journal of the mechanical behavior of biomedical materials 108 (2020) 103833

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Journal of the Mechanical Behavior of Biomedical Materials

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

Mechanical properties of native and acellular temporal muscle fascia for

surgical reconstruction and computational modelling purposes
J. Zwirner a, *, B. Ondruschka b, M. Scholze c, G. Schulze-Tanzil d, N. Hammer e, f, g, **
a
Department of Anatomy, University of Otago, Dunedin, New Zealand
b
Institute of Legal Medicine, University of Leipzig, Leipzig, Germany
c
Institute of Materials Science and Engineering, Chemnitz University of Technology, Chemnitz, Germany
d
Department of Anatomy and Cell Biology, Paracelsus Medical University, Salzburg and Nuremberg, Nuremberg, Germany
e
Department of Macroscopic and Clinical Anatomy, Medical University of Graz, Graz, Austria
f
Department of Orthopaedic and Trauma Surgery, University of Leipzig, Germany
g
Fraunhofer IWU, Dresden, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: The temporal muscle fascia (TMF) is a widely used graft material and of interest for computational simulations of
Acellularization the temporomandibular joint as well as computational and physical human head models in general. However,
Mechanical properties reliable biomechanical properties of the TMF are lacking to date. This study provides tensile data of 52 TMFs at
Osmotic stress protocol
an age range of 18 to 94 years. It further investigates, if acellular fascia scaffolds differ from native counterparts
Temporal fascia
in their biomechanical behaviour. Native TMF has a median elastic modulus of 26.2 MPa (acellular: 24.5 MPa),
Uniaxial tensile testing
an ultimate tensile strength of 2.9 MPa (acellular: 2.1 MPa), a maximum force of 12.6 N (acellular: 9.9 N) and a
strain at failure of 14.1% (acellular: 14.8%). No significant difference was found regarding the properties of
native and acellular samples. Elastic modulus and the ultimate tensile strength increased with age but only in the
acellular group (p < 0.01). Decorin and fibronectin seemed to be washed out by the acellularization procedure.
The absence of cells in acellular TMF samples is not of biomechanical relevance compared to the native state.
Acellular TMF is a biomechanically promising scaffold material for graft purposes, which can be retrieved easily
due to its superficial location.

1. Introduction tympanoplasty, it is still the most commonly used graft for that pro­
cedure (Hermann, 1960; Jalali et al., 2017). In tympanoplasty, the TMF
The temporal muscle fascia (TMF) is a frequently used transplant is used as a wet (“soft” graft, referring to the harvested state) or dry graft
material especially in the head and neck region with a high number of (“rigid” graft, which is dehydrated between harvesting and trans­
different surgical applications (Baker et al., 2005; Basterzi et al., 2009; planting), which differ in the number of fibroblasts (Alkan et al., 2009;
Bulgannawar et al., 2011; Chan et al., 2017; Jalali et al., 2017; Jiang and Singh et al., 2015). Regarding this, the contribution of cells to the
Lou, 2017; Katsuno et al., 2017; Maniu and Cosgarea, 2012; Mizuno, biomechanics of the fascia has not been investigated thoroughly to date.
2014; Neuhaus and Shorr, 1983; Park et al., 2015; Schwarz and Spinelli, Moreover, it has been stated that the TMF may have an important
2008; Singh et al., 2015; Virkkula et al., 2015; Xu et al., 2016; Yegin function in chewing kinematics, here antagonizing the forces to the
et al., 2016). From a surgical perspective, the biomechanical properties zygomatic arch caused by the masseter (Curtis et al., 2011; Eisenberg
of the fascia are of particular interest whenever a load-bearing function and Brodie, 1965). However, to date, the biomechanical properties of
of the transplant is required to positively influence the outcome of the the TMF have only been investigated by few groups with a maximum
procedure, such as in ptosis correction (Baker et al., 2005; Neuhaus and number of 16 specimens (Curtis et al., 2011; Trindade et al., 2012).
Shorr, 1983) or the correction of an upper eyelid retraction (Schwarz Given this evident lack of biomechanical data, the TMF has neither been
and Spinelli, 2008). Sixty years after the TMF has first been applied for incorporated into finite element models of the temporomandibular joint

* Corresponding author.
** Corresponding author. Department of Macroscopic and Clinical Anatomy, Medical University of Graz, Graz, Austria.
E-mail addresses: medijo@gmx.de (J. Zwirner), niels.hammer@medunigraz.at (N. Hammer).

https://doi.org/10.1016/j.jmbbm.2020.103833
Received 25 February 2020; Received in revised form 20 April 2020; Accepted 24 April 2020
Available online 4 May 2020
1751-6161/© 2020 Elsevier Ltd. All rights reserved.
J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

Fig. 1. The temporal muscle fascia (TMF) of

the left temporal area has been dissected
from an 83-year old male Thiel-embalmed
cadaver. A) The TMF becomes visible after
the temporal skin and subcutaneous fat has
been resected. B) The TMF is dissected from
the underlying temporal muscle and flipped
downwards. Thus, the inner surface of the
fascia (asterisk) becomes visible. Note that
the inner surface of the fascia reveals a
streaky pattern, which macroscopically in­
dicates an anisotropy of the tissue. The black
arrow points at the temporal muscle tendon.
s, superior; i, inferior; a, anterior; p,
posterior.

