nanometers a constant applied force in load-controlled tests. By contrast, during a
into a sample before the surface is detected) or characterize probe- displacement-controlled experiment, the displacement changes while
sample adhesion (Kohn and Ebenstein, 2013). The advantage of this the reaction force results depend on the stiffness of the material. For
displacement profile is that it captures the entire indent process from most indenters, the default setting is load-controlled as most compo­
indent approach and surface detection through full separation of the tip nents are loaded through force rather than displacement in real life
from the sample, even in the presence of adhesion, rather than starting applications. However, displacement-controlled experiments are

Fig. 6. Load (displacement)-time profile for (a)

triangular loading profile composed of a simple load-
unload cycle, (b) a trapezoidal load-hold-unload pro­
file, and (c) a trapezoidal load-hold-unload profile
incorporating a lift-off at the beginning of indentation
testing (Segment A) and at the end of indent testing
(Segment D). (d) Load-displacement response of a
viscoelastic material exhibiting a “nose” at peak load
for a viscoelastic material tested using a triangular
load-time profile (a). (e) Load-displacement response
of a viscoelastic material for trapezoidal load-time
profile (b), eliminating the “nose” during initial
unloading at peak load. (f) Load-displacement
response of a viscoelastic material for Trapezoidal II
profile (c), with the red box indicating the data that
would have been captured using the standard trape­
zoidal profile (b) without the lift-offs. Including the
lift-offs allows determination of the zero-displacement
point to correct for sink-in.

8
S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

preferred for testing compliant, adhesive and time-dependent materials.

One reason why displacement-controlled experiments are preferred
for soft biological materials is that the properties may vary with depth
because of their hierarchical structure - cartilage is a good example,
because depth can make the difference between indenting just the su­
perficial layer versus superficial to deep. When testing a soft material in
load-control, it will undergo substantially more displacement in the z-
direction in comparison to a stiffer sample for the same applied load.
Such behavior results in measuring properties at various depths based on
material stiffness, and makes it challenging to target a specific layer of a
material such as cartilage or observe variations across sample groups
when comparing load-controlled indents (Kaufman et al., 2007).
Another advantage of displacement-controlled experiments is the ability
to correct for sink-in when displacement-controlled indents are per­
formed using the time profile shown in Fig. 6c, as described in section
3.2. In addition, displacement control allows visualization and quanti­
fication of adhesion, which will be discussed in subsequent sections.
Furthermore, displacement-controlled experiments simplify the analysis
of viscoelastic materials that are strain rate dependent (Oyen et al.,
2012). An important consideration for time-dependent materials is that
displacement-controlled nanoindentation offers constant strain rate
while load-controlled experiments do not (Oyen et al., 2012). A sum­ Fig. 7. Representative load-displacement curve (this is a load-controlled
mary of the characteristics for both load-controlled and indent), illustrating the loading, unloading, hystereses and slope of unloading
displacement-controlled nanoindentation experiment is summarized in curve (dP/dh). Note that the full adhesive load-displacement curve would only
Table 5. be collected using a loading profile with initial and final liftoff, as shown in
Fig. 6c.An indent with the profile shown here would only capture the data
shown in the red box in Fig. 8.
3.4. Load-displacement curves
and unloading (pull-off) due to adhesion between the probe and the
An indentation load-displacement curve is commonly referred to as
sample. This evidence of adhesive interaction is not apparent unless
the “mechanical fingerprint” of the material. Fig. 7 shows a generalized
indenting in displacement control using a displacement-time profile
load-displacement curve based on a trapezoidal load-time profile.
incorporating lift-offs as showing in Figs. 6c and 9. In the presence of
Nanoindentation load-displacement data is commonly used to ascertain
adhesion, the probe is experiencing three balanced forces: the resistant
basic nanomechanical properties such as elastic modulus and hardness.
and adhesive forces from the sample and the externally applied force
However, it is also possible to determine creep and stress relaxation
from the nanoindentation system. When the probe is brought near
parameters from nanoindentation data of viscoelastic materials. The
enough to the surface to sense the surface energy, the probe is drawn
path of the loading and unloading in the load-displacement curve is also
into the sample until zero force is reached (non-zero displacement). At
indicative of whether a material is exhibiting elastic, elastic-plastic,
this point, the stored elastic energy and the surface energy are balanced.
brittle, viscoelastic or adhesive behavior.
This is indicative of zero external loads acting on the probe. The indent
The load-displacement curve shows distinct behavior for elastic-
then proceeds with sample loading and unloading, and finally, as the
plastic, viscoelastic and adhesive materials based on a trapezoidal load
probe is lifted off the sample, the tip and sample fully separate.
profile, as illustrated in Fig. 8. For elastic-plastic materials, the
While viscoelastic effects are typically mitigated primarily by using
unloading path does not follow the loading path as it would for a
the trapezoidal loading profiles, changes to both data collection and data
perfectly elastic material, and there is an observable hysteresis denoted
analysis methods are often employed for samples exhibiting adhesion,
by the area between the loading and unloading path. Viscoelastic ma­
and this behavior is addressed below.
terials also experience energy loss due to deformation, thereby forming a
hysteresis, and also show evidence of stress relaxation (or creep, in a
load-controlled test) during the hold period (Oyen and Cook, 2009). 3.5. Overview: contact models in nanoindentation
Adhesion offers another challenge in nanoindentation of soft mate­
rials. The indenter probe and sample interaction for a material exhib­ Nanoindentation involves penetrating the specimen using an
iting probe-sample adhesion is illustrated in more detail in Fig. 9, along indenter probe while recording the indentation load, P, and displace­
with the important points on the curves that are used during analysis. ment, h, for one complete loading and unloading cycle (Fig. 7). The
Note that negative forces are observed during both loading (snap-on) indenter penetrates the specimen at a given load or displacement rate
until it reaches the desired maximum load or displacement; subse­
quently a hold period at maximum load or displacement may be
Table 5
enforced to prevent viscoelastic effects followed by an unloading
Characteristics of load control and displacement control experimentation during
nanoindentation.
portion. The loading region undergoes a mixture of elastic and plastic
deformation with a resultant residual impression of the indenter probe
Load-Control Displacement-Control
while the unloading region exhibits elastic recovery and provides a
Characteristics • Strain rate is not • Strain rate can be held constant measure of the surface modulus (Oliver and Pharr, 2010; Oliver and
constant • Can incorporate lift-offs to accurately
Brotzen, 1992). The seminal nanoindentation papers that lay the foun­
define the initial contact point and detect
adhesion
dation for the use of load-displacement curves in determining mechan­
ical properties are summarized in Appendix A.
The Oliver and Pharr method (see section 3.5.2) is the standard
Materials • Stiff • Compliant technique for analyzing the nano-mechanical behavior of stiff materials
• Not prone to • Prone to adhesion such as metals and ceramics. This method is based on the work of
adhesion • Time-dependent behavior
Boussinesq, Hertzian contact mechanics and Sneddon’s derivation that

9
S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

Fig. 8. (a) Trapezoidal time-profile in displacement control and associated load-displacement behavior for (b) elastic-plastic and (c) viscoelastic materials. (d)
Trapezoidal II time profile in displacement control typically used for testing an adhesive material, and the associated load-displacement behavior (e). The portion of
the load-displacement curve contained in the red box is the data that would be collected using the basic trapezoidal displacement-time profile in Fig. 8a.

Fig. 9. Representative load-displacement

behavior of a material exhibiting adhe­
sion during the indentation process, with
accompanying displacement-time profile.
By including the lift-offs in segments A
and D of the displacement-time profile,
the indentation data captures the entire
indent process. First, the probe ap­
proaches the sample (1), which is fol­
lowed by a snap-on (2) process as it
makes contact with the sample. In steps
(3–5), the probe engages with the sample
and step (6) shows the probe pulling from
sample and experiencing adhesion (ma­
terial sticking to probe as it is
withdrawn).

provide the general relationships among the load, displacement and surface. The contact stiffness, S, is obtained from the force-displacement
contact area for any punch described as a solid of revolution of a smooth curve. Here Er , the reduced modulus, is related to the elastic modulus of
function. While the Oliver and Pharr method was developed and vali­ the substrate as defined by Equation 2
dated for elastic and elastic-plastic materials, it is commonly employed [( ) ( )]
1 1 − υ2s 1 − υ2i
in the analysis of soft biomaterials and polymers (Klapperich et al., = + Equation 2
Er Es Ei
2001; Malito et al., 2018). However, this method may not always yield
accurate results since adhesion is presumed to play a negligible role in
Here Es is the elastic modulus of the substrate and Ei is the elastic
the sample deformation when using the Oliver and Pharr method. For
modulus of the indenter, υs is the Poisson’s ratio of the substrate and υi is
example, using a Hertzian approach to analyze materials with adhesion
the Poisson’s ratio of the indenter. It is common practice to neglect the
underestimates the contact area as well as the effective load experienced
Poisson’s ratio and modulus of the indenter when testing soft bio­
by the substrate, resulting in overestimation of sample modulus (John­
materials, as the indenter stiffness is generally significantly larger than
son et al., 1971). Adhesion-dominated interactions between the nano­
the substrate making the indenter term negligible. The stiffness (S) of the
indentation probe and sample are best analyzed with other contact
material is measured from the unloading segment of the force-
models (discussed later).
displacement curve (Fig. 7), as shown by Equation (3):
( )
3.5.1. Hertz linear elastic analysis dP
S= Equation 3
The Hertz linear elastic analysis is based on the classical Hertzian dh h=hmax
contact theory. The hertzian reduced modulus, EHertz
r , is expressed as
Combining Equations (Equations (1)–(3)), and assuming probe
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅
S3 deformation is negligible, provides an expression for the elastic modulus
ErHertz = Equation 1 for spherical indentation (Equation (4)):
6RPmax
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
( )2
where R is the nominal radius of the probe, and Pmax is the maximum S3 1 − υ2s
Hertz
ES = Equation 4
applied force that corresponds to the total penetration depth into the 6RP

