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Contents lists available at ScienceDirect

Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio

Full length article

Correlating the microstructural architecture and macrostructural

behaviour of the brain
Mayra Hoppstädter a, Denise Püllmann a, Robert Seydewitz a, Ellen Kuhl b, Markus Böl a,∗
a
Institute of Mechanics and Adaptronics, Technische Universität Braunschweig, Braunschweig D-38106, Germany
b
Departments of Mechanical Engineering and Bioengineering, Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, United States

a r t i c l e i n f o a b s t r a c t

Article history: The computational simulation of pathological conditions and surgical procedures, for example the re-
Received 15 April 2022 moval of cancerous tissue, can contribute crucially to the future of medicine. Especially for brain surgery,
Revised 2 August 2022
these methods can be important, as the ultra-soft tissue controls vital functions of the body. However,
Accepted 16 August 2022
the microstructural interactions and their effects on macroscopic material properties remain incompletely
Available online xxx
understood. Therefore, we investigated the mechanical behaviour of brain tissue under three different
Keywords: deformation modes, axial tension, compression, and semi-confined compression, in different anatomical
Microstructural analyses regions, and for varying axon orientation. In addition, we characterised the underlying microstructure in
Histological investigation terms of myelin, cells, glial cells and neuron area fraction, and density. The correlation of these quantities
Axial tension tests with the material parameters of the anisotropic Ogden model reveals a decrease in shear modulus with
Axial compression tests increasing myelin area fraction. Strikingly, the tensile shear modulus correlates positively with cell and
Semi-confined compression tests
neuronal area fraction (Spearman’s correlation coefficient of rs = 0.40 and rs = 0.33), whereas the com-
Material modelling
Sus scrofa domesticus
pressive shear modulus decreases with increasing glial cell area (rs = −0.33). Our study finds that tissue
non-linearity significantly depends on the myelin area fraction (rs = 0.47), cell density (rs = 0.41) and glial
cell area (rs = 0.49). Our results provide an important step towards understanding the micromechanical
load transfer that leads to the non-linear macromechanical behaviour of the brain.

Statement of significance

Within this article, we investigate the mechanical behaviour of brain tissue under three different defor-
mation modes, in different anatomical regions, and for varying axon orientation. Further, we characterise
the underlying microstructure in terms of various constituents. The correlation of these quantities with
the material parameters of the anisotropic Ogden model reveals a decrease in shear modulus with in-
creasing myelin area fraction. Strikingly, the tensile shear modulus correlates positively with cell and
neuronal area fraction, whereas the compressive shear modulus decreases with increasing glial cell area.
Our study finds that tissue non-linearity significantly depends on the myelin area fraction, cell density,
and glial cell area. Our results provide an important step towards understanding the micromechanical
load transfer that leads to the non-linear macromechanical behaviour of the brain.
© 2022 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction
The brain to be part of the central nervous system is located
in the cranial cavity, where under physiological conditions it is
well protected from mechanical stress by the cerebrospinal fluid
∗
Corresponding author.
E-mail address: m.boel@tu-braunschweig.de (M. Böl).
and meninges. Abnormal loads can only occur under pathological
conditions, e.g., due to traumatic brain injuries [1–3] or diseases
such as cancer [4], the latter often requiring surgical treatment to
remove the tumor cells. Another example of such a procedure is
decompressive craniectomy, which is used to relieve the pressure
caused by brain swelling [5]. The study by Weickenmeier et al.
[6] showed that a patient-specific simulation can be used to plan
and optimise the performance of a decompressive craniectomy. To

https://doi.org/10.1016/j.actbio.2022.08.034
1742-7061/© 2022 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Please cite this article as: M. Hoppstädter, D. Püllmann, R. Seydewitz et al., Correlating the microstructural architecture and macrostruc-
tural behaviour of the brain, Acta Biomaterialia, https://doi.org/10.1016/j.actbio.2022.08.034
JID: ACTBIO
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M. Hoppstädter, D. Püllmann, R. Seydewitz et al.
use the potential of computer simulation in medicine, it crucial to
understand the mechanisms of load transfer in brain tissue. With
this knowledge, computer models of the brain can be created and
validated [7–10]. To gain this knowledge, a mechanical, experimen-
tal characterisation of the brain tissue is required.
A large number of in vitro experiments to characterise brain tis-
sue have been carried out in the past. Besides shear experiments,
which can be divided into oscillatory [11–17] and simple shear
[18–26], most studies are based on uniaxial tensile [23,26–33],
compression [19,20,23,33–45], and indentation tests [21,38,46–54].
Thereby tissue from humans [23,26,28,55], monkeys [56,57], cattles
[13,33,49,50], pigs [11,12,29,34], and rodents [32,52,58–60] were
used. Due to the different species and the fact that most studies
have developed different testing protocols, the results of the me-
chanical tests are difficult to compare. An example of this is the
determination of pre-load. Depending on the definition, there are
large differences in the maximum stress/strain. Since the extreme
softness of brain tissue implies very small forces, many studies
point to an optically determined contact point as the origin of
the force. However, this depends on many factors, which will be
discussed throughout this publication. Nevertheless, incredible in-
sights into the mechanical properties of the brain have been gained
in the last 60 years [15,61]. Most studies agree that brain tissue de-
pends on the loading rate [23,27,34,40,42,44]. Thus, an increase in
the loading rate is associated with an increase of the reaction force.
Further, the behaviour under tension and compression differs in
form and extent, which is also referred to as tension-compression
asymmetry (TCA) [23,26,27,31,33,62]. In addition, there are regional
dependencies [15,19,23,26,29,32,59], as the brain can be divided
into grey and white matter from a macroscopic point of view.
The reason for this division is the underlying microstructure, see
Section 2. The heterogeneity of this microstructure thus leads to a
change in the material properties of the brain tissue.
However, there is a lack of understanding how exactly the mi-
crostructure influences the macroscopic behaviour. It is not clear,
e.g., whether and which cells have an influence on the nonlinear
material behaviour of brain tissue. To characterise this influence,
there are micro-level studies in which individual components of
the microstructure are mechanically tested [63–69], and macrome-
chanical studies in which histological data are used to establish
a correlation between microstructural and mechanical properties
[32,50,60,70–72]. In particular, a positive correlation was found be-
tween axon volume fraction and white matter stiffness [63,64]. In
addition, Javid et al. [66] reported up to three times higher ini-
tial and long-term elastic modulus of axons compared to the ex-
tracellular matrix (ECM) in the brainstem. Studies on the mechan-
ical properties of individual neurons yielded contrasting results:
Bernick et al. [65] reported rate-dependent behaviour, while An-
thonisen et al. [69] found no statistically significant dependence.
However, these micro-level approaches are only capable of captur-
ing the mechanical properties of a single component. Studies at the
macro level, on the other hand, take into account the interaction
of the various microstructural components. E.g., Antonovaite et al.
[60] used atomic force microscopy on mouse brains in combination
with immunohistochemical staining, and found a positive correla-
tion between myelin and astrocyte content and stiffness. Further-
more, a negative correlation between general cell density, without
subdivision into the different cell types, and the shear modulus
was documented in Budday et al. [72]. However, contradictory re-
sults such as the correlation of cell density with stiffness, which
is both positive [32,71] and negative [70], suggest that a general
statement about the correlation of microstructure with mechanical
behaviour is not yet possible.
Therefore, the aim of this work is to investigate the origin of
these discrepancies by relating microstructural components to me-
chanical properties. We expect that this study will lead to a better
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understanding of the load transfer mechanisms in the brain and
will serve as a basis for the development of suitable microstruc-
tural models in the future.
2. Anatomy and microstructure of the brain
The brain is the control organ of the human body. It consists of
a three-dimensional network of about 86 billion neurons responsi-
ble for receiving and processing information [73]. The largest pro-
cessing area is the cerebral cortex, which forms the outer layer
of the cerebrum and includes the characteristic convolutions and
folds that allow for a relatively large surface area in the limited
space of the cranial cavity, see Fig. 1. It is connected to subcorti-
cal neuronal regions such as the basal ganglia, which transmit sig-
nals to and from the cortex. Transmission, in turn, occurs through
axons running through connecting regions such as the corona ra-
diata and the corpus callosum. The former connects subcortical
regions to the cortex, the latter neurons from both hemispheres.
Based on this functional difference and macroscopic appearance,
the brain is divided into grey and white matter. Fig. 1 illustrates a
coronal section of the porcine brain with the microstructural com-
positions of the above anatomical regions visualised by Klüver–
Barrera staining. The cortex, representing the grey matter, consists
mainly of neurons and the second important cell type within the
brain, the glial cells, also called neuroglia. Neuroglia are divided
into microglia and macroglia, which include astrocytes and oligo-
dendrocytes. The former take on stabilising and metabolic tasks,
the latter form a fatty sheath, called myelin, around the axons,
which enables a high transmission speed of impulses [74]. In the
staining, both cell types, neurons and glia, are highlighted in pur-
ple and differ in shape and size. The white matter is represented
by the corpus callosum and the corona radiata. Here, myelinated
axons and oligodendrocytes are arranged predominately along the
axons. This is evident from the stained samples, as the blue-stained
myelin serves as an indirect visualisation of the axons. In addition,
the purple stained glial cells line up in the direction of the myeli-
nated axons. In contrast, the basal ganglia are present in both grey
and white matter and contain both characteristic microstructures.
It is worth noting that the brain is densely packed with the cell
network and the ECM therefore occupies only 10–20% of the total
brain volume [75].
3. Materials and methods
The study was exempted from ethical committee review accord-
ing to national regulations (German Animal Welfare Act), as brains
from healthy, domestic pigs were obtained from a slaughterhouse
immediately after animal sacrifice.
3.1. Tissue sample dissection and processing
Due to the limited availability of fresh human tissue and the
anatomical similarity between human and pig brains [76,77], ex-
periments were performed on seventeen (n = 17) procine brains
(sus scrofa domestica) aged 7.2 ± 1.8 months and weighing
110 ± 10 g. To ensure damage-free transport, the brains were
left in the skull and transported to the laboratory within a max-
imum of 1 h after animal sacrifice. In the laboratory, the brains
were removed, weighed, measured, and stored at 4°C for a few
minutes until the samples were cut. During all processing steps,
the samples were continuously hydrated with Dulbecco’s phos-
phate buffered saline (DPBS). The samples for the mechanical tests
were taken from four anatomical regions, see Fig. 1: The gray mat-
ter cortex (CX), the corpus callosum (CC), and the corona radiata
(CR) of the white matter, as well the basal ganglia (BG), a mixed
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Fig. 1. Coronal section of the brain with highlighted regions from which samples were taken in the present study. The histological sections schematically show the mi-
crostructure of the brain and its components (single-arrow: neuron, double-arrow: glia cell, triple-arrows: myelin). Green lines illustrate the axon orientations. Scale bars;
black: 110 μm, white: 10 mm.