as a functionally related structure (Creuillot et al., 2013; Fricova� et al., the samples were precooled and subsequently transferred to a 80 � C
2006; Perez Del Palomar and Doblare, 2006), nor into physical or freezer for storage. When further processed, the samples were cut into a
computational skull models (Bandak et al., 1995; Kleiven, 2003; Liang ‘dog bone’ shape using a template, adapted from the ISO 527-2 standard
and Luo, 2015; Thali et al., 2002; Zwirner et al., 2017). In this study, the (International Organization for Standardization, 1996).
biomechanical parameters of fresh and chemically unfixed TMF are
investigated in a quasi-static testing setup. Recent advances to stan­
2.2. Acellularization with sodium dodecyl sulphate and water content
dardize soft tissue testing such as the use of 3D-printed clamps to avoid
adjustment using the osmotic stress technique
material slippage or the application of an osmotic stress protocol were
included (Hammer et al., 2014; Schleifenbaum et al., 2016; Scholze
The 26 samples of the acellularization group were acellularized as
et al., 2018). It will be investigated whether acellularization using so­
described previously (Hammer et al., 2014). For the removal of cells, the
dium dodecyl sulphate influences the biomechanical properties of the
samples were submerged in a sodium dodecyl sulphate (SDS) solution of
TMF. This may help increase an understanding of fascia grafts used for
1.0 wt.-% (Roth- Karlsruhe, Germany) for 7 days at ambient temperature
tympanoplasty. Derived from previous investigations of our group on
(AT) of 22 � C. The SDS solution was renewed after 3 days. The acellular
collagen-rich tissues, we stated the following hypothesis: Acellular TMF
fascia scaffolds were subsequently rinsed in distilled water for 7 days.
has similar (statistically non-different) biomechanical properties
The distilled water was renewed daily. A shaking table at AT was used
compared to the native state.
for the SDS submersion and the rinsing. The osmotic stress technique
was applied as described previously (Hammer et al., 2014; Zwirner
2. Materials and methods
et al., 2019b). A total of 40 representative fascia pieces of approximately
25 mm2 each taken from 10 cadavers were allocated to 20 mM
2.1. Retrieval and processing of human temporal fascia samples
hydroxymethyl aminomethane-buffered polyethylene glycol (Tris-PEG;
pH ¼ 7.4; Merck KGaA- Darmstadt, Germany; molecular weight 20,000)
A total of 52 human TMF samples from 35 cadavers were harvested
solutions at concentrations of 2.0, 3.0, 4.0 and 5.0 wt.-% for 24 h. A
according to Fig. 1 in a fresh and chemically unfixed condition at the
water content determination of the samples was done by means of
Institute of Legal Medicine, University of Leipzig, Germany during
lyophilization.
forensic autopsies. The samples were allocated into a native group of 26
samples (10♀, 16♂) with a post-mortem interval of 69 � 24 h (range 11
to 115 h) and an age-matched acellular group of another 26 samples (8 2.3. Mechanical testing and scanning electron microscopy
females, 18 males) with a comparable post-mortem interval of 71 � 27 h
(range 11 to 122 h). A total of 17 cadavers contributed tissue samples to Following the tapering, the TMF samples were moulded with poly­
both the acellular and native group. The mean age of both groups was 60 siloxane impression material (medium-bodied, Exahiflex; GC Corpora­
years (range 18 to 94 years). Retrieval of these tissues for the purpose of tion, Tokyo, Japan) in the area of parallel measurement length of the
mechanical testing was approved by the Ethics Committee of the Uni­ tapered specimens to determine their cross-sectional area. The cross
versity of Leipzig, Germany (protocol number 486/16-ek). All experi­ section casts were scanned at a 1200-dpi resolution (Perfection
ments were conducted according to the Declaration of Helsinki. Initially, 7V750Pro; Seiko Epson Corporation- Suwa, Japan) and calculated using
the Measure 2.1d software (DatInf- Tübingen, Germany). A randomly-

Fig. 2. Images of a representative temporal

muscle fascia sample at different stages of
uniaxial tensile testing and the related stress-
strain curve are depicted. A) The sample is
speckled and clamped into the machine at
the beginning of tensile testing with no load
applied (1). After the beginning of the uni­
axial quasi-static testing, a slightly uniform
reduction of the tapered area can be
observed (2). The reduction continues with
an increase of the applied load (3). Finally, a
localized failure appears in the tapered area
(4). B) The stress-strain curve of the spec­
imen from A is displayed. The red numbers
correspond to the different stages of tensile
testing.

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J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

distributed speckle pattern was then created at the tissues with standard
charcoal pens for strain measurement by digital image correlation (DIC).
Uniaxial tensile tests were performed at AT using a universal testing
machine (Allround Table Top Z020; Zwick Roell- Ulm, Germany). An
Xforce P load cell of 2.5 kN with testControl II measurement electronics
(all Zwick Roell) was used. To minimize material slippage, 3D-printed
clamps were used for the mounting of the samples as recently estab­
lished by our group (Scholze et al., 2018). All samples were precondi­
tioned with 20 load-unload cycles at a force range of 0.5 to 2.0 N before
a stretching until failure was performed. A crosshead displacement rate
of 20 mm/min and a sampling rate of 100 Hz was used for the force
readings. Deformation of specimen surface was recorded perpendicular
to the surface by a DIC system using a single charge-coupled camera
with a resolution of 2.8 Megapixels (Q400; Limess- Krefeld, Germany).
The ISTRA 4D software (VRS 4.4.1.354; Dantec Dynamics- Ulm, Ger­
many) was used for the evaluation of strain data of the mechanical tests
(see Fig. 2 for images of the tensile testing and a typical stress-strain
curve of the here studied cohort). Scanning electron microscopy (SEM)
was performed on representative native and acellular samples using a
JEOL 6700F field emission scanning electron microscope (JEOL- Pea­
body, USA) using a magnification of up to 5000x. Before imaging, the
SEM samples were treated in a K575X sputter coater with a 5-nm layer of
gold-palladium (Emitech Technologies- Kent, England).