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

The Hertzian assumptions are described in section 2.2. For a nano­ Equation (7).
indentation testing procedure the relevant criteria are that the sample
Equation 7
1
/ 1/

has low adhesion with the indenter tip, the sample is not loaded so much A = C1 h2c + C2 h1c + C3 hc2 + C4 hc4 + …
that it plastically deforms (i.e. the sample is stiff enough or the applied The values for C1 , C2 , C3 , C4 , etc. can be experimentally obtained by
load or displacement is sufficiently low), and indenter tip can be performing a series of indents at varying penetration depths on a stan­
approximated as spherical. dard calibrating material (as described above) and subsequently fitting
Equation (7). Many test systems use a default area function that assumes
3.5.2. Oliver and Pharr an ideal probe geometry. The ideal area function equations for Berko­
The Oliver and Pharr method utilizes Sneddon’s relationship for a vich, conical probe, spherical and cylindrical flat punch are depicted in
flat cylindrical punch and is generalized for any punch described as a Table 6, these assume ideal probe geometry. Klapperich and colleagues
solid of revolution for a smooth function (Sneddon, 1965). The mathe­ noted that the value for C1 is approximately 24.5 when using a Berko­
matical relation between contact stiffness, contact area and modulus are vich indenter to probe polycarbonate (Klapperich et al., 2001). This
based on Sneddon’s simplification from the Boussinesq equations (de­ value is equivalent to the ideal Berkovich area function
tails can be found in Appendix B) (Hay et al., 1999). Appendix B sum­ √̅̅̅
(A = 3 3 tan 2 θh2c = 24.5h2c ) for deep indents. While using the ideal
marizes seminal papers on the theory and contact mechanics associated
probe geometry instead of experimental calibration can be effective
with the theory of the Oliver and Pharr method. Important elements are
when performing deep indents with sharp probes or perfectly spherical
highlighted below.
probes (e.g., fabricated from glass or sapphire, not diamond), for
In the Oliver and Pharr technique, the unloading portion of the load-
shallow indents experimental probe calibration is needed to avoid
displacement curve (Fig. 7) is fit with a power-law, which is useful for
measurement errors due to probe imperfections. For example, sharp
materials with non-linear unloading behavior (Oliver and Pharr,
probes do not come to perfect points as assumed in the ideal probe area
1992b). The unloading slope is estimated as the first derivative of the
function; a new Berkovich probe will typically have a radius of curvature
fitted function at the maximum displacement and is the prevalent
of at least 100 nm, and this can increase over time due to wear if testing
method for analyzing indentation data (Equation (5)).
hard materials. Similarly, spherical probes fabricated from diamond
dP ( )m may diverge from their ideal shape at the nm scale due to the faceted
S= = α h − hf Equation 5
dh nature of diamond.

Here, ⍺ and m are empirically determined fitting parameters, h is the 3.5.3. Adhesion-based models (JKR, MD, MDT)
displacement and hf is the final displacement after complete unloading The models described above are valid when the interfacial adhesive
(Oliver and Pharr, 1992b). forces are much lower than the applied compressive force between
Classical indentation theory states that the contact stiffness of the indenter and sample substrate. When adhesion forces are not negligible,
material is related to the contact area, A, and reduced modulus, Er, as and spherical probes are used for indentation, it is more appropriate to
expressed in Equation (6). apply continuum mechanics models that include the adhesive effect:
dP 2 √̅̅̅
= √̅̅̅ AEr Equation 6 a3 =
6R (
f P, Fpo
)
Equation 8
dh π 8Er
The projected contact area, A, requires precise knowledge of the
Established models for adhesion include Johnson Kendall Roberts (JKR),
shape of the indenter, which is described by a shape or area function that
Maugis-Dougdale (MD), and Derjaugin-Muller-Toporov (DMT). These
relates the cross-sectional area of indenter to the distance, d, from its
are summarized in Table 7.
probe tip. Two methods exist for determining the area function and they
The most widely used contact mechanics models for capturing
both involve making a series of indentations on a calibrating material
adhesion are the Johnson-Kendall-Roberts (JKR) and Derjaguin-Muller-
over a range of indentation depth. The first method involves indentation
Toporov (DMT) theories. JKR is used for low modulus materials with
on a soft metal and using transmission electron microscopy (TEM) to
high surface energy and large radius of curvature while DMT theory is
measure the size of the impression created by indentation and the
applicable to high modulus materials with low surface energy and small
accompanying load-displacement data to establish the area function.
radius of curvature. Physically, JKR accounts for adhesion forces only
The other method involves making indentations on an isotropic material
within the expanded area of contact; DMT accounts for adhesion forces
of known modulus from which the area function can be deduced from
outside the contact area while maintaining the Hertzian gap profile.
the load-displacement data alone. The advantage of using a calibrating
Maugis-Dougdale (MD) is a transitory model that captures the in­
material to identify the area function is that it eliminates the need for
teractions between surfaces that lie between the JKR and DMT models. A
imaging the indents.
more in-depth analysis is provided in Appendix C.
The use of a suitable standard calibration material is critical. Both
Tabor observed that JKR and DMT exist as two extreme limits that
quartz and fused silica have been explored (Briscoe et al., 1998b);
can be parametrized using Tabor’s parameter, as defined by Equation
however, the use of fused silica as a standard calibrating material has
(9).
come into question when testing soft materials (Klapperich et al., 2002).
Klapperich and co-workers utilized polycarbonate as a standard cali­
brating testing material because the depths probed during calibration
were similar to their compliant materials of interest (Klapperich et al.,
Table 6
2001). To our knowledge, an alternative standard calibrating material Area function Equations for Berkovich, conical probes (Fischer-Cripps, 2011),
has not been proposed for materials with moduli that are orders of spherical and cylindrical flat punch. Where hc is the contact depth, θ is the
magnitude softer than polycarbonate. A potential standard calibrating semi-angle for a Berkovich indenter (65.27◦ ), α is the effective cone angle for a
material for use in characterization of soft materials is poly­ conical indenter, and a is the radius of the flat punch tip.
dimethylsiloxane (PDMS), an isotropic and homogenous polymeric Probe Geometry Area function equation
material whose properties can be tailored via crosslinking. More √̅̅̅
Berkovich A = 3 3h2c tan 2 θ = 24.5h2c
research is needed in the realm of creating standard calibrating mate­
Conical of half-apical angle α A = πh2c tan 2 α
rials for soft samples.
Spherical A = π(2Rhc − h2c )
The contact area is expressed a function of contact depth as shown in Cylindrical flat punch A = π(a2 )

11
S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

Table 7 curve, worked best for analyzing nanoindentation data of soft polymers
Commonly used adhesion-based models for analyzing contact mechanics data using a large diameter spherical probe (Tabor, 1977a, 1977b), but
with adhesion. Method I was used by Grunlan and in analysis of AFM force curves
Johnson, Kendall, Derjaguin, Muller, Maugis- (Grunlan et al., 2001). Curve fitting the unloading curve using equations
Model Name Roberts (JKR) and Toporov (DMT) Dougdale (MD) derived from the JKR model (Fig. 10) is the most reliable method, but is
Assumptions The initial pressure Attraction forces are Describes the more labor-intensive (Kohn and Ebenstein, 2013).
distribution between occurring outside the interaction Per review of the literature (Table 8), nano-JKR is the most
two contacting contact area and potential commonly used model for analysis of load-displacement curves that
spheres consists of these forces are too between
exhibit adhesion as measured via depth sensing indentation, while the
repulsion close to the weak to produce any surfaces
center and attraction substantial other adhesion-based contact theories are more generally employed in
at the edge of the deformation of AFM.
contact area spheres
Application • Compliant • Stiff materials
4. Experimental challenges and errors
materials • Small sphere radii
• Large sphere radii • Weak, long-range
• Strong, short-range adhesion forces This section describes challenges that are encountered during the
adhesion forces nanoindentation process that can interfere with accurately measuring
Tabor’s μ>5 μ < 0.1 0.1 < μ < 5 the elastic modulus, and poses potential solutions to those challenges.
Parameter
(μ)
The challenges discussed pertain to indenter probe and sample inter­
action, particularly, (a) surface roughness, (b) contaminated probe, (c)
adhesion, (d) false engagement, and (e) substrate effects, as depicted in
[ ]13 Fig. 11. These are all issues that can be detected by looking at the load-
d R(Δγ)2
μ : = c≈ Equation 9 displacement curves.
z0 Er2 z30