Table 1
Summary of one hundred and ninety experiments depending on the dissection region (CX, BG, CC, CR),
orientation (0°, 45°, 90°, I, II, III) and deformation state (uniaxial tension/compression, semi-confined com-
pression).

Region Orientation [°] Uniaxial tension Uniaxial compression Semi-confined compression

CX – – 14 –
BG – 18 19 –
CR 0 14 15 –
90 – 15 –
CC 0 18 14 –
45 – 6 –
90 7 15 –
I – – 6
II – – 10
III – – 10
Total 57 98 26

area of gray and white matter. For further testing, isotropic mate-
rial characteristics are assumed for the grey matter [78] and a po-
tentially anisotropic behaviour of the white matter is investigated
[21,32,79]. Consequently, samples of the CC and the CR were tested
as a function of axon alignment.
In a first step, the brains were carefully cut in the sagittal plane
to separate the two hemispheres, see small subfigure in Fig. 1.
A total of one hundred and eighty one n = 181 cuboid samples
were taken using a custom-made cutting tool, cf. Tyson et al. [80],
with an equidistant spacing of 4 mm and siliconised microtome
blades to prevent sample sticking. The corresponding sample di-
mensions can be found in the supplementary material. The num-
ber of specimen for the different deformation conditions (uniaxial
tension/compression, semi-confined compression) and loading di-
rections are listed in Table 1.
3.2. Mechanical experiments
All experiments were conducted with two axial testing ma-
chines (Zwick Z0.5, Zwick GmbH & Co. Ulm, Germany), cf. Fig. 2,
equipped with 2 N or 5 N load cells. All experiments were dis-
placement controlled regardless of the deformation state and per-
formed at a quasi-static deformation rate of 0.05 %/s. In addition,
a camera was positioned in front of the specimens during the
test, providing images with a resolution of 2048 × 2048 pixels
at a frequency of 5 Hz and recording the transverse deformation
of the specimen. This camera was used to assess the correct per-
formance of the experiments, e.g., correct contact between upper
plate/plunger and tissue. To mimic physiological conditions and
prevent the samples from drying out, all experiments were per-
formed in transparent basins filled with 37 ± 2°C tempered DPBS
solution. Due to the use of the solution, no change in sample shape
due to gravity could be observed. Finally, to minimise the effects
of tissue degradation, all experiments were performed within 12 h
post-mortem.
3.2.1. Axial tension experiments
Fifty-seven specimens (n = 57, Table 1) were subjected to an
orientation-dependent axial tensile test (index: at), see Fig. 2(a).
Specifically, two axon orientations (0°, 90°) were considered, with
the axons oriented parallel and perpendicular to the loading di-
rection. Samples were glued with superglue (Loctite® 406, Henkel
Ireland Operations and Research Ltd. Dublin, Ireland) to the up-
per stamp and the lower plate, which were covered with sandpa-
per (grain size 320) for better adhesion. More precisely, we first
glued one side of the sample to the sandpaper of the lower plate
and moved the upper stamp down to glue the opposite side of
the sample to the stamp. After a curing period of 2–3 min, the
basin was filled with tempered DPBS solution. The stamp was then
moved to the original height of the sample as measured before