2.4. Histology (Hematoxylin and eosin, Alcian blue and Weigert’s

resorcin-fuchsin stain)

Six representative samples of three cadavers (one native sample and

one acellularized each) were fixed in 4% neutrally buffered para­
formaldehyde solution (Sigma-Aldrich- Munich, Germany), dehydrated
and then embedded in paraffin. Subsequently, 7-μm sections were
deparaffinized with xylene and rehydrated in a descending ethanol se­
ries. For hematoxylin and eosin (H&E) staining, the deparaffinized
sections were incubated for 6 min in Harry’s hematoxylin (Sigma-
Aldrich), then rinsed in tap water and counterstained for 4 min in eosin
(Roth). For the Alcian blue (AB) stain, rehydrated sections were incu­
bated for 3 min in 1% acetic acid before immersed for 30 min in 1% AB Fig. 3. Flow chart of the experimental setup. PEG, polyethylene glycol.
staining solution (Roth). After rinsing in 3% acetic acid and a 2-min
lasting washing step in distilled water, cell nuclei were counterstained
corresponding isotype antibody (mouse IgG1; Abcam- Cambridge,
for 5 min using nuclear fast red aluminium sulphate solution (Roth). To
United Kingdom), which were used in the same concentrations as the
detect the elastic fibres, Resorcin-fuchsin stain was used. Rehydrated
original primary antibodies. Samples were rinsed with TBS before in­
sections were stained in Weigert’s Resorcin-fuchsin solution (Waldeck
cubation with donkey-anti-goat (Invitrogen- Carlsbad, USA), anti-
GmbH- Münster, Germany) for 5 min. Afterwards, the sections were
rabbit-Alexa-488 (Invitrogen) or donkey-anti-mouse-cyanine-3 (Cy3,
differentiated in hydrochloric acid/ethanol for 1 min. After washing
Invitrogen) coupled secondary antibodies (diluted 1:200 in TBS),
with tap water for 10 min, an additional washing step in distilled water
respectively, for 1 h at AT. Cell nuclei were counterstained using 10 μg/
followed. Nuclear fast red aluminium sulphate solution was used to
mL 40 ,60 -diamidino-2-phenylindol (DAPI; Roche- Mannheim, Germany).
counterstain the cell nuclei in the Resorcin-fuchsin stain. All stained
Labelled sections were washed three times for 5 min with TBS before
sections were covered with entellan (Merck-Millipore- Darmstadt, Ger­
mounting with fluoromount mounting medium (Southern Biotech-
many). Photos were taken using a DM1000 LED light microscope (Leica-
Eching, Germany) and examined by using a SPEII confocal laser scan­
Wetzlar, Germany).
ning microscope (Leica).

2.5. Immunolabeling (collagen types I/III, decorin and fibronectin)

2.6. Data processing and statistical analysis
The sections used here of the samples mentioned above were washed
three times with Tris-buffered saline (TBS: 0.05 M Tris, 0.15 M NaCl, pH The mechanical properties of the TMF samples were calculated from
¼ 7.6), before being incubated with a 0.1% pronase ready to use solution the DIC data and synchronized force readings using MATLAB R2017b
(BioLogo- Kronshagen, Germany) to demask epitopes for 15 min at 37 software (Mathworks- Natick, USA). Elastic modulus, ultimate tensile
�
C. Subsequently, they were rinsed again with TBS before being incu­ stress, maximum force, strain at maximum force and strain at failure
bated with protease-free donkey serum (5% diluted in TBS with 0.1% were assessed. As samples tend to swell during the SDS treatment
Triton X100 for cell permeabilization) for 20 min at AT. Sections were (Zwirner et al., 2019a), a separate ‘acellular corrected group’ was
incubated overnight with primary antibodies for type I collagen (1:80, created for evaluation purposes, in which the individual sample cross
goat-anti-human; Abcam- Cambridge, UK), type III collagen (1:80, section was corrected for swelling. Therefore, the overall swelling of the
mouse-anti-human; Acris Laboratories), decorin (1:50, rabbit-anti- acellular group was determined by calculating the proportion of the
human; Acris Laboratories) and fibronectin (1:80, mouse-anti-human; overall cross section difference in percent. The individual cross sections
Dianova- Hamburg, Germany) at 4 � C in a humid chamber. As con­ of the acellular group were then corrected by this factor to prevent a bias
trols, the primary antibodies were omitted or replaced with the of the results caused by a potential SDS-related tissue swelling. Excel

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J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

Fig. 4. The biomechanical temporal muscle fascia data that depend on the Fig. 5. Force and strain data of temporal muscle fascia independent of the
cross-sectional area of the tested samples and the related cross sections are cross-sectional area. No statistically significant differences were obtained. The
depicted. The left darker grey boxes display the 26 native samples age-matched left darker grey boxes display the 26 native samples age-matched to the acel­
to the acellular group, light grey boxes the acellular group and white boxes the lular group, light grey boxes the acellular group and white boxes the acellular
acellular group corrected for the cross sections. The outlines of the boxes group corrected for the cross sections. The outlines of the boxes indicate the
indicate the 25% and 75% percentile, the solid black line the median. Whiskers 25% and 75% percentile, the solid black line the median. Whiskers indicate the
indicate the minimum and maximum. The dotted line separates the left and minimum and maximum. The dotted line separates the left and right y-axis.
right y-axis. Emod, elastic modulus; CS, cross section; UTS, ultimate ten­ Fmax, maximum force.
sile strength.

ranges (IQRs) are provided in the text. A flow chart of the experimental
Version 16.15 (Microsoft Corporation- Redmond, USA) and GraphPad setup is shown in Fig. 3.
Prism software version 7 (GraphPad Software- La Jolla, USA) were used
for the statistical evaluation. The D’Agostino & Pearson normality test
3. Results
was used to test the Gaussian distribution of the samples. Parametric
data of samples were then tested using an ordinary one-way ANOVA. A
3.1. Ultimate tensile strength of acellular TMF was lower compared to
Kruskal-Wallis test was used for nonparametric sample data. Presuming
native TMF; altered cross sections were found for acellular TMF. Those
the hypothesis-generating nature of this study no post hoc test has been
tensile data of native and acellular TMF independent of the samples’ cross
applied (Fisher’s least significant difference test for ANOVA and un­
sections were similar and non-different
corrected Dunn’s test for Kruskal-Wallis). Pearson and Spearman cor­
relation coefficients were reported for normally and non-normally
The median ultimate tensile strength of 2.1 MPa (IQR ¼ 2.0 MPa;
distributed values, respectively. P values equal to or smaller than 0.05
mean � standard deviation (STDEV) ¼ 2.3 � 1.2 MPa) of acellular
were considered statistically significant. Medians and interquartile
samples was significantly lower than of the native ones with 2.9 MPa

Table 1
Overview of biomechanical data of native and acellular temporal muscle fascia samples in age and post-
mortem interval matched groups. The acellular group of 26 samples was corrected for the higher cross
sections compared to the native group to increase comparability of the parameters, that are based on cross
sections (elastic modulus and ultimate tensile strength). The colours used for the different groups (red, dark
green and light green) match the colours for these groups used in Fig. 3. Age and post-mortem interval data
are provided as means � standard deviations, all other data as medians with interquartile ranges in brackets
(Taylor, 1997).