Here z0 is the equilibrium separation between the two contacting sur­ 4.1. Surface roughness
faces, Δγ is the work of adhesion, Er is the reduced modulus, and R is the
probe radius being used for testing. The work of adhesion represents the Surface roughness can lead to a wide variability in indent load-
amount of energy needed to pull two surfaces apart and is a function of displacement response, as shown in Fig. 11. Surface roughness can
probe size and adhesion forces (Tabor, 1977a, 1977b). The Tabor have a significant effect on nanomechanical measurements, particularly
parameter is calculated to identify whether the contact conditions fall if the arithmetical mean deviation (Ra) of the assessed roughness profile
within the JKR (μ > 5), DMT (μ < 0.1) or MD (0.1 < μ < 5) regimes. is on the same scale as the probe radius (Fig. 11a). Chen and co-workers
The most commonly utilized theory to analyze nanoindentation used a computational approach to investigate the effects of surface
load-displacement data of soft materials with adhesion is the nano-JKR roughness on nanomechanical properties. They found a decrease in
method (Ebenstein and Wahl, 2006). Ebenstein and Wahl compared the mechanical properties with increased surface roughness (Chen et al.,
results of load-displacement data using several methods based on the 2013). Since their simulation findings analyze elastic-plastic materials,
JKR adhesion model (Ebenstein and Wahl, 2006). The nano-JKR the computational analysis and results may potentially differ for soft
methods used to analyze the mechanical properties of materials exhib­ biomaterials. Nevertheless, in general, the surface roughness affects the
iting adhesion are captured in Fig. 10. These methods rely on several scatter of load-displacement curves and can lead to deviation of hard­
important points in the load-displacement curves and are derived by ness and reduced modulus (load-displacement curves illustrated in
evaluating the JKR equations at specific points. Table 8 summarizes Fig. 11a and b). Researchers have proposed that large variations of the
relevant research in the area of adhesive-based models and nano­ measured mechanical properties are in part produced by the surface
indentation on soft and hydrated materials. Ebenstein and Wahl found roughness (Farine, 2013).
that displacement method I, which used points from the unloading To minimize the impact of surface roughness on your indentation
results, the surface should be prepared such that it is free of

Fig. 10. Nano-JKR method used to analyze samples

experiencing adhesion. There are three approaches:
Displacement Method I, Displacement Method II, and
curve fitting. In all equations, R is the radius of the
conospherical probe. The other variables used to
calculate the reduced modulus, Er, in Displacement
Methods I and II are labeled on the figure. In the
curve fit equation, Padh is the maximum negative load
reached during pull-off (at δadhesion), a0 is the pro­
jected contact radius between the probe and the
sample at zero load (at δ0), and δcontact is a variable
included to rezero the displacement to optimize the
fit. Padh, a0, and δcontact are solved for using a non-
linear curve fit of the load-displacement (P-δ) data
from the unloading curve.

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

Table 8
Research utilizing JKR theory to analyze nanoindentation load-displacement curves and research that advances the nanoindentation research for soft materials.
Reference Summary

C. Jin, Z. Wang, A. Volinsky, A. Sharfeddin, N. Gallant (2016) (Jin Mechanical characterization of crosslinking effect in PDMS using nanoindentation
et al., 2016) The nano-JKR method was implemented to analyze the load-displacement curves of polydimethylsiloxane
(PDMS).
D. Ebenstein (2011) (Ciavarella et al., 2019) Nano-JKR force curve method overcomes challenges of surface detection and adhesion for
nanoindentation of a compliant polymer in air and water
Addresses the challenges of testing soft and hydrated samples, provides alternative hydrating fluids to minimize
adhesion effects. The research focused on characterizing polydimethyl siloxane (PDMS) and showed the JKR to be
a suitable method to account for adhesion.
S. Gupta, F. Carrillo, C. Li, L. Pruitt, C. Puttlitz (2006) (Gupta Adhesive forces significantly affect elastic modulus determination of soft polymeric materials in
et al., 2007) nanoindentation
Investigated the nanomechanical properties of PDMS elastomers, and used the JKR contact model to analyze the
data and compared the results to data analyzed from Hertz contact model. This paper highlighted the importance
of utilizing the appropriate contact model when a sample experiences non-negligible adhesion effects.
F. Carrillo, S. Gupta, M. Balooch, SJ. Marshall, G Marshall, L. Nanoindentation of PDMS elastomers: Effect of crosslinking, work of adhesion, and fluid environment on
Pruitt, C. Puttlitz (2005) (Carrillo et al., 2005) elastic modulus
This research used nanoindentation to characterize elastic moduli of soft, elastomeric polydimethylsiloxane
(PDMS) with varying degrees of crosslinking. Adhesion contact mechanics was implemented on the indentation
measurements and determined the effects of adhesion. The research highlighted the importance of considering
adhesion in the calculation for determining the elastic modulus, especially for soft materials.
C. Jin, D. Ebenstein (2016) (Jin and Ebenstein, 2017) Nanoindentation of compliant materials using Berkovich probes and flat probes
The paper extends the nano-JKR analysis to include Berkovich and flat indenter probes. The researchers
performed numerical simulations by applying adhesive interactions as interaction potential and the surface
deformations coupled by half-space Green’s functions discretized on the surface.
J. Kohn, D. Ebenstein (2013) (Kohn and Ebenstein, 2013) Eliminating adhesion errors in nanoindentation of compliant polymers and hydrogels
This research provides two methods for considering adhesion in the nanoindentation analysis. The first method is
the nano-JKR curve method and the second method is the surfactant method.
D. Ebenstein, K J. Wahl (2006) (Ebenstein and Wahl, 2006) A comparison of JKR-based methods to analyze the quasi-static and dynamic indentation force curves
This research compares five approaches used to analyze quasi-static and dynamic load-displacement data
obtained from instrumented indentation. The data was analyzed by direct curve fitting, three different simplified
methods that are based on the JKR theory.
Q. Liao, J. Huang, T Zhu, C. Xiaong, J. Fang (2010) (Liao et al., A hybrid model to determine mechanical properties of soft polymers by nanoindentation
2010) The development of a hybrid model – JKR and Hertz- to analyze the elastic modulus of soft polymers (PDMS)
using nanoindentation techniques.
Y. Cao, D. Yang, W Soboyejoy (2005) (Cao et al., 2005) Nanoindentation method for determining the initial contact and adhesion characteristics of soft
polydimethylsiloxane (PDMS)
This research illustrates the method that combines JKR and Maugis-Dugdale adhesion theories and nonlinear
least squares fitting to predict the load-indentation depth characteristics of PDMS.

contamination and sufficiently smooth to allow for high quality inden­ 4.2. Probe contamination
tation data. According to ISO 14577 (standard for nanoindentation of
metals and ceramics), the arithmetic roughness of the surface should be Probe contamination can also affect load-displacement data, often
20 times lower than the maximum indentation depth (Chang et al., presenting as a low stiffness toe region at the start of the load-
2016). This is in part to reduce the scatter in the measurements (ISO displacement curves, as illustrated in Fig. 11b. To avoid the likelihood
14577). Since this standard was developed for metals and ceramics, of using a contaminated probe in nanoindentation, researchers
surface roughness guidelines may vary for biological materials and the encourage gently cleaning the probe after every use with isopropyl
specific probe geometry and size. On a similar note, Farine cautions alcohol or acetone.
researchers performing nanomechanical measurements on biological
tissues, as they cannot be considered flat, that the effects of surface
4.3. Adhesion
roughness should not be disregarded (Farine, 2013). Farine performed
experimental and computational work to understand the effects of sur­
Soft biomaterials (i.e. synthetic and biological) are prone to adhesion
face topography, and showed that finite element simulations exhibited
effects, a phenomena in which the material adheres to the probe, pre­
significantly lower stiffness when indenting on rough materials than on
senting itself in the load-displacement curve as negative forces during
smooth (reduced surface roughness) samples. Since the effect of surface
sample approach (snap-to-contact) and withdrawal (pull-off) (Fig. 11c).
roughness is significantly impacting the measurements, Farine
If modulus measurements are extracted using traditional analytical
concluded that it warrants addressing experimentally or through
methods (i.e. Oliver and Pharr) for compliant materials with dominant
computational methods.
adhesion interactions, an overestimation of properties has been
To this end, it is pivotal to implement best experimental practices to
observed (Ebenstein, 2011). Despite the demonstrated errors in
reduce surface roughness. As such, researchers have also employed the
modulus, many studies of soft tissues and other compliant biomaterials
following techniques to minimize surface roughness when possible:
ignore the effects of adhesion. Adhesion phenomena is often not
microtoming (Klapperich et al., 2001), lap polishing (Klapperich et al.,
recognized in a load-controlled experiment or when unloading data is
2001), or cryo-microtoming (Ebenstein and Pruitt, 2006). When it is not
not recorded past zero displacement (e.g., no lift-off). One solution to
possible to reduce the roughness (i.e., testing cartilage in situ), an
verify whether adhesion is occurring is to continue collecting the data
alternative is to increase the indent depth to improve the ratio of indent
until the probe fully separates from the surface by including a lift-off at
depth to arithmetic roughness or to increase the ratio of the indenter tip
the end of the displacement-time profile (Fig. 6c).
radius to roughness (Farine, 2013).
As described in Section 3.5.3, there are contact models that account
for adhesion; however, nanoindentation software typically does not
include these models and hence adhesion analysis needs to be performed
using other software. An alternative to implementing JKR analysis to