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Fig. 2. Experimental setup for (a) axial tension, (b) axial compression, and (c) semi-confined compression experiments. Note, (i) and (ii) illustrate the undeformed and
deformed state, respectively. Scale bars: 10 mm.
gluing, and the mechanical test started. While the displacement u
of the upper platen was predefined, the resulting force F was mea-
sured and converted to the mean engineering stress via P = F /Aat
by division through the axial cross-sectional area Aat , measured
from a digital image which was recorded before testing. The tensile
stretch λat = 1 + u/h0 was calculated from u and the undeformed
sample height h0 . All experiments were performed up to λat = 1.5
or until tissue failure occurring prior to this.
3.2.2. Axial compression experiments
Ninety-eight (n = 98) samples were subjected to orientation-
dependent (0°, 45°, 90°) axial compression (index: ac) tests. To di-
minish friction effects, polytetrafluoroethylene (PTFE) platens were
glued on the upper custom-made light-weighted stamp and on
the lower platen. The engineering stress P = F /Aac was determined
as ratio between the measured force F and the cross-sectional
area Aac , measured from the undeformed sample. The compres-
sive stretch λac = 1 − u/h0 was calculated from the displacement
u of the upper stamp and the undeformed sample height h0 , cf.
Fig. 2(b).
3.2.3. Semi-confined compression experiments
Based on the anisotropic results under compressive loading
shown in Section 3.2.2, we additionally performed twenty-six (n =
26) semi-confined compression experiments (index: sc), sometimes
called ’plane strain compression’ [81] within the CC, see Fig. 2(c).
The experimental protocol followed established protocols for mus-
cle and tendon tissue [82–84]. Mounting the brain specimens such
that the axons were parallel to the x-, y-, or z-direction, the axons
were compressed (mode I), stretched (mode II), or kept constant
in length (mode III), respectively. To account for variability in sam-
ple dimensions, two identical test devices were used with 3 mm
spacing between the back and front plates.
Remark 1 (Homogeneous deformations - effects of specimen ge-
ometry:). Generally, it is very difficult to realise ideal homoge-
neous deformations on the very soft brain tissue, as boundary ef-
fects such as friction between the sample and the tool can influ-
ence the deformation. Thus, slight deviations from the ideal homo-
geneous state may occur. In order to approach these idealised con-
ditions, we have reduced the influence of the boundary conditions
by choosing a diameter-thickness ratio of ≤ 1 for the axial tension
experiments as proposed by Rashid et al. [42]. In case of the ax-
ial compression and semi-confined compression experiments, we
reduced the friction between the tools and between the tools and
the tissue by using materials with a smooth surface (polytetraflu-
oroethylene and polymethyl methacrylate) and by treating the sur-
faces with silicone spray before inserting the tissue sample. In an
early study [85], we showed that the friction between muscle tis-
sue and tools can be significantly reduced in this way.
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3.3. Histological investigations
To correlate microstructural architecture and mechanical be-
haviour, histological examinations were carried out on seventy-
four (nhist = 74) samples. We histologically examined the tissue ad-
jacent to the mechanical sample. However, as the anatomical re-
gions are quite small, the adjacent tissue was limited and histo-
logical samples could not be taken for each mechanical sample.
First, the preparations for the histological examinations were taken
from the immediate vicinity of the samples taken for the mechan-
ical tests. Samples were cryofixed by rapid freezing in isopentane
(−160°C) cooled with liquid nitrogen (−196°C) and and thereafter
stored in an ultra-low freezer at −80°C. For the preparation of the
actual histological sections (10 μm thickness), these were cut us-
ing a cryomicrotome at −15°C and stored in formal saline for up to
3 h. To visualise myelin, glial cells, and neurons, the sections were
stained according to the Klüver-Barrera protocol, see Supplemen-
tary material, and preserved by a mounting medium and a cover
glass. Finally, all histological sections were digitised in two steps
using a microscope (Nikon Eclipse Ni, Tokyo, Japan), equipped
with a 20× objective: First, overview images of the sample were
scanned and second, areas of interest (ROI) of 0.65 × 0.65 mm
were randomly excised to avoid artifacts such as holes that would
distort the analysis.
3.4. Data processing
3.4.1. Analysis of the mechanical data
The measured force-displacement data were stored as raw data
with a frequency of 10 Hz. Furthermore, the additional forces
caused by the hydrostatic pressure in the DPBS solution were taken
into account by calculating the force-displacement data using a
reference curve representing the movement of the plunger in the
solution. To avoid local peaks that would potentially distort post-
processing, the raw data were smoothed depending on the noise of
the acquired data. Consequently, the smoothing (function smooth,
MATLAB® R2019b, The MathWorks, Inc.) was realised by calculat-
ing the mean value over 5%, 4%, and 4% strain for axial tension,
compression, and semi-confined compression, respectively.
Pre-load used plays a special role, especially when sampling
soft, biological tissue, since the curves are shifted both horizon-
tally and vertically. In previous experiments of brain tissue, differ-
ent methods were used to account for pre-load: While most stud-
ies do not specify a specific pre-load [e.g. 12,15,22,45,86,87], other
studies define the force origin as the point when the tool comes
into contact with the tissue [e.g. 11,19,20,34,35,42,51,88]. Other au-
thors use previously defined specific pre-load levels [37,46] or glue
the sample to the tool [89]. Basically, it can be stated that all these
methods have advantages and disadvantages and that a uniform
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Fig. 3. Clustering of (a) histological sections into (b) myelin, (c1 ) cells, and (d) unstained components. Further, (c2 ) illustrates the cell cluster with counted cells highlighted
by blue circles. Scale bars: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
and comparative sampling of brain tissue is only possible to a very
limited extent without the development of new methods. How-
ever, since in the present study the experiments were performed
in DPBS solution, which caused additional noise, the use of a pre-
load is not advantageous. Therefore, the origin of the force/stress
is defined as the point where the plunger comes into contact with
the tissue sample. For this purpose, the point of contact was de-
fined in a post-processing step based on the camera and force-
displacement data as the point at which the sample experiences
a transverse strain (axial compression, semi-confined compression)
with a simultaneous significant change in the force signal. In the
tensile experiments, the origin of the force was defined by the ini-
tial sample height, which was measured optically before the test.
Finally, the elastic regions of the force-displacement curves
were determined by successively computing tangents Ti by finite
differences at the points ui , i = 1, 2, . . . , m, where m indicates the
maximum number of measuring points. The elastic range of the
curve is defined as the region where the slope is monotonically
increasing, i.e., where Ti+1 ≥ Ti . The point u j at which T j+1 < T j
marks the end point of the elastic region.
In general, measurements were discarded from the analysis if
operational disturbances occurred, e.g., the sample slipping under
pressure or no force development despite contact and incomplete
adhesion of the sample under tensile load. Furthermore, we fol-
lowed Tukey [90] and identified outliers based on the interquartile
range. Considering the maximum stresses per deformation state,
anatomical region, and axon orientation, we defined values that
were more than 1.5 interquartile ranges above the 75% and below
the 25% quartile of the respective maximum stress as outliers.
3.4.2. Processing of the histological data
Using the histological sections described in Section 3.3, we de-
termined the tissue components (myelin, cells) and the cell num-
bers (glia, neurons) in a post-processing step. To this end, the im-
ages of Klüver–Barrera stained sections were subjected to cluster-
ing based on a k-means clustering algorithm of Lloyd [91] with
seeds generated by the k-means++ algorithm of [92], which al-
lowed colour differentiation between myelin, cells, and unstained
components. In more detail, the RGB images were converted to
the L × a × b colour space, resulting in a m × n× 3 matrix. By ex-
cluding the lightness (L), the matrix reduces to m × n× 2 and the
segmentation is based only on the colour spaces (a × b). This ma-
trix of colour pixels was converted to greyscale and used as data
points for k-means clustering, which involves the following steps:
Selecting the cluster number c, randomly selecting the first cluster
centre, selecting c − 1 cluster centres with D2 weighting, determin-
ing the closest data points in the cluster using the usual Euclidean
norm, recalculating the centre based on the data points in the clus-
ter, and repeating the clustering until the changes in the centre
are less than the threshold of 10-6 [92–94]. This procedure was re-
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peated three times to avoid local minima and the result with the
lowest variance was retained. The image was divided into 12 clus-
ters using the steps described, which were manually assigned to
microstructural components. Following Fig. 3, blue portions were
assigned to myelin, purple to cells, and white or yellow portions to
unstained components. In (a), the ROI of a white matter sample is
shown with the characteristic high proportion of myelin and glial
cells aligned in the axon direction. In (b-d), the data points associ-
ated with the corresponding cluster are highlighted in the original
colour, while the others are covered with a black mask. Thus, in
(b) only the blue elements are visible as the myelin portion, in (c1 )
the cell cluster of the ROI is visible and in (d) the other uncoloured
components. Based on the clustered data points, the area fraction
of the myelin is calculated.
In addition, the cell clusters were used to determine the area
fraction ( f ) and density (ρ ) of cells, glial cells, and neurons per
area. For this purpose, the clustered cell images were converted to
greyscale in (c1 ) and binarised with rgb2gray or imbinarize, respec-
tively. Further, clustering noise (small individual pixels that are not
identified as cells) was removed with bwareaopen (maximum pixel
size of 60) and truncated cells were deleted with imclearborder. Fi-
nally, imfill and regionprops were used to fill the cells and evalu-
ate the regional properties, respectively. In (c2 ) the resulting bina-
rised image is shown. The counted cells are highlighted by blue cir-
cles, with the corresponding equivalent diameter determined from
the regional properties. To distinguish between glia and neurons,
we introduced a threshold diameter dt . This simplified approach is
based on the mean cell density ρcells of the CX and the CC, repre-
sentative of grey and white matter. We evaluated cell diameters for
each value between dt = 5–20 μm and calculated the cell density
by counting only cells whose equivalent diameter is smaller, equal
(ρcells≤dt = ρglial cells ), or larger (ρcells>dt = ρneurons ). Based on these
two cell densities, we calculated the difference Qt between the cor-
responding mean values of the CC and the CX for the tested diame-
ters, hypothesising that the maximum difference marks the thresh-
old diameter. As shown in Fig. 4, the maximum is at a diameter of
9.5 μm, indicating that the proportion of cells larger than 9.5 μm
is significantly lower in the CC compared to the CX. Consequently,
the cells larger than dt = 9.5 μm were counted as neurons and the
rest as glial cells. The corresponding area fraction was determined
by summing the counted cell areas and dividing by the ROI area.
In a final step, we calculated the mean value of the area fractions
(myelin, cells, glial cells, neurons) and densities (cells, glial cells,
neurons) over the defined ROIs.
3.5. Statistics
For the experimental stress-strain curves, mean and standard
deviation as well as coefficients of variance (CV) were determined
as the ratio of the standard deviation to the mean value depending
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4.1. Mechanical material model for brain tissue

As described in Section 3.1, depending on the region, we char-

Fig. 4. Cell density difference Qt for cells > dt (black line) and ≤ dt (green line) as a
function of the investigated border diameters dt (circles). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version
of this article.)
acterise brain tissue as either isotropic or transversely isotropic
material. Furthermore, the tissue exhibits strongly non-linear ma-
terial characteristics in combination with incompressible proper-
ties. Based on the well-known Ogden material model [96], Mero-
dio and Ogden [97] have developed a model that provides reliable
results [29] as well as a physical interpretation for the material pa-
rameters. The strain-energy function of this model
 +ψ
ψ =ψ iso aniso + U (1)
is decomposed into an isotropic, isochoric (ψ  ) and an anisotropic
iso
(ψaniso ) contribution. Further, the purely volumetric part
Kvol 2
U= (J − 1 − 2 ln(J )) (2)
4
depends on J = det F , the determinant of the deformation gradient
F , and the parameter Kvol , controlling the degree of incompressibil-
ity. The isotropic, isochoric part