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J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

Fig. 6. Scanning electron microscopy images

of the acellular temporal muscle fascia. A) At
a 1000x magnification (scale bar 10 μm) of
the temporal muscle fascia several unorga­
nized areas (white arrows) can be depicted.
However, the majority of the collagen fibres
appear to take a preferred course making the
fascia an anisotropic structure. Several larger
multiaxially aligned bundles of jointly
running collagens are observed (1þ2). A
“wavy” collagen pattern can be seen in
numerous sites throughout the whole field of
vision (dotted white line). B) The white
dotted square shown in A is depicted at a
5000x magnification (scale bar 1 μm). Note
the “wavy” course of several collagen bun­
dles exemplary shown with the dotted white
line. The unorganized areas indicated with a
white arrow seem to be superficial to the
predominantly well-organized collagens
running underneath.

(IQR ¼ 2.9 MPa; mean � STDEV ¼ 3.6 � 1.8 MPa; p ¼ 0.01). The median 3.5. Histology indicates a complete removal of cells by the acellularization
cross sections of the acellular TMF were significantly larger than the procedure
native ones with 5.3 mm2 (IQR ¼ 2.3 mm2; mean � STDEV ¼ 5.9 � 2.3
mm2) and 4.0 mm2 (IQR ¼ 1.7 mm2; mean � STDEV ¼ 4.2 � 1.1 mm2; p Acellularization by means of SDS led to a complete removal of
¼ 0.01), respectively. Following correction for the altered cross sections cellular components in all performed histological stains (see Fig. 7A–F).
induced by the acellularization, statistically significant differences were In the H&E stain a ‘wavy’ collagen pattern can be detected (see Fig. 7A).
not observed anymore. Although the median elastic modulus of acellular Fig. 7F shows that elastic fibres can be found preferably in the areas,
samples of 24.5 MPa (IQR ¼ 24.9 MPa; mean � STDEV ¼ 25.7 � 15.8 where the extracellular matrix piles up, creating a ‘wavy’ pattern of the
MPa) was smaller compared to the native ones 26.2 MPa (IQR ¼ 36.4 overall sample. The detection of elastic fibres is enhanced in the acel­
MPa; mean � STDEV ¼ 36.0 � 22.6 MPa), no difference on a statistically lular samples. The AB stain does not show sulphated glycosaminogly­
significant level was observed (p ¼ 0.09), see Fig. 4 and Table 1 for a cans (GAGs) in both the native and acellular TMF.
data overview. The median maximum force of native TMF samples of
12.6 N (IQR ¼ 13.9 N; mean � STDEV ¼ 14.9 � 8.6 N) was similar to
3.6. Immunostainings yield an increased detectability of type III collagen,
acellular TMF with 9.9 N (IQR ¼ 5.5 N; mean � STDEV ¼ 11.4 � 4.1 N;
but a decreased one for decorin and fibronectin in the acellular samples
p ¼ 0.07). Strain at maximum force and strain at failure of native and
compared to the native state
acellular TMF samples were also similar and non-significantly different
(p ¼ 0.28 and p ¼ 0.32), see Fig. 5 and Table 1 for data illustration.
Type I and type III collagen were detected throughout the entire
extracellular matrix in the related immunostainings. However, the
3.2. Ultimate tensile stress and elastic modulus of acellular TMF samples staining result of type III collagen was more intense for the acellular
show moderate to strong correlations with age. Contrary, an age-related samples compared to the native ones (see Fig. 8A–F). Therefore, in the
decrease of the elastic modulus was depicted for native samples overlay image of the collagen type I and III in the native state, type I
collagen was very dominant in the field of vision, whereas the depiction
The elastic modulus of the acellular samples strongly increases with of the two components was evenly distributed in the acellular state (see
age in both the uncorrected (r ¼ 0.72, p < 0.01) and corrected group (r Fig. 8EþF). Decorin and fibronectin stainings were fainter in the acel­
¼ 0.75, p < 0.01). However, native samples (n ¼ 26) showed a moderate lular samples compared to the native ones (see Fig. 9A–F). The streaky
negative correlation of the elastic modulus with age (r ¼ - 0.50, p < distribution of fibronectin in areas, where the extracellular matrix is
0.01). The ultimate tensile strength of acellular samples showed mod­ discontinued, which can observed in the native state (see Fig. 9E), was
erate positive correlations with ageing in both the uncorrected (r ¼ 0.46, not visible in the acellular state (see Fig. 9F).
p ¼ 0.02) and corrected group (r ¼ 0.56, p < 0.01).
4. Discussion
3.3. Native TMF samples showed weak correlations of biomechanical
parameters with the post-mortem interval, which was not observed in the The biomechanical properties of the TMF have been investigated by
acellular group two groups before with only 4 (Curtis et al., 2011) and 16 (Trindade
et al., 2012) samples. However, the 16 investigated samples in the study
In the native group, moderate positive correlations were found for of Trindade et al. were harvested from only eight cadavers in total
the ultimate tensile strength (r ¼ 0.37, p < 0.05) and the maximum force (Trindade et al., 2012). Trindade et al. reported a significantly higher
(r ¼ 0.44, p ¼ 0.02) with the post-mortem interval. maximum stress and a significantly lower stretch at maximum stress for
the “old” group (51 to 70 years) compared to the “young” group (20 to
3.4. The TMF is an anisotropic structure 50 years), which indicates an age-dependent stiffening of the TMF
(Trindade et al., 2012). Although it was increasing, the investigated
SEM revealed that the TMF collagen fiber bundles were aligned in a secant modulus in their study did not reach statistical significance
preferential orientation according to an anisotropic material (see (Trindade et al., 2012).
Fig. 6A). Several bundles of numerous jointly running collagens can be The ultimate tensile strength was the only of the here studied tensile
depicted over the entire surface of the TMF (see Fig. 6 A). Multiple parameters, that was significantly different in the native compared to
collagen fibres of the TMF take a “wavy” course (see Fig. 6 A þ B). the acellular group. This was caused by the significantly higher cross