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Fig. 11. Illustration of potential challenges that may be encountered during nanoindentation, (a) surface roughness, (b) contaminated probe, (c)adhesion, (d) false
probe-sample engagement, and (e) substrate effects, accompanied by their respective load-displacement behavior.

analyze nanoindentation data when adhesion is present is to utilize affect the mechanical properties by producing an artificial drift from the
experimental approaches to mitigate adhesion during testing. Several reference point for probe displacement and surface-tension on the probe
researchers have submerged samples in a surfactant solution to cut (Tang and Ngan, 2007). To mitigate capillary forces on the indenter, a
adhesion - a surfactant is an amphiphilic molecule that binds to hy­ common practice when testing in fluids is the use of probes with longer,
drophilic and hydrophobic materials to reduce the surface tension be­ small diameter shafts (Mann and Pethica, 1996); yet, the movement of
tween the probe and sample (Kohn and Ebenstein, 2013). Some partially submerged shafts can still affect the accuracy in the force
researchers have utilized surfactants with dilauryl sulfate (SDS) to measurements during indentation (Ebenstein, 2011). Alternatively, re­
mitigate adhesion effects during indentation of hydrated bone (Rho and searchers also proposed coating the probe or using surfactants (Kohn
Pharr, 1999) and silicone elastomers (Kohn and Ebenstein, 2013). Kohn and Ebenstein, 2013; Haque, 2003a) to reduce capillary forces. How­
and Ebenstein used OptiFree ExpressⓇ contact lens solution and others ever, as previously mentioned, the challenge with using surfactant is the
have utilized F108 pluronic surfactant to reduce adhesion during potential effect on the nanomechanical properties, which is especially
indentation of hydrogels (Kohn and Ebenstein, 2013; Kalcioglu et al., problematic in hydrogels (Kohn and Ebenstein, 2013). As for coating
2010). The use of surfactants is a potential avenue for eliminating probes to aid in reducing capillary forces, Slaboch and colleagues have
adhesion during testing; however, submerging samples in surfactants been one of the few researchers to functionalize the nanoindentation
may result in changes in mechanical properties, especially for hydrogels probe. This highlights the need for future studies to focus on coating and
(Kohn and Ebenstein, 2013). Therefore there is a need for more research functionalizing probes to reduce or eliminate capillary forces.
on the types of surfactants that can be used to minimize adhesion during A final hack to minimize the likelihood of false engagement is to
nanoindentation of soft polymers, hydrogels, and tissues without increase the setpoint force to reduce the possibility that capillary forces
changing the properties of the material. Coating of probes is another alone will trigger the indenting process. Setpoint values as high as 10 μN
option that can help mitigate adhesion, as mentioned in Section 3. Sla­ (vs. typical values of 0.1–2 μN (Buffinton et al., 2015b)) have been used
boch and colleagues pioneered the use of keratose-functionalized probes (Kohn and Ebenstein, 2013), but this leads to increased sink-in and must
in a nanoindentation study of blood clots (Slaboch et al., 2012). Other be used in conjunction with lift-offs in the displacement-time profile to
chemistries could also be investigated for glass indenter probes, as there allow re-zeroing of the displacement. This hack is also often used with
is much research on functionalization of glass. Probe functionalization is other methods such as long shafts and surfactants.
a common practice in the AFM community (Slaboch et al., 2012), but it Although researchers have introduced many mitigating measures,
is less commonly used in nanoindentation. As such, more research is false engagement from capillary forces when testing hydrated samples is
needed to standardize a solution to eliminate adhesion during nano­ an ongoing challenge that merits more research. This novel challenge
indentation in hydrated and soft systems. arises because nanoindentation primarily characterizes hard, dry ma­
terials, not soft, hydrated materials. Innovations in indentation in­
4.4. False engagement struments and experimental methodology will likely be needed to tackle
this problem entirely.
Issues of false probe-to-sample engagement, when the indenter set­
point force is triggered by capillary forces and data collection is initiated 4.5. Substrate effects (and composite effects)
without the probe being in contact with the sample, can be a con­
founding factor when indenting soft, hydrated materials. Meniscus Performing nanoindentation on small-dimensioned specimens (i.e.,
forces or capillary forces during indentation can arise when testing hy­ thin-films) poses a challenge in nanoindentation data acquisition, as
drated materials, as a capillary neck forms between the probe and substrate effects may be pronounced and influence the nanomechanical
sample surface. More commonly these forces act on the indenter shaft properties. The indentation response of a thin film on a substrate is
when indenting submerged samples (Rho and Pharr, 1999; Mann and complex since it involves the mechanical response of both the film and
Pethica, 1996; Tang and Ngan, 2007), and result in detection of negative substrate. Hence, when indenting a thin soft material on a stiff substrate,
forces by the nanoindenter, leading to false engagement. The the substrate effect manifests in nanoindentation data through an arti­
load-displacement behavior for false engagement may resemble the one ficial stiffening of the material with decreasing thickness of sample, as
illustrated in Fig. 11d (Farine, 2013), as the indent is performed in the illustrated in Fig. 11e.
submerging liquid rather than in the sample. As a result, properties of Numerous investigators have used experimental and theoretical ap­
the sample material cannot be extracted from the load-displacement proaches to study the problem of extracting “true” film properties from
data. In other cases, mechanical properties may be extractable from nanoindentation of film/substrate composites. For example, Saha and
the load-displacement curve, but they may be inaccurate due to the Nix examined the influence of substrate on the properties, exploring the
varying capillary forces. effects of soft films on hard substrates and hard films on soft substrates
Tang et al. suggests that the presence of liquid film may significantly and providing potential models to account for substrate in the analysis

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

(Saha and Nix, 2002). In order to measure “sample-only” properties, analysis methods.
researchers recommend limiting the indentation depth to less than 10% The first step in designing a nanoindentation experiment is usually
of the film thickness (Chang et al., 2016). While this may be feasible for deciding on an appropriate probe based on the material composition and
some samples, for others, particularly irregularly shaped biological properties (Fig. 12). Two important factors to consider when selecting a
materials, it may be more challenging to obtain samples thick enough to probe are the stiffness of the sample material and the size scale of in­
minimize substrate effects. In those cases, coupling indentation with terest in the study. Blunt probes (e.g., large diameter spherical or flat
analytical (Saha and Nix, 2002) or computational models can allow punch probes) are preferred when testing soft materials both to avoid
extraction of “true” properties of the soft material. In addition, when puncturing the material and to ensure a large contact size/stiffness to
measuring the properties of a specific constituent in a heterogeneous facilitate detecting the surface before too much sink-in occurs (Fig. 3).
composite material, other researchers have shown that the maximum But keep in mind that there is a tradeoff between probe diameter (or
recommended indent depth depends on the relative properties of the indent contact size) and spatial resolution of testing – the larger the
different constituents, and is often less than 10% of the heterogeneity contact size, the lower the spatial resolution of your sample mapping
size (Chang et al., 2016). (Figs. 12-1a). The size of the probe should be selected based on the
As such, various researchers developed protocols to identify the length-scale of the research question (e.g., are you interested in measuring
“true” modulus of the sample in a composite half-space. They suggest the properties of the individual constituents, or in capturing the combined
utilizing a displacement-control experiment to control the depth and microstructural behavior of the material?) and knowledge of the sample’s
know which portion of the sample they are characterizing, whether they microstructure or the size of the various constituents of interest. The
are characterizing onto the substrate or, in the case of a heterogenous literature advises to choose an indenter size that is less than 10% of the
sample, another structure with different properties (Ma et al., 2017). In expected length-scale of interest, as this enables accurate probing of the
many research fields using nanoindentation, it is common to implement mechanical variations in the materials with significant modulus het­
nanomechanical characterization with ultrasonic testing, modeling, erogeneity (Figs. 13-1b) (Armitage and Oyen, 2017). A summary of
chemical analysis and statistical analysis. Ultrasonic testing enables probes utilized in the nanoindentation of biological materials, hydrated
characterization of the “macroscopic” elastic properties for composites. biomaterials and soft materials with varying stiffnesses are given in
This technique is based on longitudinal waves in the kHZ to MHz fre­ Table 4.
quency. Implementing statistical analysis with nanoindentation may As for selecting the type of probe material (Figs. 13-1c), there are a
provide insight into the properties of composite materials and account few options. Diamond-based probes are the gold-standard for nano­
for substrate effects (Arevalo and Pruitt, 2020; Dohnalík et al., 2021; indentation; yet, many researchers diverge from diamond-based in­
Kariem et al., 2015). denters when indenting soft materials because (as explained in the
earlier sections) soft materials do not require overly stiff probes and
5. Framework for designing a nanoindentation experiment some alternative probe materials, such as glass and tungsten, are better
suited for mitigating adhesion. Glass could be a particularly advanta­
This review focuses on developing a framework (illustrated in geous material for mitigating both probe-sample adhesion and capillary
Figs. 13–14) to be used for designing a nanoindentation experiment to forces on the indenter when testing hydrated samples, given the large
accurately measure the nanomechanical properties of soft and hydrated body of research on functionalizing glass. It is also sufficiently stiff to
materials. This framework can serve as a guide for novice researchers in make a negligible contribution to reduced modulus for samples with
the field of nanoindentation. As such, it provides guidance on the moduli as high as 50 MPa. Lastly, these alternative probe materials may
following topics in the context of testing soft, hydrated materials: probe be preferred for soft material testing as they are cheaper and easier to
selection, experimental set-up, data collection challenges, and data manufacture into ideal conospherical geometries than faceted diamond.