 = 2μ  α 
ψ λ1 + 
λα2 + 
λα3 − 3 (3)
iso
α2
on the anatomical regions and axon orientations. In addition, the
experimental data were statistically analysed for significant differ- is represented by the isotropic Ogden material model. Herein, λ ˆα =
i
ences between the maximum stresses of the investigated anatom- J −1 / 3 α
λi with i = 1, 2, 3 represent the isochoric parts of the princi-
ical regions and axon orientations. The analyses were performed pal stretches λα i
, the shear modulus is given by μ, and α defines a
for all three deformation modes by first performing a Shapiro- dimensionless constant. Finally, the anisotropic contribution of the
Wilk test for the maximum tensile, compressive and shear stresses, strain-energy function
followed by a Wilcoxon rank sum test. In addition, the Wilcoxon
2kμ  α /2 
rank sum test was used again to determine if there was a signif- ψaniso = I4 + 2I4−α /2 − 3 (4)
icant change in both microstructural properties (myelin, cell, glial α2

cell and neuron area fraction, and cell, glial cell and neuron den- depends on the anisotropy factor k and on the fourth invariant
sity) and material properties (μ, α ) for the different brain regions
I4 = tr[C M ]. (5)
and axon orientations. To quantify the relationship between mi-
crostructure and mechanics, the non-parametric Spearman rank Herein, C = FT F
defines the right Cauchy–Green tensor and M =
correlation coefficient rs was calculated. Thus, it can be determined m0  m0 is the second-order structural tensor, the dyadic product
to what extent a linear function can represent the correlation be- of the directional tensor m0 , representing the direction of the ax-
tween microstructure properties and material parameters. Accord- ons. In dependence on the various brain regions and their depen-
ing to the classification of Cohen [95], values |rs | ≥ 0.10 indicate a dence on axon orientation described in Section 5.2.2, we consider
weak, |rs | ≥ 0.30 a medium, and |rs | ≥ 0.50 a strong effect. For all the following strain-energy combinations
statistical tests, we calculated the p values and considered values 
ψ +ψ
aniso + U for CC
smaller than 0.05 to be significant. ψ = iso (6)
ψiso + U for CX, BG, CR.
4. Material modelling and parameter identification

4.2. Model parameter identification

Basically, the goal of a mathematical modelling approach is the
phenomenological description of the mechanical behaviour. In do-
ing so, the determined model parameters must ultimately apply
to all deformation states. Here, the relationship between the mi-
crostructural information of the brain tissue and the mechanical
behaviour represented by the model parameters is investigated. For
this purpose, a minimalist model, see also Remark 2, was used that
directly links the microstructural properties with the mechanical
properties, but cannot necessarily describe all deformation states
with the same accuracy.
Remark 2 (Model selection:). Note, the choice of material model
is limited to the objective of this study, which is to directly corre-
late the mechanical properties with the microstructural properties.
Consequently, a model with a small number of material parame-
ters representing the most important properties of the mechanical
behaviour, the strength, and the waveform was needed. Further-
more, the fit of the experimental data should be accurate to avoid
errors due to an inappropriate approximation. Finally, we wanted
to use a model that could be compared with the current literature
to reveal possible similarities and discrepancies.
To successfully identify the material parameters and thus cor-
relate the mechanical properties with the microstructural compo-
nents, an inverse numerical optimisation method was used. Here,
a forward finite element analysis is coupled with an optimisation
algorithm that determines the optimal parameter vector p by min-
imising the error O ( p) between the measured and simulated re-
sponses.
For the special case of an isotropic material behaviour, the
anisotropy factor k is zero, cf. Eq. (4). To identify the parameter
vector p = (μ, α , k = 0 ), the objective function
1  sim
m
O CX,BG,CR ( p) = |Fj − Fjexp | (7)
m
j=1
minimises the error between simulated (Fjsim ) and experimental
exp
(Fj ) data. This optimisation was carried out for each individual
measurement.
For the identification of the material parameters of the
anisotropic region CC, a two-step identification approach was

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M. Hoppstädter, D. Püllmann, R. Seydewitz et al.
utilised: In the first step, the anisotropy factors were optimised us-
ing the following objective function
O CC ( p) = O0CC◦ + O45
CC CC
◦ + O90◦ .
Herein, the sub-objective functions
1  sim
m
OiCC ( p) = |Fj,i − F̄j,iexp | for i = 0◦ , 45◦ , 90◦
m
j=1
are functions of the orientation-dependent, mean experimental
exp sim . This first identifica-
curves F̄j,i and the simulated results Fj,i
tion step was performed for all three deformations modes, leading
to kCC CC CC
ac = 10.22, kat = 0.42, and ksc = 0.38. In a second step, these
parameters were fixed and the remaining parameters in p (μ, α )
were optimised via
1  sim
m
OiCC ( p) = |Fj,i − Fj,iexp | for i = 0◦ , 45◦ , 90◦
m
j=1
for each single measured force curve. The number of increments
are m = 20, 100, and 100 for the tension, compression, and semi-
confined compression experiments, respectively. For both compres-
sion and shear tests, we assume homogeneous deformation. For
tension tests, this condition is not reasonable due to the glued sur-
faces. Therefore, we used one finite element for compression and
shear, and 250 elements for tension to discretise the samples.
5. Results
In the following, the mechanical behaviour within the investi-
gated anatomical regions is examined for the influence of the mi-
crostructure and thus the microstructural and mechanical proper-
ties are related to each other.
5.1. Microstructural components
The studied anatomical regions (CX, BG, CC, CR) consist of dif-
ferent microstructural components, cf. Section 2. Using the meth-
ods described in Section 3.4.2, we quantified two main compo-
nents in terms of area fraction f and density per area ρ , namely
myelin and cells (glia, neurons). While the former evaluates the
area covered by the components, the latter provides information
on the number of cells per area. Fig. 5(a) illustrates the myelin sur-
face area fmyelin within the anatomical regions. As can be seen, the
gray matter tissue CX has no measurable myelin content. The min-
imum of the mixed gray and white matter region BG is also 0%.
Fig. 5. Region-dependent area fraction of (a) myelin, (b) cells, glial cells, and neurons as well as (c) density distribution of cells, glial cells, and neurons. Mean values are
shown as black crosses, medians as red straight lines, outliers as red plus, and the boxes as interquartile. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
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However, the BG exhibits the highest variability, showing a maxi-
mum of 89.7%. In contrast, the white matter regions (CR, CC) con-
sist mainly of myelinated axons, resulting in the highest propor-
(8) tions of myelin surface (mean: 78.1% in CC, 76.9% in CR). Statisti-
cal studies show that the CC and the CR contain significantly more
myelin than the BG and the CX.
Following Section 3.4.2, we further differentiated between glial
(9) cells and neurons using a threshold diameter (dt = 9.5 μm). In (b),
the distribution of the area fractions of glial cells and neurons
( fglial cells , fneurons ) within the anatomical regions and the corre-
sponding sum as cell area fraction ( fcells ) is shown. Within the lat-
ter, region BG shows the highest variability with a minimum of
fcells = 2.4% and a maximum of fcells = 8.3%.
Also, fcells decreases from the CX to the CR. While in the CX
an average of 5.6% of the total area is covered by cells, in the CR
it is only 3.2%. Both white matter regions (CC, CR) differ signifi-
cantly from the CX, and in addition the CR also from the BG. This
(10) decreasing trend of fcells can be attributed to the cell area cov-
ered by neurons, since fneurons is also highest in the CX and lowest
in the CR. Furthermore, the proportion of the neuron area of fcells
is 70% in the CX and only 23% in the CR and 18% in the CC. The
glial cells feature an opposite trend with the highest mean value
of fglial cells = 3.1% in CC. The differences in the area fractions of
glial cells and neurons are statistically significant between white
matter (CC, CR) and the CX. Within fneurons the BG has a signif-
icantly higher area percentage than the CC and the CR. The glial
cells in turn cover a significantly larger area in the CC compared
to the BG and the latter compared to the CX.
The cell network densities ρcells , ρglialcells , and ρneurons are
shown in (c) as a function of the different regions. Similar to fcells ,
the region BG shows the highest variability, with a range from
ρcells = 688 to 1861 1/mm2 . In contrast, the CX has the lowest with
a mean of ρcells = 893 1/mm2 and the CC with ρcells = 1477 1/
mm2 features the highest number of cells per area. These region-
dependent differences are statistically significant. Specifically, the
cells density of the CX is significantly lower than the other three
regions, and the BG and CR are also significantly different from the
CC. Comparing the proportion of glial and neurons within ρcells , it
becomes clear that small glial cells make up the largest proportion
in all anatomical regions. Thus, the number of glial cells in par-
ticular influences the cell density. Consequently, the CC contains
the highest density of glial cells with a mean value of ρglial cells =
1415 1/mm2 . In addition, the statistical differences in glial cells be-
tween the anatomical regions are identical to those in cell density.
As a result, the neuron density in gray matter (CX) is significantly
higher than in white matter (CC, CR). In addition, the BG contains
significantly more neurons than the CC and the CR, with a 53.5%
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Fig. 6. Orientation- and region-dependent stress-stretch behaviour from the tension experiments for the (a) BG, (b) CR0°, (c) CC0°, and (d) CC90°. Black curves including
circles indicate the mean experimental values, shaded areas the standard deviations, light gray the individual test curves, and the green curves indicate the fitted model
response. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and 52.3% higher mean. Interestingly, the CX features a higher ab-
solute density of glia (ρglial cells = 675 1/mm2 ) compared to neu-
rons (ρneurons = 218 1/mm2 ), although fglial cells is smaller than
fneurons . These results suggest that the white matter regions have a
more densely packed cellular network than the gray matter. How-
ever, the area percentage of the cells in the gray matter is larger.
5.2. Mechanical behaviour
5.2.1. Axial tension
Fig. 6 shows the experimental stress-strain curves and model
responses of the three regions (BG, CC, CR) as a function of axon
orientation. While the light grey curves represent the individual
experiments with their individual maximum stretches, the black
curves show the mean value of all experiments, the shaded areas
the standard deviations, and the green curve the model response.
Both the experimental mean curves and the model responses are
plotted up to the minimum stretch level, i.e., the level reached
by each sample. This level of strain, given by λat,max , is listed
in Table 2. Regardless of region and axon orientation, all curves
are characterised by a pronounced nonlinear stress-strain response.
The associated stresses for the comparable strain of λat = 1.071
are listed in Table 2. There are high variances (maximum CV of
50%) and similar stress magnitudes, with a difference between
the highest (BG) and lowest voltage (CC90°) of only 36%. The lat-
ter was statistically compared to CC0° at the comparative stretch
of 1.091 to investigate the degree of anisotropy. We observed no
significant difference and conclude that the tensile behaviour of
the CC is independent of axon orientation. Similarly, the stresses
of the anatomical regions were compared by considering the CC
as isotropic to quantify the influence of the different microstruc-
tural compositions, see Section 5.1. Despite the heterogeneous mi-
crostructure, were no significant differences between CC, BG, and
CR, indicating a homogeneous material behaviour of the brain tis-
sue. Furthermore, we fitted the mechanical behaviour with the ma-
terial model presented in Section 3 and the mean curve of the
simulated stress-strain response agrees well with the experimental
curves, see Fig. 6. Table 2 contains the mean material parameters
μat , αat , and kat as well as the coefficients of determination R2 .
Similar to the stress, the BG has the highest shear modulus with
μat = 0.59 kPa. Note that the anisotropy factor kat of the CC is not
zero, although the stresses imply an isotropic behaviour. The sta-
tistical comparison of the material parameters (μat , αat ) shows no
significant differences.