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J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

Fig. 7. Histological investigations of native

and acellular human temporal muscle fascia
(TMF). A) The H&E stained sample of native
TMF shows several cell nuclei (black ar­
rows). The purple stained collagens seem to
run in a ‘wavy’ pattern (dotted black line).
The asterisk illustrates a cutting artefact. B)
The cell nuclei are not detectable anymore
after the TMF has been acellularized using
sodium dodecyl sulphate. C) The Alcian blue
(AB) stain does not indicate the presence of
sulphated glycosaminoglycans. The black
arrows indicate the cell nuclei. D) The
absence of sulphated glycosaminoglycans
can be confirmed in the AB-stained acellu­
larized TMF, cell nuclei are not visible any
longer. E) The Resorcin-fuchsin stain of
native TMF displays several elastic fibres
throughout the entire TMF (black arrows).
The red arrow indicates a cell nucleus in the
native sample. The asterisk illustrates a
cutting-related collagen fibre damage. F) The
detection of elastic fibres is improved in the
acellularized TMF sample (black arrows).
The ‘wavy’ pattern (black dotted line) of the
TMF according to A is still visible. The tissue
appears to be piled up in the areas of the
elastic fibres. Magnification: 400x; scale
bars: 50 μm; *, cutting artefact.

sections of the acellular TMF samples compared with the native ones. biomechanical perspective the acellular scaffold of TMF could be used as
After the acellular samples were adjusted for the native cross sections, a transplant material for the same indications as the native TMF with a
no significant difference was observed between the two groups. An commonly decreased rejection risk due to a lower immune response risk
increased tissue hydration as a consequence of the SDS treatment is most (Mirmalek-Sani et al., 2013; Protzman and Brigido, 2015; Song et al.,
likely the reason for the increased cross sections of the acellular samples. 2014). Regarding the recent developments in tissue engineering,
Therefore, the difference in ultimate tensile strength between native and allowing to 3D bioprint multi-layered membranes (Tayebi et al., 2018),
acellular TMF samples has to be attributed to the used acellularization the extracellular matrix of the TMF could potentially be processed and
procedure rather than the cell removal itself. Consequently, we accept then, bioprinted and used as a transplant material. This study underlines
our hypothesis and conclude that the acellular TMF has similar tensile that a seeding of the bioprinted scaffold may not be required from a
properties compared to their native counterparts. Taking this into ac­ biomechanical perspective. The TMF, in general, is a collagen
count, the reduced number of cells of dry compared to wet TMF grafts in rich-tissue, mostly organized in parallel lamellae interconnected by
tympanoplasty may unlikely influence the tensile properties of the some crossing fibers as shown in the H&E stainings. Most of the fibro­
transplant. However, it has to be considered that wet and dry TMF grafts blastic cells containing elongated or ovoid cell nuclei are arranged
could potentially have different biomechanical properties due to ex­ parallel to the lamellae or even in rows. Only a few small blood vessels
pected differences in the water content of the tissues, but differences in and capillaries can be detected. The high collagen content of the
cell count do not contribute to that according to our study (Fosse et al., extracellular matrix is indicated by the immunohistochemical stains for
2006; Hammer et al., 2014). The results of our study indicate that from a collagen type I and III in this study, as well as by the performed SEM.

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J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

Fig. 8. Anti-collagen type I and III immu­

nostaining of temporal muscle fascia (TMF).
A) In the native TMF type I collagen can be
detected throughout the entire field of
vision. The white arrow displays the blue
chromatin stained with DAPI representing
the cells of a small blood vessel in the native
sample. B) Type I collagen can equally to A
be detected in the acellularized sample
throughout the entire field of vision. C) Type
III collagens are represented by a faint red
staining in the native TMF. The white arrow
displays the blue DAPI-stained chromatin
representing the cells in the native sample.
D) The visibility of type III collagens is
improved in the acellular TMF compared to
the native one displayed in C. E) An overlay
of type I collagen (A) and type III collagen
(C) is displayed. The type I collagens are
predominant throughout the entire field of
vision compared to type III collagens. The
white arrow reflects the blue chromatin
stained with DAPI representing the cells in
the native sample. F) The overlay of immu­
nostaining for type I (B) and III (D) collagens
shows an equal distribution of the two
collagen types throughout the entire field of
vision in acellular samples. Scale bars: 50
μm.

Whereas the collagen type I detectability was similar in native and by a destruction or washing out of the components, albeit we cannot
acellular samples, type III collagen seemed to be uncovered by the exclude, that the components are masked in some way by the acellula­
acellularization. This might be caused by the removal of cells and per­ rization procedure itself. The small leucine-rich proteoglycan decorin
icellular proteoglycans and glycoproteins such as decorin and fibro­ plays a major role in collagen fibrillogenesis and is therefore crucial for
nectin, which allow an improved accessibility of the epitopes by the the integrity of collagen-rich tissues such as the here investigated TMF
antibodies in the acellular state. Contrary, the accessibility of the epi­ (Reed and Iozzo, 2002). The treatment with SDS seems to decrease the
topes could be identical, but the cells and pericellular extracellular amount of decorin according to the immunostainings in this study,
matrix components could mask the same and therefore result in a fainter indicated by a less intense staining result compared to the native sample.
staining of type III collagen in the native state. We suspect the fainter The strong positive correlation between ageing and the elastic
staining for decorin and fibronectin in the acellular samples to be caused modulus of the acellular TMF samples indicates a progressive age-

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J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