Fig. 12. Part I: Framework provides a guideline for

selecting a nanoindentation probe (geometry, size and
material). The tip geometry is important to indenta­
tion testing; for example, sharp probes are preferred
to use on stiff materials to maximize spatial resolu­
tion, while blunt probes are preferred to use on soft
materials to avoid sample puncture and facilitate
sample detection. However, the use of blunt probes
comes at the expense of spatial resolution. As for tip
material, there is a variety of materials to choose from
when testing soft materials and many researchers
might choose a tip material that mitigates adhesion
and is cheap and easy to manufacture rather than the
traditional diamond probes.

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

Fig. 13. Part II: Framework provides the experi­

mental set-up for nanoindentation testing. First, it
provides a guide for identifying the appropriateness of
using a displacement-controlled vs load-controlled
experiment. Second, it provides a guide for identi­
fying the maximum load or displacement to apply
during indentation testing. Third, it provides a general
framework for developing the time-profiles based on
whether a material is viscoelastic, whether the indent
will need re-zeroing from sink-in effects or whether
the material is prone to adhesion.

The experimental set-up of nanoindentation involves creating a load and probe radius should satisfy the criteria shown in Fig. 3 to ensure that
or displacement profile (for load-controlled or displacement controlled the indent loads and displacements fall within the range of the nano­
experiments, respectively). This profile defines the indent parameters indentation instrument.
such as loading/displacement rate, maximum load/displacement and As illustrated in Figs. 13-2c, many features of the time profile depend
hold period. As illustrated in Figs. 13-2a, the choice of load-controlled on the behavior of the material. For example, a hold period is recom­
vs. displacement-controlled experimentation is dependent on the sam­ mended on the time-profile when testing viscoelastic materials. This is
ple (i.e. adhesion, stiffness and structure). For example, displacement- done to reduce the creep related artifacts manifesting in nano­
controlled experiments are preferred for testing compliant, adhesive indentation as a “nose” on the unloading curve, as observed by Briscoe
and time-dependent materials, or materials that may have depth- and colleagues (Briscoe et al., 1998a). Almost all polymers and bio­
dependent properties due to complex microstructures (e.g., cartilage). materials will exhibit sufficient time-dependent properties to mandate
Displacement-controlled experiments can aid in detecting the surface of the trapezoidal time profile, but the time at peak load/displacement will
soft materials and simplify the analysis of viscoelastic materials that are vary by material (from a few seconds to a few minutes). If the hold time
strain rate dependent (Oyen et al., 2012). is too short, the “nose” will be visible in the load-displacement data and
The next step is choosing the maximum applied load or displace­ inhibit data analysis (Figs. 14-3B).
ment, which depends on the research question being asked and the If the material exhibits adhesion to the probe (as observed as nega­
material being characterized (Figs. 13-2b). Shallow indentations require tive forces on the indenter probe during unloading), the sample should
smooth surfaces to avoid artifacts of surface roughness while substrate be tested in displacement control using a modified displacement profile
effects can become an issue when performing deep indentations into the that includes a lift-off at the start and end of the indent (Figs. 13-2c).
sample. Additionally, the desired penetration depth may also depend on This allows collection of the full interaction between the probe and the
the sample, as some materials can exhibit depth dependent properties. sample, starting from snap-to-contact and ending after pull-off
Finally, the combination of peak load or displacement, sample stiffness, (Figs. 14–4). This full data is required to extract accurate modulus

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

Fig. 14. Part III: This portion of the framework illustrates the potential challenges manifested in load-displacement curves. Further, the framework provides solutions
for addressing these challenges. Finally, this framework focuses on the data analysis methods that should be employed after indentation testing to ascertain the
mechanical properties, showing that the type of model used will depend on whether adhesion is present.

values during data analysis using the nano-JKR method. Alternatively, the indent before the probe is in contact with the sample). The high
the researcher can engage measures to reduce or eliminate the adhesive set-point method should only be used in conjunction with a displace­
interaction between the probe and sample, such as testing in a surfactant ment profile with an initial lift-off as a high set-point force will lead to
solution or coating the probe with a material that reduces adhesion. substantial sink-in, sometimes greater than 1 μm in a soft sample. Hence,
Accurate surface detection is a prevalent challenge when charac­ the initial lift-off will be needed for accurate determination of the zero
terizing the surface properties of soft (or hydrated) samples, due to the point of displacement for data analysis.
low contact stiffness when testing soft materials, and, in the case of In addition to surface detection challenges, experienced nano­
hydrated samples, capillary forces acting on the indenter probe. As a indentation researchers have acknowledged other sources of errors
result, some researchers have implemented the high-set point method to when characterizing soft and hydrated biomaterials – surface roughness,
find the surface and avoid false engagement under these conditions contaminated probe, adhesion, false engagement, and substrate effects.
(Figs. 13-2c). Using the set-point method to detect the sample surface, The experimental errors are manifested in the load-displacement
the indenter approaches the surface until a small predetermined set- behavior (illustrated in Figs. 14–3). Suggestions of changes to make in
point force is detected, typically 0.1–2 μN (Kaufman and Klapperich, sample preparation and/or experimental set-up to overcome these
2009; Ebenstein, 2011; Deuschle et al., 2007; Fischer-Cripps, 2000; challenges are explained in detail in the text (Section 3 and Section 4,
VanLandingham et al., 2001). Since transient capillary forces can exceed Fig. 11) and summarized in Figs. 14-3B.
these typical values, when testing in fluid some researchers have used As nanoindentation continues to rise in utility for testing soft poly­
set-point loads as high as 10 μN to avoid false engagement (triggering mers, it becomes imperative to recognize the assumptions and

17
S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

limitations in the traditional methods for analyzing load-displacement materials.

curves, and consider other contact mechanics models for analyzing
data. The presence of adhesion requires application of adhesion models CRediT authorship contribution statement
for analysis of nanoindentation data, as Oliver-Pharr and Hertz models
all assume negligible adhesion (Fig. 14). Choosing between the MD, Sofia E. Arevalo: Writing – review & editing, Writing – original
DMT and JKR adhesion models depends on the value calculated for draft, Visualization, Methodology, Investigation, Formal analysis,
Tabor’s parameter. Per review of the literature (Table 8), nano-JKR is Conceptualization. Donna M. Ebenstein: Writing – review & editing,
the most commonly used model for analyzing load-displacement curves Visualization, Conceptualization. Lisa A. Pruitt: Writing – review &
that exhibit adhesion (measured via depth sensing indentation), while editing, Supervision.
the other adhesion-based contact theories are more generally employed
in AFM. More details on how to implement nano-JKR analysis can be Declaration of competing interest
found in the text (Section 3.5.3, Fig. 10).
In summation, as nanoindentation continues to rise in utility and The authors declare that they have no known competing financial
expand across many disciplines from medicine to materials science, it interests or personal relationships that could have appeared to influence
remains of utmost importance to understand the multifactorial param­ the work reported in this paper.
eters involved in nanoindentation of soft or hydrated materials. This
decision framework can serve as a guide for novice researchers in the Data availability
field of nanoindentation to facilitate determination and dissemination of
reliable, accurate nanoindentation moduli when testing soft or hydrated No data was used for the research described in the article.