Table 2
Maximum tensile stresses Pat,max at stretches λat,max , minimum stresses Pat,min at λat,min = 1.071, mean
material parameters (μat , αat , kat ) and coefficients of determination R2 in dependence on the region
(BG, CC, CR) and axon orientation.

Region Orient. [°] λat,max [−] Pat,λat =1.071 [kPa] μat [kPa] αat [−] kat [−] R2

BG – 1.073 0.11 ± 0.05 0.59 −8.6 0.00 0.925

CC 0 1.091 0.10 ± 0.05 0.34 −1.9 0.42 0.946
CC 90 1.106 0.07 ± 0.02 0.39 −7.6 0.42 0.915
CR 0 1.071 0.08 ± 0.03 0.42 −10.0 0.00 0.883

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Fig. 7. Orientation- and region-dependent stress-stretch behaviour from the compression experiments for the (a) CX, (b) BG, (c) CR, and the (d) CC. Solid curves including
circles indicate the mean experimental data, shaded areas the standard deviations, light coloured curves the individual tests, and the green curves indicate the fitted model
response. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

5.2.2. Axial compression entation, the Wilcoxon rank sum test was applied to statistically
Fig. 7 shows the stress-strain behaviour of the investigated compare the maximum stresses of 0°, 45°, and 90°. There is a
anatomical regions (CX, BG, CR, CC) as a function of axon orienta- trend in anisotropy within the CC region, as the differences be-
tion (0°, 45°, 90°) under compressive loading. All curves are shown tween all tested axon alignments of the CC, except between CC0°
up to a compression of λac,max = 0.55. Overall, the brain tissue and CC45°, are significantly different. When axons are loaded in
shows a non-linear, exponential behaviour with different character- tension (90°), the tissue responded 245% stiffer than in compres-
istics depending on region and axon orientation. The greatest vari- sion (0°) and 215% stiffer than in with axons under an angle of
ability is found at the point of maximum load. The corresponding 45°. The CR, in turn, exhibited a 15.4% stiffer response with axons
mean stresses and standard deviations are listed in Table 3. The oriented in the direction of loading, see (c). However, this differ-
anatomical region BG has the lowest coefficient of variation (CV) ence was not significant, indicating an isotropic material behaviour.
at 31.0%, while the region CC0° has the highest at 54.8%. CC90° Therefore, in the following, both axon orientations of the CR are
reaches the maximum mean stress of 1.45 kPa, and CR0° in turn combined and statistically compared to the other isotropic regions
the second highest value (1.05 kPa), see Fig. 7(d) and (c), respec- (CX, BG). The CR and BG are significantly stiffer than the CX with
tively. In contrast, CC45° and CC0° exhibit the softest response with 66.1% and 47.5% higher maximum stresses, respectively. These re-
maximum stresses of 0.46 and 0.42 kPa, see (d). To investigate sults suggest that the heterogeneous microstructure influences the
these stiffness differences concerning a dependence on axon ori- material behaviour of brain tissue under axial compression.
Table 3 summarises the mean material parameters and coef-
ficients of determination. The agreement between simulated and
Table 3
Maximum compression stresses Pac,max at λac,max = 0.55, mean material parameters
experimental stress-strain curves is satisfactory with a lowest R2
(μac , αac , kac ) and coefficients of determination R2 in dependence on the region (CX, of 0.994 and overlapping mean curves in Fig. 7. In terms of the
BG, CC, CR) and axon orientation. shear modulus μac , the mixed region BG of grey and white matter
Region Orient. [°] Pac,max [kPa] μac [kPa] αac [−] kac [−] R2
is the stiffest. Interestingly, the CC has the lowest shear moduli,
although the CC90° is characterised by the highest stresses. This
CX – 0.59 ± 0.19 0.11 8.7 0.00 0.997
is due to the high anisotropy factor kac , which reverses the stress
BG – 0.87 ± 0.27 0.16 8.9 0.00 0.994
CR 0 1.05 ± 0.44 0.14 10.2 0.00 0.996 trend. Conversely, the nonlinearity factor is highest in the white
CR 90 0.91 ± 0.35 0.14 10.2 0.00 0.996 matter regions (CC, CR) except in CC45°. Statistical comparisons of
CC 0 0.42 ± 0.23 0.05 11.2 10.22 0.995 the anisotropic (CC0,45,90°) and isotropic (CX, CR, BG) material pa-
CC 45 0.46 ± 0.24 0.09 8.4 10.22 0.994 rameters show significant differences for μac between CX and BG.
CC 90 1.45 ± 0.76 0.06 10.2 10.22 0.999
In addition, αac is significantly different for all orientations and re-

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M. Hoppstädter, D. Püllmann, R. Seydewitz et al. Acta Biomaterialia xxx (xxxx) xxx

Fig. 8. Semi-confined compression stress-stretch response as in dependence on axon direction represented by (a) mode I, (b) mode II, and (c) mode III. Black curves including
circles indicate the mean experimental values, shaded areas the standard deviations, light gray curves the individual tests, and the green curves indicate the fitted model
response. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