Fig. 9. Anti-fibronectin and anti-decorin

immunostainings of the native and acellular
temporal muscle fascia (TMF). A) Decorin,
stained in green colour, can be detected
throughout the entire field of vision in the
native TMF. The white arrow displays the
blue chromatin stained with DAPI repre­
senting the cells in the native sample. B) The
anti-decorin staining is weaker in the acel­
lular sample compared to the native one
depicted in A. C) Fibronectin, stained in red,
can be seen throughout the entire field of
vision of the native TMF. The white arrow
displays the blue DAPI-stained chromatin
representing the cells in the native sample.
D) The anti-fibronectin immunostaining is
fainter in the acellular sample compared to
the native sample displayed in C. E) The
overlay of decorin and fibronectin shows the
distribution of the two matrix components in
the human TMF. Whereas decorin is distrib­
uted throughout the entire matrix, fibro­
nectin is most prominently seen in a streaky
pattern in transition areas (red arrow) be­
tween the extracellular matrix and ‘empty
spaces’, which are filled with fluid in the
immunohistochemistry slices. The white
arrow displays the blue chromatin stained
with DAPI representing the cells in the native
sample. F) The acellular sample shows an
evenly distributed decorin (green staining)
and several red fibronectin containing
streaks. However, the streaky fibronectin
pattern seen in the native sample displayed
in E cannot be depicted in the acellular state.
Scale bars: 50 μm.

related stiffening of the tissue, which was, for example, stated for other anymore or their regulatory influence in extracellular matrix homeo­
soft tissues such as the aorta or lung before but was yet not shown for the stasis may become ineffective. Thus, the age-dependent stiffening of the
TMF on a statistically significant level (Lai-Fook and Hyatt, 2000; extracellular matrix can be detected biomechanically as shown in this
Trindade et al., 2012; Wuyts et al., 1995). One possible explanation is study. Interestingly, our results yield that an increased post-mortem
that cells, proteoglycans or glycosaminoglycans compensate for an interval (time between death of the cadaver and harvesting of the tis­
age-dependent stiffening of the collagens in the TMF’s native state. sue during forensic autopsies) leads to increased elastic moduli of the
Another one might be the smaller number of samples investigated in TMF samples, too. For both variables, normal ageing and the
former studies which potentially do not allow to reach statistical rele­ post-mortem interval, higher values increase the probability of tissue
vance. If the cells, proteoglycans or glycosaminoglycans are removed, deterioration (Jellinghaus et al., 2019; MacNee et al., 2014). Deduced
the stiffening character of the ageing collagens cannot be balanced from the elastic moduli obtained in the given study, the deterioration of