APPENDIX
APPENDIX A
Seminal papers that provide the basis for utilizing the main equations enabling for analysis of load-displacement curves to determine the mechanical properties using
nanoindentation

Reference Summary

I.N. Sneddon, 1986 (Poon et al., 2008) The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile
Derives a solution of the axisymmetric Boussinesq problem that deduces simple formulae for the depth of penetration of the
probe for a punch of arbitrary profile and the total applied load to achieve this penetration.
M.F. Doerner, W.D. Nix, 1986 (Doerner and Nix, 1986) A method for interpreting the data from depth-sensing indentation instruments
Describes an early method for determining the hardness and young’s modulus from depth-sensing indentation data. The
initial one-third of the unloading region is used to calculate the stiffness of the material.
J.L Loubet, J.M Georges, O. Marchesini, G. Meille, Vickers Indentation Curves of Magnesium Oxide
1984 (Loubet et al., 1984) Investigation of the load-displacement curve to understand the role of plasticity and elasticity, and determining the physical
meaning of Vickers hardness by using residual indentation measurements
A.E.H. Love, 1939 (Love, 1939) Boussinesq’s problem for a rigid cone
Using Boussinesq’s equations to find a solution for the cases when the surface of the rigid body is a surface of revolution
N.A. Stillwell, D. Tabor, 1961 (Stilwell and Tabor, Elastic recovery of conical indentations
1961) This paper discusses the formation of plastic indentation when a hard conical indenter is pressed into a metal. Further, an
analytical expression in terms of material’s elastic constants shows an agreement between the theoretical and observed depth
changes. The calculated energy released when the indenter is withdrawn is shown to account for the elastic rebound, which
is observed when a conical probe indents an elasto-plastic metal.

APPENDIX B
Seminal papers for the development and advancement of the Oliver and Pharr indentation theory.

Reference Summary

G.M Pharr, A. Bolkashov (Pharr and Understanding Nanoindentation unloading curves

Bolshakov, 2002) Developing a conceptual framework to explain the mathematical form of experimental nanoindentation unloading curves obtained
using a sharp, geometrically self-similar indenter (pyramids and cones)
G.M Pharr, W.C Oliver (Oliver and Pharr, Measurement of thin film mechanical properties using nanoindentation
1992a) Discusses the method for analyzing and procuring mechanical properties of thin-films
JC. Hay, A. Bolshakov, GM. Pharr (Hay et al., A critical examination of the fundamental relations used in the analysis of nanoindentation data
1999) An examination of Sneddon’s solution for indentation by a rigid cone to address the largely ignored features that affect
nanoindentation properties. A Finite element and analytical results are presented to show correction to Sneddon’s equations.
GM. Pharr, WC. Oliver, FR. Brotzen (Oliver and On the generality of the relationship among contact stiffness, contact area, and elastic modulus during indentation
Brotzen, 1992) Using the result of sneddon’s analysis, this paper shows a relationship between contact stiffness, contact area and the elastic
modulus that is independent on the geometry of the punch.
TY. Tsui, WC. Oliver, GM. Pharr (Tsui et al., Indenter geometry effects on the measurement of mechanical properties by nanoindentation with sharp indenters
1996) Discussion on the application of sharp indenters in the measurement of hardness and elastic modulus using nanoindentation
methods and addresses some of the challenges

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S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

APPENDIX C
Seminal papers for the development and advancement of the adhesion based nanoindentation theory.

Reference Summary

K.L Johnson, K. Kendall, Surface energy and the contact of elastic solids
A.D. Roberts, 1971 (Johnson et al., A discussion on the influence of surface energy on the contact between elastic solids. This paper derives the equations to determine the
1971) effect of contact size and the force of adhesion between two spherical solid surfaces. Experiments on rubber and gelatine spheres were
performed to support theoretical findings.
B.V. Derjaguin, V.M. Effect of contact deformations on the adhesion of particles
Muller, Y.P.Toporov, 1975 (Derjaguin Develops a theory that identifies the influence of the contact deformation and molecular attraction of a ball and a plane. The theory shows
et al., 1975) that forces required to overcome the molecular forces when the contact is broken does not increase, regardless of the van der Waals forces
being capable of increasing the elastic contact area. Rather, it remains equal to the attraction force value between the point contact of a
non-deformed ball with a plane.
D. Tabor. J., 1977 (Tabor, 1977a, 1977b) Surface forces and surface interactions
A discussion on the theoretical problems relating to surface problems, particularly the calculation of interaction energies between
condensed phases in atomic contact. The paper provides an experiment on the adhesion between soft elastic solids, and highlights the
importance of implementing concepts from surface energies with principles of contact mechanics.
D. Maugis, 1992 (Maugis, 1992) Adhesion of spheres: The JKR-DMT transition using a Dugdale model
A brief overview of the development and derivation of the adhesion based contact mechanics that are essential to the analysis of
nanoindentation data.