gions except for CX and BG, and for CC0° and CC90°. These re-
sults suggest that compressive stress waveforms depend on axonal
alignment and the different microstructural compositions of gray
and white matter.
5.2.3. Semi-confined compression
Fig. 8 shows the stress-strain relations of the semi-confined
compression experiments as a function of axon orientations. All ex-
periments were performed up to a maximum stretch of λsc,max =
0.55 and the stress-stretch relations are characterised by a non-
linear, exponential material behaviour. Axons are oriented in (a)
x-, (b) y-, and (c) z-directions, resulting in compression (mode I),
extension (mode II), or constant length (mode III) of the ax-
ons under stress. The CV at the maximum load value is highest
for mode I at 51.9%, followed by 46.8% and 35.4% for mode III
and mode II, respectively. The corresponding mean stresses are
1.31 ± 0.68, 3.25 ± 1.15, and 3.44 ± 1.61 kPa for mode I, II and
III, from lowest to highest. These differences are statistically sig-
nificant, with modus I showing significantly softer response than
modi II and III, with the former showing a 148.1% stiffer response
and the latter a 162.6% stiffer response. In contrast, no significant
difference is found between modes II and III, suggesting that ad-
ditional microstructural components besides axons contribute to
tissue stiffness. Finally, the responses of the model presented in
Section 4 can also be seen for all three modes. It can be seen that
the model response agrees well with the experimental response
with a mean coefficient of determination of R2 = 0.992 ± 0.008.
Similar to the maximum stress, mode III has the highest mean
shear modulus and mode I has the lowest value, with μsc = 0.31
and μsc = 0.27 kPa. In contrast, mode I has a mean nonlinearity
factor αsc of -4.41 and the other two modes 3.0 (mode II) and −1.8
(mode III). The statistical comparison of these parameters did not
reveal any significant differences.
5.2.4. Comparison of deformation states with respect to region and
axon orientation
Finally, the results of the different deformation states are com-
pared with each other. For this purpose, Table 4 lists the mean
stresses and standard deviations at a comparable deformation
(minimum elastic stress strain) for all deformation states, regions,
and axon orientations. When comparing the CVs, it is noticeable
that the BG region has comparable values in tension and com-
pression with 45.5% and 41.2%. However, the CC0° shows higher
variability in compression (114.3%) and semi-confined compression
(75.0%) compared to tension (50.0%). CC90° and CR0° show a sim-
ilar trend.
Furthermore, there are differences in the stiffness of the tissue
between the deformation states. Brain tissue shows a stiffer be-
haviour when loaded under semi-confined compression than un-
der compression. In the CC0°, the maximum compressive stress of
0.007 kPa corresponds to only 17.5% of the stress in semi-confined
compression. Furthermore, the tensile stress is significantly higher
than the semi-confined compression and compression stresses. The
latter behaviour is also referred to as TCA and indicates different
microstructural load transfer mechanisms in the two deformation
modes. To illustrate the TCA, Fig. 9 shows the ratio (Pat /|Pac |) be-
tween the magnitudes of tensile stress Pat and compressive stress
|Pac | as a function of the absolute strain |u/h0 | for BG, CC0°, and
CR0°. In general, the degree of TCA decreases with increasing de-
formation. This is particularly evident in the CC0° region. Smaller
deformations show tensile stresses that are 180 times larger than
their compressive counterparts. This ratio decreases within 9.1%
strain to a TCA ratio of about 11. A similar behaviour is observed
for the CR0°, albeit less pronounced with a maximum TCA ratio of
90 at smaller deformations. Finally, the BG also shows a decreas-
ing TCA behaviour, albeit rather linear, with a maximum TCA ratio
of 12 for smaller and 6 for larger deformations. In a further step,
it was investigated whether the statistical differences discussed in

Table 4
Stresses of the three deformation states at comparable deformations (minimum elastic
tensile stretch) in dependence on region (CX, BG, CC, CR) and axon orientation.

Region Orientation [°] Pat,λat =1.071 [kPa] Pac,λac =0.929 [kPa] Psc,λsc =0.929 [kPa]

CX – – 0.015 ± 0.008 –
BG – 0.11 ± 0.05 0.017 ± 0.007 –
CR 0 0.08 ± 0.03 0.016 ± 0.007 –
CR 90 – 0.013 ± 0.004 –
CC 0 (modus I) 0.10 ± 0.05 0.007 ± 0.008 0.04 ± 0.03
CC 45 – 0.014 ± 0.008 –
CC 90 (modus II) 0.07 ± 0.02 0.016 ± 0.010 0.06 ± 0.02
CC 90 (modus III) – – 0.05 ± 0.03

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M. Hoppstädter, D. Püllmann, R. Seydewitz et al.
Fig. 9. Development of tension-compression asymmetry. Tensile-to-compressive
stress ratio versus absolute strain for the BG, CC0°, and CR0°.
Sections 5.2.2 and 5.2.3 still hold for small deformations by sta-
tistically comparing the stresses listed in Table 4. There were no
significant differences between the CX, BG, and CR under com-
pression, nor between the modes tested under semi-confined com-
pression. However, the CC90° remained significantly stiffer under
compression than the CC0° with a 128.6% higher stress. These re-
sults indicate that, at small deformations, the influence of a hetero-
geneous microstructure on the mechanical behaviour can be ne-
glected. However, for compressive loading, the axon orientation in
the CC needs to be considered, even for small deformations.
5.3. Correlation of microstructure and mechanics
To establish a direct correlation between the microstructural
architecture and the mechanical behaviour, the properties deter-
mined in the previous sections were correlated with each other.
Specifically, Spearman’s rank correlation coefficients rs were cal-
culated between the mechanical properties in terms of material
parameters (μ, α ) and the microstructural components. A distinc-
tion was made according to the deformation states uniaxial stress
(nhist,at = 28) and compression (nhist,ac = 46), as we assume dif-
ferent load transfer mechanisms as evidenced by the observed
TCA, see Section 5.2.4. For reasons of clarity, only the correlations
between the material parameters and the area fractions (myelin,
cells, glial cells, neurons) are highlighted in Fig. 10. The correla-
tion of the material parameters with the densities (cells, glial cells,
neurons) and the table with Spearman’s rank correlation coeffi-
cients rs can be found in the Supplementary material. Fig. 10(a)
shows that the tensile and compressive shear modulus decreases
with increasing myelin area fraction. The compressive correlation
coefficient correspond to a moderate effect and is statistically sig-
nificant. In contrast, the nonlinearity factor αat correlates nega-
tively with fmyelin (rs = −0.18), while αac has a significant posi-
tive correlation (rs = 0.47 with p = 0.0 0 09), see (b). However, con-
sidering |αat |, tensile and compressive nonlinearity show a sim-
ilar trend. Regardless of the deformation state, the stiffness de-
creases and the nonlinearity increases as the area covered with
myelin increases. Regarding the cell density and area fraction, a
positive correlation with the tensile shear modulus (rs = 0.36 for
ρcells , rs = 0.40 for fcells ) can be observed. The latter correlation is
significant at p = 0.04 and shown in (c). Although the maximum
stress in Section 5.2.1 has no regional dependencies, the stiffness
increases with the number of cells and the area covered by the
cells. In contrast, neither the tensile and compressive non-linearity
factor nor the compressive stiffness show significant correlations
with cell area fraction, cf. (c) and (d). However, the compressive
shear modulus significantly decreases with increasing cell den-
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sity (rs = −0.36). Furthermore, there is a mean effect between αac
and ρcells with rs = 0.41 ( p = 0.004). Interestingly, this correlations
arise from the number of glial cells, as both μac and αac correlate
significantly with ρglial cells (rs = −0.32 for μac , rs = 0.43 for αac )
and not significantly with ρneurons (rs = 0.15 for μac , rs = −0.23 for
αac ). Moreover, the area covered by glial cells correlates moder-
ately with the non-linearity factor αac , as highlighted in (f). Con-
versely, (e) shows the significant ( p = 0.03) negative correlation
between μac and fglial cells . The decrease in shear modulus with in-
creasing glial area and density is consistent with the negative cor-
relation with myelin area fraction, as glial cells form the myelin
sheath. Remarkably, the correlation of tensile stiffness with neu-
ronal area fraction shows a slightly non-significant (rs = 0.33 with
p = 0.09 for fneurons ), medium effect. These results imply that, al-
though compressive stiffness decreases with increasing glial cell
area, non-linearity increases. The latter also increases with increas-
ing number of cells and glial cells. In contrast, tensile stiffness in-
creases with increasing area covered by cells.
6. Discussion
6.1. Microstructural properties
In the present study, the area fractions of myelin and cells (glial
cells, neurons) in the makeup of brain tissue microstructure were
quantified. We found that the area fraction of myelin is highest in
the white matter regions (CC, CR), as these are mainly composed
of myelinated axons. This result is in line with studies of Budday
et al. [72] and Antonovaite et al. [60]. Moreover, the mean values
of the CC and CR (78.5 and 79.6%) are in the range of 56–81% re-
ported for bovine white matter tissues [50]. Budday et al. [72], on
the other hand, have measured a value of only 15% in human tis-
sue. The different processing of the histological data may cause this
discrepancy. In [72] a colour threshold was used, whereas in the
present study k-means clustering was used.
Strikingly, this study revealed an opposite tendency of the cell
area percentage compared to cell density: On the one hand, the
area covered with cells is largest in the grey matter and smallest
in the white matter. On the other hand, the cell density is lowest
in the grey matter and highest in the white matter. These differ-
ences result from the predominant cell type in each region, be-
cause most of the neurons that predominate in the grey matter
are larger than the glial cells and occupy a larger area. However,
the absolute number of glial cells is higher than the number of
neurons. Therefore, the cell density is higher in the white matter,
where glial cells predominate. There are no studies that examine
both the area fraction and the density of the cells. Begonia et al.
[37] and Koser et al. [32] reported comparable area fractions in
mixed pig tissue (7.85%) and mouse spinal cord (between 1.4% and
12.7%), respectively. The former is within the range of 2.4 to 8.3%
we found for BG, and the latter is slightly higher than the white
matter minimum (1.7%) and grey matter maximum (7.0%). Antono-
vaite et al. [60], in turn, determined values between 5 and 95%
for different subareas of the mouse hippocampus. The maximum
was measured in the densely packed granule cell layer, which is
not present in any of the anatomical regions we studied here. Bud-
day et al. [72] also found that cell density was high in white mat-
ter and low in grey matter. However, comparing the mean value
of ρcells measured for BG, the present study shows a value 56.4%
higher than documented in Budday et al. [72]. This difference is
due to the sampling location, as the BG includes several sub-areas
with different proportions of grey matter. Budday et al. [72] chose
the caudate nucleus, an area with a higher proportion of grey mat-
ter, than the putamen we chose for the present study. Therefore,
the number of neurons is higher and the overall cell density is
lower.
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M. Hoppstädter, D. Püllmann, R. Seydewitz et al. Acta Biomaterialia xxx (xxxx) xxx

Fig. 10. Shear modules μat and μac and non-linearity factors αat and αac as a function of area fractions f of (a)-(b) myelin, (c)-(d) cells, (e)-(f) glial cells and (g)-(h)
neurons. Solid lines characterise linear regressions. The corresponding Spearman’s rank correlation coefficients are shown in each case in terms of uniaxial tension rs,at and
compression rs,ac . Note: ∗ marks p < 0.05.