8
J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

the TMF appears as an increased stiffening of the tissue most likely transplant material from a biomechanical perspective as they show
caused by changes in collagens (Panwar et al., 2015). The stiffening of similar tensile properties compared to their native counterparts.
the TMF with age could be a consequence of a decreased mechanical Therefore, no biomechanical difference is expected between wet and dry
stimulation caused by a lower food intake of older people (Drewnowski tympanoplasty grafts regarding the different number of cells. Treatment
and Shultz, 2001) in general. Consequently, the decreased total chewing of human TMF with SDS seems to wash out proteoglycans (decorin) and
motions could lead to changes in the collagens, which may result in a glycoproteins (fibronectin).
stiffer TMF. Frequently, elderly people have an incomplete denture,
which is associated with lower biting forces (Peyron et al., 2017; Shala
Declaration of competing interest
et al., 2018). During the chewing motion, forces are applied to the TMF
by the temporal muscle (Herring et al., 2011) and the masseter (Eisen­
None.
berg and Brodie, 1965). One could assume that lower biting forces most
likely lead to lower pressures to impact on the TMF. The missing me­
References
chanical stimulus of the TMF due to lower peak loads could then equally
lead to collagen changes, which result in an increased stiffness. Alkan, S., Baylancicek, S., Sozen, E., Basak, T., Dadas, B., 2009. Effect of the use of dry
The SEM performed in this study confirmed the anisotropic character (rigid) or wet (soft) temporal fascia graft on tympanoplasty. J. Otolaryngol. Head
of the TMF and its arrangement in collagen bundles. The amount of these Neck Surg. 38, 126–132.
Baker, R.H., de Silva, J.D., Henderson, H.W., Kirkpatrick, N., Joshi, N., 2005. A novel
bundles and their course is highly variable, which can be detected based technique of harvesting temporalis fascia autografts for correction of recurrent
on both the SEM findings and the large inter-individual differences in blepharoptosis. Ophthalmic Plast. Reconstr. Surg. 21, 298–300.
cross sections. These factors account for the high scatter of individual Bandak, F.A., Vander Vorst, M.J., Stuhmiller, L.M., Mlakar, P.F., Chilton, W.E.,
Stuhmiller, J.H., 1995. An imaging-based computational and experimental study of
results of the biomechanical tests obtained in this study. As collagens skull fracture: finite element model development. J. Neurotrauma 12, 679–688.
resist higher forces when stretched longitudinal rather than perpendic­ Basterzi, Y., Sari, A., Sari, A., 2009. Surgical treatment of an exposed orbital implant with
ular to the bundles (Reilly and Burstein, 1975), it can be concluded that vascularized superficial temporal fascia flap. J. Craniofac. Surg. 20, 502–504.
Bulgannawar, B.A., Rai, B.D., Nair, M.A., Kalola, R., 2011. Use of temporalis fascia as an
the orientation in which the TMF graft is transplanted can be utilized to
interpositional arthroplasty in temporomandibular joint ankylosis: analysis of 8
optimally use the resistance of the tissue. This study showed that the cases. J. Oral Maxillofac. Surg. 69, 1031–1035.
anisotropic course of the collagens of the TMF can already be recognized Chan, H., Delyfer, M.N., Pechmeja, J., Andrebe, C., Mercier, A.E., Dutheil, C.,
Korobelnik, J.F., Paya, C., 2017. Spontaneous rupture of chorioretinal coloboma in
macroscopically and therefore easily be utilized by the surgeon.
an 8-year-old child is treated by temporal fascia graft. J AAPOS 21, 73–75.
Furthermore, the “wavy” course of several collagen bundles seen in the Creuillot, V., Areiza, D.A., de Brosses, E.S., Bonnet, A.S., Lipinski, P., 2013. Finite
SEM and histology images indicates the potential of the fascia to stretch, element analysis of temporomandibular joints during opening-closing motion:
when forces are applied. Furthermore, we found that the ‘wavy’ pattern asynchronous case report. Comput. Methods Biomech. Biomed. Eng. 16 (Suppl. 1),
300–301.
of the fascia is most likely caused by elastic fibres, indicated by the Curtis, N., Witzel, U., Fitton, L., O’Higgins, P., Fagan, M., 2011. The mechanical
preferable detection of those fibres in areas, where the fascia appeared significance of the temporal fasciae in Macaca fascicularis: an investigation using
to be piled up. In this regard, the TMF could be pre-strained in the native finite element analysis. Anat. Rec. 294, 1178–1190.
Drewnowski, A., Shultz, J.M., 2001. Impact of aging on eating behaviors, food choices,
state and contract after the harvesting, resulting in a corrugated nutrition, and health status. J. Nutr. Health Aging 5, 75–79.
appearance of the extracellular matrix in histology and the here per­ Eisenberg, N.A., Brodie, A.G., 1965. Antagonism of temporal fascia to masseteric
formed SEM. Also, the collagens of the fascia could be corrugated in vivo, contraction. Anat. Rec. 152, 185–192.
Fosse, L., Ronningen, H., Benum, P., Sandven, R.B., 2006. Influence of water and fat
with the potential to strain for example in biting movements. According content on compressive stiffness properties of impacted morsellized bone: an
to the here obtained data, the fascia can be strained approximately 16% experimental ex vivo study on bone pellets. Acta Orthop. 77, 15–22.
of its initial length before the tissue fails. The straining potential of the Fricov� a, M., Zdenek, H., Konvickov� a, S., 2006. Modelling of the temporomandibular joint
and FEM analysis. Acta Bioeng. Biomech. 8, 35–43.
TMF has to be regarded when it is argued that the TMF antagonizes the
Hammer, N., Huster, D., Fritsch, S., H€ adrich, C., Koch, H., Schmidt, P., Sichting, F.,
forces to the zygomatic arc applied by the masseter (Curtis et al., 2011; Wagner, M.F., Boldt, A., 2014. Do cells contribute to tendon and ligament
Eisenberg and Brodie, 1965). This study furthermore highlights the biomechanics? PloS One 9, e105037.
Hermann, H., 1960. Tympanic membrane plastic with temporalis fascia. Hals Nas.
TMF’s tendency to strain and thus elongate in loading situations.
Ohrenh. 9, 136–139.
Therefore, it has to be elucidated, if the TMF can provide a significant Herring, S.W., Rafferty, K.L., Liu, Z.J., Lemme, M., 2011. Mastication and the postorbital
contribution to antagonize the masseter forces in forceful biting ligament: dynamic strain in soft tissues. Integr. Comp. Biol. 51, 297–306.
(Eisenberg and Brodie, 1965) or if the zygomatic arch would preferably International Organization for Standardization, 1996. Plastics-determination of Tensile
Properties (ISO Standard No. 527-2).
fracture before a counterforce could be reached by the TMF. Potentially, Jalali, M.M., Motasaddi, M., Kouhi, A., Dabiri, S., Soleimani, R., 2017. Comparison of
the TMF is involved in the force-regaining capacity of the masticatory cartilage with temporalis fascia tympanoplasty: a meta-analysis of comparative
region (Curtis et al., 2011; Eisenberg and Brodie, 1965). Equally, the studies. Laryngoscope 127, 2139–2148.
Jellinghaus, K., Urban, P.K., Hachmann, C., Bohnert, M., Hotz, G., Rosendahl, W.,
bulging of the temporal muscle in forceful biting could pre-strain the Wittwer-Backofen, U., 2019. Collagen degradation as a possibility to determine the
TMF. Thus, the TMF might reach higher forces, which could aid as post-mortem interval (PMI) of human bones in a forensic context - a survey. Leg.
counterforces to the masseter. Med. 36, 96–102.
Jiang, Z., Lou, Z., 2017. Impact of the nature of the temporalis fascia graft on the
outcome of type I underlay tympanoplasty. J. Laryngol. Otol. 131, 472–475.
4.1. Limitations Katsuno, M., Uchida, K., Matsuno, A., 2017. A temporofrontal fascia flap that penetrated
temporal muscle for the reconstruction of an anterior skull base bone and dura: a
technical case report. Br. J. Neurosurg. 1–3.
The performed tensile testing of the TMF samples was conducted Kleiven, S., 2003. Influence of impact direction on the human head in prediction of
under uniaxial loading conditions in a quasi-static condition. Load- subdural hematoma. J. Neurotrauma 20, 365–379.
bearing forces under native conditions are likely of a multiaxial and Lai-Fook, S.J., Hyatt, R.E., 2000. Effects of age on elastic moduli of human lungs. J. Appl.
Physiol. 89, 163–168, 1985.
dynamic nature. Despite a very careful separation of the TMF from the
Liang, Z., Luo, Y., 2015. A QCT-based nonsegmentation finite element head model for
strongly attached temporal muscle with no caused defects visible to the studying traumatic brain injury. Appl. Bionics. Biomech. 2015, 837585.
naked eye, a slight disruption of the extracellular matrix during har­ MacNee, W., Rabinovich, R.A., Choudhury, G., 2014. Ageing and the border between
health and disease. Eur. Respir. J. 44, 1332–1352.
vesting with a potential effect on the tensile properties of the tissue
Maniu, A., Cosgarea, M., 2012. Mastoid obliteration with concha cartilage graft and
cannot be excluded totally. temporal muscle fascia. ORL J. Otorhinolaryngol. Relat. Spec. 74, 141–145.
Mirmalek-Sani, S.H., Sullivan, D.C., Zimmerman, C., Shupe, T.D., Petersen, B.E., 2013.
5. Conclusions Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue. Am.
J. Pathol. 183, 558–565.
Mizuno, T., 2014. Subciliary augmentation of the lower eyelid in Asians using a deep
Acellular TMF scaffolds could be an interesting and promising temporal fascia graft: a preliminary report. Aesthetic Plast. Surg. 38, 303–308.