References Ebenstein, D.M., Pruitt, L.A., 2004. Nanoindentation of soft hydrated materials for
application to vascular tissues. J. Biomed. Mater. Res., Part A 69, 222–232. https://
doi.org/10.1002/jbm.a.20096.
Arevalo, S.E., Pruitt, L.A., 2020. Nanomechanical analysis of medical grade PEEK and
Ebenstein, D.M., Pruitt, L.A., 2006. Nanoindentation of biological materials. Nano Today
carbon fiber-reinforced PEEK composites. J. Mech. Behav. Biomed. Mater. 111
1, 26–33. https://doi.org/10.1016/S1748-0132(06)70077-9.
https://doi.org/10.1016/j.jmbbm.2020.104008.
Ebenstein, D.M., Wahl, K.J., 2006. A comparison of JKR-based methods to analyze quasi-
Armitage, O.E., Oyen, M.L., 2017. Indentation across interfaces between stiff and
static and dynamic indentation force curves. J. Colloid Interface Sci. 298, 652–662.
compliant tissues. Acta Biomater. 56, 36–43. https://doi.org/10.1016/j.
https://doi.org/10.1016/j.jcis.2005.12.062.
actbio.2016.12.036.
Ebenstein, D.M., Chapman, J.M., Li, C., Saloner, D., Rapp, J., Pruitt, L.A., 2002. Assessing
Barnoush, A., Welsch, M.T., Vehoff, H., 2010. Correlation between dislocation density
structure-property relations of diseased tissues using nanoindentation and FTIR.
and pop-in phenomena in aluminum studied by nanoindentation and electron
Mater. Res. Soc. Symp. Proc. 711, 47–52. https://doi.org/10.1557/proc-711-ff4.2.1.
channeling contrast imaging. Scripta Mater. 63, 465–468. https://doi.org/10.1016/
Ebenstein, D.M., Coughlin, D., Chapman, J., Li, C., Pruitt, L.A., 2009. Nanomechanical
j.scriptamat.2010.04.048.
properties of calcification, fibrous tissue, and hematoma from atherosclerotic
Briscoe, B.J., Fiori, L., Pelillo, E., 1998a. Nano-indentation of polymeric surfaces. J. Phys.
plaques. J. Biomed. Mater. Res., Part A 91, 1028–1037. https://doi.org/10.1002/
D Appl. Phys. 31, 2395–2405. https://doi.org/10.1088/0022-3727/31/19/006.
jbm.a.32321.
Briscoe, B.J., Fiori, L., Pelillo, E., 1998b. Nano-indentation of polymeric surfaces. J. Phys.
Edward Hoffler, C., Edward Guo, X., Zysset, P.K., Goldstein, S.A., 2005. An application of
D Appl. Phys. 31, 2395–2405. https://doi.org/10.1088/0022-3727/31/19/006.
nanoindentation technique to measure bone tissue lamellae properties. J. Biomech.
Buffinton, C.M., Tong, K.J., Blaho, R.A., Buffinton, E.M., Ebenstein, D.M., 2015a.
Eng. 127, 1046–1053. https://doi.org/10.1115/1.2073671.
Comparison of mechanical testing methods for biomaterials: pipette aspiration,
Farine, M., 2013. Instrumented Indentation of Soft Materials and Biological Tissues. ETH
nanoindentation, and macroscale testing. J. Mech. Behav. Biomed. Mater. 51,
Zurich. https://doi.org/10.3929/ethz-a-010051403.
367–379. https://doi.org/10.1016/j.jmbbm.2015.07.022.
Fischer-Cripps, A., 2000. A review of analysis methods for sub-micron indentation
Buffinton, C.M., Tong, K.J., Blaho, R.A., Buffinton, E.M., Ebenstein, D.M., 2015b.
testing. Vacuum 58, 569–585. https://doi.org/10.1016/S0042-207X(00)00377-8.
Comparison of mechanical testing methods for biomaterials: pipette aspiration,
Fischer-Cripps, A.C., 2006. Critical review of analysis and interpretation of
nanoindentation, and macroscale testing. J. Mech. Behav. Biomed. Mater. 51,
nanoindentation test data. Surf. Coating. Technol. 200, 4153–4165. https://doi.org/
367–379. https://doi.org/10.1016/j.jmbbm.2015.07.022.
10.1016/j.surfcoat.2005.03.018.
Cao, Y., Yang, D., Soboyejoy, W., 2005. Nanoindentation method for determining the
Fischer-Cripps, A.C., 2011. Nanoindentation, third ed. Springer-Verlag New York, New
initial contact and adhesion characteristics of soft polydimethylsiloxane. J. Mater.
York. https://doi.org/10.1007/978-1-4419-9872-9.
Res. 20 https://doi.org/10.1557/JMR.2005.0256, 2004–2011.
Franke, O., Göken, M., Meyers, M.A., Durst, K., Hodge, A.M., 2011. Dynamic
Carrillo, F., Gupta, S., Balooch, M., Marshall, S.J., Marshall, G.W., Pruitt, L., Puttlitz, C.
nanoindentation of articular porcine cartilage. Mater. Sci. Eng. C. 31, 789–795.
M., 2005. Nanoindentation of polydimethylsiloxane elastomers: effect of
https://doi.org/10.1016/j.msec.2010.12.005.
crosslinking, work of adhesion, and fluid environment on elastic modulus. J. Mater.
Galli, M., Comley, K.S.C., Shean, T.A.V., Oyen, M.L., 2009. Viscoelastic and poroelastic
Res. 20, 2820–2830. https://doi.org/10.1557/JMR.2005.0354.
mechanical characterization of hydrated gels. J. Mater. Res. 24, 973–979. https://
Chang, A.C., Der Liao, J., Liu, B.H., 2016. Practical assessment of nanoscale indentation
doi.org/10.1557/jmr.2009.0129.
techniques for the biomechanical properties of biological materials. Mech. Mater.
Grunlan, J.C., Xia, X., Rowenhorst, D., Gerberich, W.W., 2001. Preparation and
98, 11–21. https://doi.org/10.1016/j.mechmat.2016.03.005.
evaluation of tungsten tips relative to diamond for nanoindentation of soft materials.
Chen, L., Ahadi, A., Zhou, J., Ståhl, J.E., 2013. Modeling effect of surface roughness on
Rev. Sci. Instrum. 72, 2804–2810. https://doi.org/10.1063/1.1370564.
nanoindentation tests. Procedia CIRP 8, 334–339. https://doi.org/10.1016/j.
Gupta, S., Carrillo, F., Li, C., Pruitt, L., Puttlitz, C., 2007. Adhesive forces significantly
procir.2013.06.112.
affect elastic modulus determination of soft polymeric materials in nanoindentation.
Ciavarella, M., Joe, J., Papangelo, A., Barber, J.R., 2019. The role of adhesion in contact
Mater. Lett. 61, 448–451. https://doi.org/10.1016/j.matlet.2006.04.078.
mechanics. J. R. Soc. Interface 16. https://doi.org/10.1098/rsif.2018.0738.
Gupta, S., Lin, J., Ashby, P., Pruitt, L., 2009. A fiber reinforced poroelastic model of
Cuy, J.L., Mann, A.B., Livi, K.J., Teaford, M.F., Weihs, T.P., 2002. Nanoindentation
nanoindentation of porcine costal cartilage: a combined experimental and finite
mapping of the mechanical properties of human molar tooth enamel. Arch. Oral Biol.
element approach. J. Mech. Behav. Biomed. Mater. 2, 326–338. https://doi.org/
47, 281–291. https://doi.org/10.1016/S0003-9969(02)00006-7.
10.1016/j.jmbbm.2008.09.003.
Derjaguin, B.V., Muller, V.M., Toporov, Y.P., 1975. Effect of contact deformation on the
Haque, F., 2003a. Application of nanoindentation development of biomedical to
adhesion of elastic solids. J. Colloid Interface Sci. 53, 314–326. http://linkinghub.
materials. Surf. Eng. https://doi.org/10.1179/026708403322499173.
elsevier.com/retrieve/pii/0021979775900181%
Haque, F., 2003b. Application of nanoindentation to development of biomedical
0Apapers3://publication/doi/10.1016/0021-9797(75)90018-1.
materials. Surf. Eng. 19, 255–268. https://doi.org/10.1179/026708403322499173.
Deuschle, J., Enders, S., Arzt, E., 2007. Surface detection in nanoindentation of soft
Hay, J.C., Bolshakov, A., Pharr, G., 1999. A critical examination of the fundamental
polymers. J. Mater. Res. 22, 3107–3119. https://doi.org/10.1557/jmr.2007.0394.
relations used in the analysis of nanoindentation data. J. Mater. Res. 14, 2296–2305.
Dey, A., Chakraborty, R., Mukhopadhyay, A.K., 2011. Nanoindentation of soda lime-
Hodzic, A., Stachurski, Z., Kim, J., 1999. Nano-indentation of polymer-glass interfaces
silica glass: effect of loading rate. Int. J. Appl. Glass Sci. 2, 144–155. https://doi.org/
PartI. Experimental and mechanical Analysis. Polymer (Guildf) 6895–6905.
10.1111/j.2041-1294.2011.00046.x.
il Jang, J., Pharr, G.M., 2008. Influence of indenter angle on cracking in Si and Ge during
Doerner, M.F., Nix, W.D., 1986. A method for interpreting the data from depth-sensing
nanoindentation. Acta Mater. 56, 4458–4469. https://doi.org/10.1016/j.
indentation instruments. J. Mater. Res. 1.
actamat.2008.05.005.
Dohnalík, P., Pichler, B.L.A., Zelaya-Lainez, L., Lahayne, O., Richard, G., Hellmich, C.,
Jin, C., Ebenstein, D.M., 2017. Nanoindentation of compliant materials using Berkovich
2021. Micromechanics of dental cement paste. J. Mech. Behav. Biomed. Mater. 124,
tips and flat tips. J. Mater. Res. 32, 435–450. https://doi.org/10.1557/
104863 https://doi.org/10.1016/j.jmbbm.2021.104863.
jmr.2016.483.
Ebenstein, D.M., 2011. Nano-JKR force curve method overcomes challenges of surface
Jin, C., Wang, Z., Volinsky, A.A., Sharfeddin, A., Gallant, N.D., 2016. Mechanical
detection and adhesion for nanoindentation of a compliant polymer in air and water.
characterization of crosslinking effect in polydimethylsiloxane using
J. Mater. Res. 26, 1026–1035. https://doi.org/10.1557/jmr.2011.42.