12
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M. Hoppstädter, D. Püllmann, R. Seydewitz et al.
Furthermore, we introduced a threshold diameter of dt =
9.5 μm to distinguish between neurons and glial cells. The sig-
nificant differences between the neuron density of the grey and
white matter underline the applicability of the chosen diameter,
see Section 5.1. Since neurons are almost absent in the white mat-
ter, the significantly higher number of cells larger than dt in the CX
and BG indicates that the cells counted are neurons. To the best of
our knowledge, no study to date has reported an equivalent di-
ameter to distinguish between neurons and glial cells. We would
like to point out that this approach cannot capture all sizes of glial
and neuronal cells present in the brain. This is partly due to the
limited amount of histological data and partly due to the simpli-
fication of choosing one diameter for two main cell groups, which
themselves consist of several subtypes. Instead, it serves as an ef-
ficient first approximation to distinguish cell types based on their
size.
6.2. Mechanical brain tissue properties
Generally, before the actual mechanical testing, tissues are sub-
jected to various mechanical and thermal procedures. While in
some cases cadaver tissue is selected [16,18,26,51,86,98,99], in
other cases the tissue is taken as soon as possible after dissection
[e.g. 12,36,50,59,79,100] in order to come as close as possible to
the in vivo situation. Another important factor that significantly
affects the mechanical response of the material is the loading
rate, which in the relevant literature varies between 0.0 0 064%/s
[34] and 30 0,0 0 0%/s [36,39]. In addition, a variety of areas in the
brain have been mechanically examined, with some studies sam-
pling only a single region [11,13,41,54,79,98,101–103], while oth-
ers examined up to seven regions [104]. Finally, the starting point
of the stress-strain relationship has a significant influence on the
maximum stress, but also on the elastic stretch range. Here, too,
there are different approaches in the literature. Some studies do
not use a pre-load [e.g. 12,15,22,45,86,87, while others choose it
arbitrarily [37,46] or select them visually depending on the defor-
mation [e.g., 11,19,20,34,35,42,51,88]. It is therefore not surprising
that the results obtained in these studies are highly dependent
on the different protocols and vary accordingly. In Figure S1 (see
Supplementary material), the maximum stresses reached at stretch
of λ = 1.071 of the tensile (a) and at λ = 0.55 of the compressive
(b) stretch range from approximately 0.035 kPa [78] to 1.049 kPa
[105] and −0.577 kPa [45] to −10.010 kPa [36], respectively. The
mean tensile and compressive stresses of 0.091 ± 0.018 kPa and
−0.811 ± −0.202 kPa determined in the present study are thus
within the range of values reported in the literature. The com-
parison is based on the mean value of isotropic anatomical areas
(CX, BG, CR), as a large proportion of studies have investigated
mixed tissue [e.g. 34,37,45,86] or white matter without consider-
ing different axon orientations [e.g. 20,28,35,44]. In the present
study, the elastic stretch of brain tissue under tensile loading was
found to range from λ = 1.071 (CR) to λ = 1.106 (CC90°). This
range is consistent with the study by Bain and Meaney [106], re-
porting a conservative failure strain of λ = 1.14 for optical nerve
tissue. However, the elastic stretches observed in this study are
lower than those of Miller and Chinzei [27] (λ = 1.21), Franceschini
et al. [28] (λ = 1.91 ± 0.42) and Velardi et al. [29] (λ = 1.27 for the
CR90° and λ = 1.63 for the CX). This discrepancy could be due to
the use of mixed white and grey matter, a different failure criterion
and tissue clamping instead of gluing. In addition, the CC with-
stands the highest tensile strain, with loading in the axon direction
leading to earlier failure than perpendicular to it, suggesting that
axons fail earlier than the network of cells and the ECM in which
they are embedded. These results mirror those of Velardi et al.
[29] who also found that the CR0° fails at λ = 1.27,earlier than
CR90° (λ = 1.46) and CC0°, resists the highest strain (λ = 1.52) ex-
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cept for the CX, which is not considered in the present study. In
the other studies in Figure S1 (a) (see Supplementary material), the
brain tissue was loaded without defining an elastic range.
We also found a tension-compression asymmetry with up to
180 times higher tensile stresses. However, other studies that ex-
amined tension and compression identified higher compressive
stresses [23,26,27,33]. Two obvious reasons could lead to these
differences: Firstly, boundary conditions that lead to a deviation
from the assumed homogeneous deformation can cause discrepan-
cies in the results, see also Remark 1. Consequently, Rashid et al.
[42] have shown that the ratio of diameter to thickness of the
specimen tested in tension has a significant influence on the as-
sumption of a homogeneous deformation state. The authors stated
that a diameter to thickness ratio ≤ 1 should be aimed for in or-
der to achieve a homogeneous stress distribution. Otherwise, the
influence of inhomogeneous deformation due to boundary condi-
tions such as bonding or clamping can no longer be neglected. In
this case, the measured forces, normalised to the initial surface, no
longer represent the stress ratios within the specimen. In the stud-
ies of Miller and Chinzei [27], Jin et al. [23], Budday et al. [26], and
Eskandari et al. [33], the TCA ratios are 3, 2.8, 1, and 1.3, respec-
tively. In turn, we used a thickness/height ratio of 0.5. In addition,
Böl et al. [85] discusses the effect of friction between the speci-
men and the tool in uniaxial tests. The study reports an increase
in stress response with increasing friction. Therefore, the degree
of inhomogeneity is higher for glued samples as in Budday et al.
[26] than in the current study with smooth contact surfaces that
were additionally wetted with silicone oil. Another possible rea-
son is the loading rate. In the present study a rate of 0.05 %/s was
used, which is lower than in the studies mentioned above. Assum-
ing incompressible material behaviour, the tissue exhibits higher
resistance at high strain rates than at low rates under compressive
loading. This is due to poroelasticity, because the lower the strain
rate, the more fluid is pushed out of the tissue. Furthermore, the
studies of Miller and Chinzei [34] and Miller and Chinzei [27] have
shown that the dependence on strain rate is more pronounced un-
der compression than under tension. Therefore, as shown in the
supplementary material, we measured lower compressive stresses
than most studies in the literature.
Our study shows that the material behaviour can be consid-
ered homogeneous for small deformations, but becomes heteroge-
neous as deformations increase. For large deformations, the CR of
the white matter behaves significantly stiffer than the CX and BG
regions. These results confirm the findings of most previous stud-
ies that report higher stiffness of white matter compared to grey
matter [16,23,29,36,43]. In contrast, Li et al. [44] found no signifi-
cant differences between the CX and the CR. A possible explanation
for this could be that the regional difference is age-dependent, as
eight-week-old porcine brain tissue was used in the study. Another
study testing human brain tissue under compression and tension
showed an opposite trend with stiffer grey matter than white mat-
ter [26]. This inconsistency could also be due to age effects, as
samples with an average age of 66 years were used, and Elkin et al.
[38] and MacManus et al. [52] reported a significant increase in
stiffness with age.
Interestingly, the present study showed anisotropic behaviour
of the CC for the deformation modes of uniaxial compression and
semi-confined compression, where axons loaded in tension dis-
played a higher stiffness than those loaded in compression. This
finding is in contrast to previous studies that assumed that white
matter behaves isotropically under compressive loading [23,26,36].
However, these studies used tissues from 18-month-old cattle
to 70-year-old humans. Therefore, differences in species and age
could be the cause of the less pronounced anisotropy. In contrast,
results on brain tissue under shear stress confirm the anisotropic
behaviour found in the present study [19,21,23].
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It remains unclear whether axons are only loaded in tension,
because the semi-confined compression tests show a significant
difference between mode I and III, where axons are loaded in com-
pression or kept at a constant length. Therefore, the classical ap-
proach for transversely isotropic cardiovascular tissue [107], where
fibres are only considered in tension, seems to be less appropri-
ate for nervous tissue. Unfortunately, to our knowledge, there is
no study that tests brain tissue under semi-confined compression
conditions which we could use for comparison. There exists studies
[82–84] that investigate this deformation mode on muscle and ten-
don tissue, but as the microstructure and stiffness of the brain dif-
fers significantly from these, a comparison is only of limited value.
6.3. Relationship between microstructural architecture and
mechanical properties
In the present study, we correlated microstructural architec-
ture and mechanical properties to explore the mechanisms of load
transfer for different deformation modes. Other studies already
investigated the correlation between mechanics and microstruc-
ture using cell area fraction [32,37,60,70], myelin area fraction
[50,60,72,77], cell density [72], neuron area fraction [60], and as-
trocyte area fraction [60]. So far, however, there is no information
on the influence of glial cells and neurons on the mechanical be-
haviour at more than one deformation state, as Antonovaite et al.
[60] has only investigated the influence at indentations. Further-
more, correlation studies in the literature are mostly limited to one