9
J. Zwirner et al. Journal of the Mechanical Behavior of Biomedical Materials 108 (2020) 103833

Neuhaus, R.W., Shorr, N., 1983. Use of temporal fascia and muscle as an autograft. Arch. Tayebi, L., Rasoulianboroujeni, M., Moharamzadeh, K., Almela, T.K.D., Cui, Z., Ye, H.,
Ophthalmol. 101, 262–264. 2018. 3D-printed membrane for guided tissue regeneration. Mater. Sci. Eng. C.
Panwar, P., Lamour, G., Mackenzie, N.C., Yang, H., Ko, F., Li, H., Bromme, D., 2015. Mater. Biol. Appl. 84, 148–158.
Changes in structural-mechanical properties and degradability of collagen during Taylor, J.R., 1997. An Introduction to Error Analysis : the Study of Uncertainties in
aging-associated modifications. J. Biol. Chem. 290, 23291–23306. Physical Measurements, second ed. University Science Books, Sausalito, Calif.
Park, S.W., Kim, J.H., Choi, C.Y., Jung, K.H., Song, J.W., 2015. Various applications of Thali, M.J., Kneubuehl, B.P., Zollinger, U., Dirnhofer, R., 2002. The "skin-skull-brain
deep temporal fascia in rhinoplasty. Yonsei Med. J. 56, 167–174. model": a new instrument for the study of gunshot effects. Forensic Sci. Int. 125,
Perez Del Palomar, A., Doblare, M., 2006. Finite element analysis of the 178–189.
temporomandibular joint during lateral excursions of the mandible. J. Biomech. 39, Trindade, V.L., Martins, P.A., Santos, S., Parente, M.P., Natal Jorge, R.M., Santos, A.,
2153–2163. Santos, L., Fernandes, J.M., 2012. Experimental study of the influence of senescence
Peyron, M.A., Woda, A., Bourdiol, P., Hennequin, M., 2017. Age-related changes in in the biomechanical properties of the temporal tendon and deep temporal fascia
mastication. J. Oral Rehabil. 44, 299–312. based on uniaxial tension tests. J. Biomech. 45, 199–201.
Protzman, N.M., Brigido, S.A., 2015. Recent advances in acellular regenerative tissue Virkkula, P., Makitie, A.A., Vento, S.I., 2015. Surgical outcome and complications of
scaffolds. Clin. Podiatr. Med. Surg. 32, 147–159. nasal septal perforation repair with temporal fascia and periosteal grafts. Clin. Med.
Reed, C.C., Iozzo, R.V., 2002. The role of decorin in collagen fibrillogenesis and skin Insights Ear, Nose Throat 8, 7–11.
homeostasis. Glycoconj. J. 19, 249–255. Wuyts, F.L., Vanhuyse, V.J., Langewouters, G.J., Decraemer, W.F., Raman, E.R.,
Reilly, D.T., Burstein, A.H., 1975. The elastic and ultimate properties of compact bone Buyle, S., 1995. Elastic properties of human aortas in relation to age and
tissue. J. Biomech. 8, 393–405. atherosclerosis: a structural model. Phys. Med. Biol. 40, 1577–1597.
Schleifenbaum, S., Prietzel, T., Aust, G., Boldt, A., Fritsch, S., Keil, I., Koch, H., Xu, M., He, Y., Bai, X., 2016. Effect of temporal fascia and pedicle inferior turbinate
M€ obius, R., Scheidt, H.A., Wagner, M.F., Hammer, N., 2016. Acellularization- mucosal flap on repair of large nasal septal perforation via endoscopic surgery. ORL
induced changes in tensile properties are organ specific - an in-vitro mechanical and J. Otorhinolaryngol. Relat. Spec. 78, 303–307.
structural analysis of porcine soft tissues. PloS One 11, e0151223. Yegin, Y., Celik, M., Koc, A.K., Kufeciler, L., Elbistanli, M.S., Kayhan, F.T., 2016.
Scholze, M., Singh, A., Lozano, P.F., Ondruschka, B., Ramezani, M., Werner, M., Comparison of temporalis fascia muscle and full-thickness cartilage grafts in type 1
Hammer, N., 2018. Utilization of 3D printing technology to facilitate and pediatric tympanoplasties. Braz. J. Otorhinolaryngol. 82, 695–701.
standardize soft tissue testing. Sci. Rep. 8, 11340. Zwirner, J., Bayer, R., Japes, A., Eplinius, F., Dressler, J., Ondruschka, B., 2017. Suicide
Schwarz, G.S., Spinelli, H.M., 2008. Correction of upper eyelid retraction using deep by the intraoral blast of firecrackers - experimental simulation using a skull simulant
temporal fascia spacer grafts. Plast. Reconstr. Surg. 122, 765–774. model. Int. J. Leg. Med. 131, 1581–1587.
Shala, K., Tmava-Dragusha, A., Dula, L., Pustina-Krasniqi, T., Bicaj, T., Ahmedi, E., Zwirner, J., Ondruschka, B., Scholze, M., Schulze-Tanzil, G., Hammer, N., 2019.
Lila, Z., 2018. Evaluation of maximum bite force in patients with complete dentures. Mechanical and morphological description of human acellular dura mater as a
Open Access Maced. J Med. Sci. 6, 559–563. scaffold for surgical reconstruction. J. Mech. Behav. Biomed. Mater. 96, 38–44.
Singh, M., Kruse, C., Eriksson, E., Caterson, E.J., 2015. Deep temporal fascia coverage of Zwirner, J., Scholze, M., Waddell, J.N., Ondruschka, B., Hammer, N., 2019b. Mechanical
the loading weight in paralytic lagopthalmos patients. J. Craniofac. Surg. 26, properties of human dura mater in tension - an analysis at an age range of 2 to 94
1631–1633. years. Sci. Rep. 9, 16655.
Song, L., Murphy, S.V., Yang, B., Xu, Y., Zhang, Y., Atala, A., 2014. Bladder acellular
matrix and its application in bladder augmentation. Tissue Eng. B. Rev. 20, 163–172.

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