19
S.E. Arevalo et al. Journal of the Mechanical Behavior of Biomedical Materials 134 (2022) 105384

nanoindentation. Polym. Test. 56, 329–336. https://www.sciencedirect.com/scienc Oliver, W.C., Pharr, G.M., 1992b. An improved technique for determining hardness and
e/article/pii/S0142941816309680. elastic modulus using load and displacement sensing indentation experiments.
Johnson, K.L., Kendall, K., Roberts, A.D., 1971. Surface energy and the contact of elastic J. Mater. Res. 7, 1564–1583.
solids. Proc. R. Soc. A. 301–313. https://doi.org/10.1088/0022-3727/6/9/304. Oliver, W.C., Pharr, G.M., 2010. Nanoindentation in materials research: past, present,
Kalcioglu, Z., Qu, M., Van Vliet, K.J., Strawhecker, K.E., VanLandingham, M.R., 2010. In: and future. MRS Bull. 35, 897–907. https://doi.org/10.1557/mrs2010.717.
Multiscale Characterization of Relaxation Times of Tissue Surrogate Gels and Soft Oral, E., Wannomae, K.K., Hawkins, N., Harris, W.H., Muratoglu, O.K., 2004.
Tissues, 27th Army Conf, pp. 1–7. http://vvgroup.scripts.mit.edu/WP/wp-content/ α-Tocopherol-doped irradiated UHMWPE for high fatigue resistance and low wear.
uploads/2010/06/27thArmyScienceConferenceManuscript_FP001.pdf. Biomaterials 25, 5515–5522. https://doi.org/10.1016/j.biomaterials.2003.12.048.
Kariem, H., Pastrama, M.I., Roohani-Esfahani, S.I., Pivonka, P., Zreiqat, H., Hellmich, C., Oyen, M.L., 2011. Handbook of Nanoindentation with Biological Applications, first ed.
2015. Micro-poro-elasticity of baghdadite-based bone tissue engineering scaffolds: a Taylor & Francis.
unifying approach based on ultrasonics, nanoindentation, and homogenization Oyen, M.L., 2015. Nanoindentation of hydrated materials and tissues. Curr. Opin. Solid
theory. Mater. Sci. Eng. C. 46, 553–564. https://www.sciencedirect.com/science/ State Mater. Sci. 19, 317–323. https://doi.org/10.1016/j.cossms.2015.03.001.
article/pii/S0928493114006924. Oyen, M.L., Cook, R.F., 2009. A practical guide for analysis of nanoindentation data.
Katti, K.S., Mohanty, B., Katti, D.R., 2006. Nanomechanical properties of nacre. J. Mater. J. Mech. Behav. Biomed. Mater. 2, 396–407. https://doi.org/10.1016/j.
Res. 21, 1237–1242. https://doi.org/10.1557/jmr.2006.0147. jmbbm.2008.10.002.
Kaufman, J.D., Klapperich, C.M., 2009. Surface detection errors cause overestimation of Oyen, M.L., Shean, T.A.V., Strange, D.G.T., Galli, M., 2012. Size effects in indentation of
the modulus in nanoindentation on soft materials. J. Mech. Behav. Biomed. Mater. hydrated biological tissues. J. Mater. Res. 27, 245–255. https://doi.org/10.1557/
https://doi.org/10.1016/j.jmbbm.2008.08.004. jmr.2011.322.
Kaufman, J.D., Song, J., Klapperich, C.M., 2007. Nanomechanical analysis of bone tissue Perepelkin, N.V., Borodich, F.M., Kovalev, A.E., Gorb, S.N., 2020. Depth-sensing
engineering scaffolds. J. Biomed. Mater. Res., Part A 81A, 611–623. https://doi.org/ indentation as a micro and nanomechanical approach to characterisation of
10.1002/jbm.a.30976. mechanical properties of soft, biological, and biomimetic materials. Nanomaterials
Kaufman, J.D., Miller, G.J., Morgan, E.F., Klapperich, C.M., 2008. Time-dependent 10. https://doi.org/10.3390/nano10010015.
Mechanical Characterization of Poly(2-Hydroxyethyl Methacrylate) Hydrogels Using Pharr, G.M., Bolshakov, A., 2002. Understanding nanoindentation unloading curves.
Nanoindentation and Unconfined Compression, 23, pp. 1472–1481. https://doi.org/ J. Mater. Res. 17, 2660–2671. https://doi.org/10.1557/JMR.2002.0386.
10.1557/jmr.2008.0185. Poon, B., Rittel, D., Ravichandran, G., 2008. An analysis of nanoindentation in linearly
Klapperich, C., Komvopoulos, K., Pruitt, L., 2001. Nanomechanical properties of elastic solids. Int. J. Solid Struct. https://doi.org/10.1016/j.ijsolstr.2008.07.021.
polymers determined from nanoindentation experiments. J. Tribol. https://doi.org/ Qian, L., Zhao, H., 2018. Nanoindentation of soft biological materials. Micromachines 9.
10.1115/1.1330736. https://doi.org/10.3390/mi9120654.
Klapperich, C., Pruitt, L., Komvopoulos, K., 2002. Nanomechanical properties of Rho, J.Y., Pharr, G.M., 1999. Effects of drying on the mechanical properties of bovine
energetically treated polyethylene surfaces. J. Mater. Res. https://doi.org/10.1557/ femur measured by nanoindentation. J. Mater. Sci. Mater. Med. 10, 485–488.
JMR.2002.0059. https://doi.org/10.1023/A:1008901109705.
Kohn, J.C., Ebenstein, D.M., 2013. Eliminating adhesion errors in nanoindentation of Saha, R., Nix, W.D., 2002. Effects of the substrate on the determination of thin film
compliant polymers and hydrogels. J. Mech. Behav. Biomed. Mater. 20, 316–326. mechanical properties by nanoindentation. Acta Mater. 50, 23–38. https://doi.org/
https://doi.org/10.1016/j.jmbbm.2013.02.002. 10.1016/S1359-6454(01)00328-7.
Korhonen, R.K., Laasanen, M.S., Töyräs, J., Helminen, H.J., 2002. Jurvelin, superficial Simha, N.K., Jin, H., Hall, M.L., Chiravarambath, S., Lewis, J.L., 2007. Effect of indenter
collagen network modifies differently equilibrium response of articular cartilage in size on elastic modulus of cartilage measured by indentation. J. Biomech. Eng. 129,
unconfined compression and indentation. Trans. Orthop. Res. Soc. 27, 903–909. htt 767–775. https://doi.org/10.1115/1.2768110.
p://luotain.uef.fi/content/abstracts/ORS/0079.pdf. Slaboch, C.L., Alber, M.S., Rosen, E.D., Ovaert, T.C., 2012. Mechano-rheological
Lakes, R., 1993. Materials with structural hierarchy. Nature 361, 511–515, 511–515. htt properties of the murine thrombus determined via nanoindentation and finite
ps://www.nature.com/articles/361511a0.pdf. element modeling. J. Mech. Behav. Biomed. Mater. 10, 75–86. https://doi.org/
Liao, Q., Huang, J., Zhu, T., Xiong, C., Fang, J., 2010. A hybrid model to determine 10.1016/j.jmbbm.2012.02.012.
mechanical properties of soft polymers by nanoindentation. Mech. Mater. https:// Sneddon, I.N., 1965. The relation between load and penetration in the axisymmetric
doi.org/10.1016/j.mechmat.2010.09.005. boussinesq problem for a punch of arbitrary profile. Int. J. Eng. Sci. 3, 47–57.
Liu, K., VanLandingham, M.R., Ovaert, T.C., 2009. Mechanical characterization of soft https://doi.org/10.1016/0020-7225(65)90019-4.
viscoelastic gels via indentation and optimization-based inverse finite element Stilwell, N.A., Tabor, D., 1961. Elastic recovery of conical indentations. Proc. Phys. Soc.
analysis. J. Mech. Behav. Biomed. Mater. 2, 355–363. https://doi.org/10.1016/j. 78, 169–179. https://doi.org/10.1088/0370-1328/78/2/302.
jmbbm.2008.12.001. Tabor, D., 1977a. Surface forces and surface interactions. J. Colloid Interface Sci. 58,
Loubet, J.L., Georges, J.M., Marchesini, O., Meille, G., 1984. VICKERS INDENTATION 2–13. https://doi.org/10.1016/0021-9797(77)90366-6.
CURVES OF MAGNESIUM OXIDE (MgO). J. Tribol. Trans. ASME 106, 43–48. Tabor, D., 1977b. Surface Forces and Surface Interactions. ACADEMIC PRESS, INC.
Love, A.E.H., 1939. Boussinesq’s problem for a rigid cone. Q. J. Math. os- 10, 161–175. https://doi.org/10.1016/b978-0-12-404501-9.50009-2
https://doi.org/10.1093/qmath/os-10.1.161. Tang, B., Ngan, A.H.W., 2007. Nanoindentation measurement of mechanical properties
Ma, Y., Ye, G., Hu, J., 2017. Micro-mechanical properties of alkali-activated fly ash of soft solid covered by a thin liquid film. Soft Mater. 5, 169–181. https://doi.org/
evaluated by nanoindentation. Construct. Build. Mater. 147, 407–416. https://doi. 10.1080/15394450701538919.
org/10.1016/j.conbuildmat.2017.04.176. Tong, K.J., Ebenstein, D.M., 2015. Comparison of spherical and flat tips for indentation
Malito, L.G., Arevalo, S., Kozak, A., Spiegelberg, S., Bellare, A., Pruitt, L., 2018. Material of hydrogels. JOM (J. Occup. Med.) 67, 713–719. https://doi.org/10.1007/s11837-
properties of ultra-high molecular weight polyethylene: comparison of tension, 015-1332-9.
compression, nanomechanics and microstructure across clinical formulations. Tsui, T.Y., Oliver, W.C., Pharr, G.M., 1996. Indenter geometry effects on the
J. Mech. Behav. Biomed. Mater. https://doi.org/10.1016/j.jmbbm.2018.03.029. measurement of mechanical properties by nanoindentation with sharp indenters.
Mann, A.B., Pethica, J.B., 1996. Nanoindentation studies in a liquid environment. Mater. Res. Soc. Symp. Proc. 436, 147–152. https://doi.org/10.1557/proc-436-147.
Langmuir 12, 4583–4586. https://doi.org/10.1021/la950901z. VanLandingham, M.R., Villarrubia, J.S., Guthrie, W.F., Meyers, G.F., 2001.
Mao, W.G., Shen, Y.G., Lu, C., 2011. Nanoscale elastic-plastic deformation and stress Nanoindentation of polymers: an overview. Macromol. Symp. 167, 15–44. https://
distributions of the C plane of sapphire single crystal during nanoindentation. J. Eur. doi.org/10.1002/1521-3900(200103)167:1<15::AID-MASY15>3.0.CO;2-T.
Ceram. Soc. 31, 1865–1871. https://doi.org/10.1016/j.jeurceramsoc.2011.04.012. Wang, Z., Volinsky, A.A., Gallant, N.D., 2015. Nanoindentation study of
Maugis, D., 1992. Adhesion of spheres: the JKR-DMT transition using a dugdale model. polydimethylsiloxane elastic modulus using berkovich and flat punch tips. J. Appl.
J. Colloid Interface Sci. https://doi.org/10.1016/0021-9797(92)90285-T. Polym. Sci. 132 https://doi.org/10.1002/app.41384.
Oliver, W.C., Brotzen, F.R., 1992. On the generality of the relationship among contact Zysset, P., Guo, X., Hoffler, C., Moore, K., 1999. Elastic modulus and hardness of cortical
stiffness, contact area, and elastic modulus during indentation. J. Mater. Res. 7, and trabecular bone lmamellae measured by nanoindentation in the human femur.
613–617. https://doi.org/10.1557/JMR.1992.0613. J. Biomech. 32, 1005–1012.
Oliver, G.M., Pharr, W.C., 1992a. Measurement of thin film mechanical properties using
nanoindentation. MRS Bull. 17, 28–33.

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