or two anatomical regions, such as the white matter in Weicken-
meier et al. [50,77], hippocampus and cortex in Antonovaite et al.
[60], or spinal cord in Koser et al. [32]. An exception is the study
by Budday et al. [72], investigating CX, BG, CR, and CC, but based
the correlation on the anatomical regions, only. To fill this gap, we
investigated the direct influence of glial cells and neurons on me-
chanical behaviour in different deformation states and independent
of the anatomical region.
We found a negative correlation of compressive shear mod-
ulus with myelin area fraction, suggesting that myelin does not
contribute critically to the mechanical strength of the tissue. This
result is consistent with a mouse brain study in which stiffness
measured by dynamic indentation was correlated with myelin
area fraction [60]. In contrast, the studies by Weickenmeier et al.
[50] and Budday et al. [72] document a positive correlation. How-
ever, both only looked at white matter tissue, whereas the present
study included all anatomical regions. Furthermore, the studies as-
sumed an isotropic material behaviour, whereas here, we observed
a pronounced anisotropy of the CC region. Therefore, our correla-
tion with myelin was negative, as the CC has a high proportion of
myelin, but low shear moduli compared to the isotropic anatomical
regions.
Interestingly, we found that the tensile shear modulus increases
with increasing area covered by cells, independent of cell type.
Since this correlation only exists under tensile stress, we suspect
that the cell connections and not the cells themselves contribute
to the mechanical response. Under tension, the cell connections to
neighbouring cells and nerve fibres can act as load-transfer chains,
and the more chains are activated, the stiffer the response. The dis-
solution of these connections could explain the non-linear strain-
stress response under tension, because after a high initial stiffness,
the curve flattens with increasing deformation. However, further
studies of the inter-cellular network and its behaviour under ten-
sile loading need to be carried out in the future to support this
assumption. Koser et al. [32], Thompson et al. [71], and Antono-
vaite et al. [60] investigated the correlation between tissue stiff-
ness measured by indentation and cell area fraction. Koser et al.
[32] and Thompson et al. [71] found a positive correlation (both
with a Pearson correlation coefficient of 0.97), which is consis-
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Acta Biomaterialia xxx (xxxx) xxx
tent with the present study. In contrast, Antonovaite et al. [60] re-
ported a negative correlation for both nuclei and neurons (Pear-
son correlation coefficient of −0.73 and −0.81). To date, however,
no study has investigated the correlation for uniaxial tension. The
pressure shear modulus, in turn, correlates negatively with the
area fraction and number of glial cells. Since glial cells form the
myelin around axons, this result corresponds to the negative cor-
relation with myelin area fraction. Conversely, Antonovaite et al.
[60] showed an increase in memory modulus with increasing area
covered by astrocytes, a specific type of glial cells (Pearson corre-
lation coefficient of 0.56). In this study, hippocampal white matter
subregions were excluded from the correlation, while we exam-
ined the relationship for all anatomical regions. In addition, we ex-
amined all glial cells without distinguishing between specific cell
types such as astrocytes.
Finally, the factor of compression non-linearity increases with
increasing myelin content. Importantly, the factor correlates signif-
icantly with cell density, glial cell density, and area fraction. Con-
sequently, white matter has higher non-linearity factors than grey
matter. This suggests that the compressive curve shape is directly
influenced by the underlying myelin content and cell number. So
far, only Budday et al. [72] has investigated a comparable relation-
ship and also found a positive correlation of the non-linearity fac-
tor with cell density.
6.4. Limitations of this study
In this study, all tests were performed in vitro within 12 h
post-mortem. While some studies [e.g. 45,49] report no effects
on mechanical properties during this post-mortem period, others
[e.g. 14,108,109] do. As in vivo testing methods such as magnetic
resonance elastography are not able to characterise the full com-
plexity of the mechanical response of the brain, in vitro testing
currently remains a viable method to account for regional differ-
ences and behaviour under large deformations. However, in vitro
studies like ours are limited to the staining protocol chosen by
Klüver Barrera. Since there is no staining method that makes all
components of the complex brain microstructure visible in one
section, we decided to focus on the main components, cells and
myelin. Consequently, the chosen protocol makes cell bodies and
myelin visible, while cell connections and synapses are not. Yet,
image segmentation to identify cells and myelin is based on the
choice of the regions of interest. Here we used a large number of
regions of interest to represent differences in microstructure. Due
to the limited anatomical size, it was not possible to histologi-
cally examine every mechanical sample, so the number of samples
was reduced from nmech = 181 to nhist = 74. Another limitation is
the selection of the anisotropic Ogden model, a phenomenologi-
cally motivated model that serves as a minimalist model to inves-
tigate the relationship between microstructural components and
mechanical behaviour. The experiments revealed different proper-
ties such as the significant anisotropy of CC under compressive
loading, which is not significant under tensile loading, and the
tensile-compressive asymmetry. These different properties lead to
significantly different material parameters, such as the anisotropy
factor k, which is higher under compression than under tension.
Therefore, the deformation state was adjusted separately to deter-
mine a minimum error between experiment and simulation. De-
spite the phenomenological nature of the present modelling ap-
proach, the material parameter takes into account the mechanical
behaviour of the brain tissue. In this sense, μ stands for the gen-
eral material stiffness, α for the non-linearity of the stress-strain
response, and k for the degree of anisotropy due to structural ori-
entations. Although they are able to predict the stress-strain re-
sponse under tension, some parameter configurations are physi-
cally problematic to interpret [110]. In particular, the stress re-
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M. Hoppstädter, D. Püllmann, R. Seydewitz et al.
sponse in the corresponding strain energy function in Eq. (3) de-
creases for negative values of α . In addition, the stress response
in tension results from the volume-preserving component in the
strain energy function in Eq. (1), as the material resistance in-
creases perpendicular to the direction of tension. Therefore, both
the tensile and compressive behaviour is controlled by the com-
pressive resistance of the material. To overcome this problem, Mo-
erman et al. [110] has presented an alternative approach with sep-
arate control of resistance to tension and compression, which could
be used in future studies. However, the present implementation of
the Ogden model is a commonly used formulation to represent
the mechanical properties of brain tissue. Therefore, choosing a
different model formulation would limit comparability with other
studies and possible variations would remain undetected. Since es-
pecially in biomechanics with complex experimental setups, high
tissue variability, and complex microstructures, comparison with
existing studies is a crucial issue, we accepted this limited inter-
pretability of α in tension. Furthermore, the experiments revealed
different properties such as the significant anisotropy of CC under
compressive loading, which is not significant under tensile loading,
and the TCA. These different properties lead to significantly differ-
ent material parameters, such as the anisotropy factor k, which is
higher under compression than under tension. In order to obtain
the most accurate model prediction for the stress-strain relation-
ship, we optimised a set of material parameters for each individ-
ual test, allowing us to investigate the influence of the microstruc-
ture on the material response and its variability. Consequently, the
determined material parameters are only able to represent the be-
haviour within the respective deformation state.
7. Conclusion
The present study shows that the strongly non-linear mechan-
ical behaviour of nervous tissue is a direct result of the hetero-
geneous tissue microstructure. Although white matter tissue con-
tains more myelin and cells than grey matter, its shear moduli are
smaller. This suggests that there is a negative correlation between
the area fraction of myelin and glial cells and the shear stiffness.
In contrast, the area fraction of cells, neurons and glial cells cor-
relates positively with the tensile shear stiffness, suggesting that
the number of cell connections is responsible for the tissue stiff-
ness. Our study also found that myelin and glial cell area and cell
density influence the non-linearity of the stress-strain response, as
the non-linearity of white matter differs significantly from that of
grey matter. Understanding the microstructural load transfer mech-
anisms in the brain is a crucial step towards developing reliable
computational models to simulate surgical procedures, e.g., cancer
tissue removal. To make brain models more accurate in the future,
our study suggests that it is critical to include microstructural in-
formation into the material characterisation of nervous tissue.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgements
Partial support for this research was provided by the Deutsche
Forschungsgemeinschaft (DFG) under Grants no. 404568779.
Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.actbio.2022.08.034.
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