morphological studies of the toe pads of the rock frog,...
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ResearchCite this article: Drotlef DM, Appel E, Peisker
H, Dening K, del Campo A, Gorb SN, Barnes
WJP. 2015 Morphological studies of the toe
pads of the rock frog, Staurois parvus ( family:
Ranidae) and their relevance to the
development of new biomimetically inspired
reversible adhesives. Interface Focus 5:
20140036.
http://dx.doi.org/10.1098/rsfs.2014.0036
One contribution of 15 to a theme issue
‘Biological adhesives: from biology to
biomimetics’.
Subject Areas:biomaterials, biomimetics
Keywords:rock frog, adhesive toe pad, functional
morphology, cell shape, electron microscopy,
biomimetics
Author for correspondence:W. Jon. P. Barnes
e-mail: [email protected]
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsfs.2014.0036 or
via http://rsfs.royalsocietypublishing.org.
Morphological studies of the toe pads ofthe rock frog, Staurois parvus ( family:Ranidae) and their relevance to thedevelopment of new biomimeticallyinspired reversible adhesives
Dirk M. Drotlef1, Esther Appel2, Henrik Peisker2, Kirstin Dening2,Aranzazu del Campo1, Stanislav N. Gorb2 and W. Jon. P. Barnes3
1Max Planck Institut fur Polymerforschung, Mainz, Germany2Functional Morphology and Biomechanics, University of Kiel, Kiel, Germany3Centre for Cell Engineering, University of Glasgow, Scotland, UK
The morphology of the toe epithelium of the rock frog, Staurois parvus (Family
Ranidae), was investigated using a variety of microscopical techniques. The
toe pad epithelium is stratified (four to five cell layers), the apical parts of
the cells of the outermost layer being separated by fluid-filled channels. The
surface of these cells is covered by a dense array of nanopillars, which also
cover the surface of subarticular tubercles and unspecialized ventral epi-
thelium of the toes, but not the dorsal epithelium. The apical portions of the
outer two layers contain fibrils that originate from the nanopillars and are
oriented approximately normal to the surface. This structure is similar to the
pad structure of tree frogs of the families Hylidae and Rhacophoridae, indi-
cating evolutionary convergence and a common evolutionary design for
reversible attachment in climbing frogs. The main adaptation to the torrent
habitat seems to be the straightness of the channels crossing the toe pad,
which will assist in drainage of excess water. The presence of nanopillar
arrays on all ventral surfaces of the toes resembles that on clingfish suckers
and may be a specific adaptation for underwater adhesion and friction. The rel-
evance of these findings to the development of new biomimetically inspired
reversible adhesives is discussed.
1. IntroductionIn addition to tree frogs that are largely arboreal (though smaller species may be
found in the ground flora), there are also frogs that live around freshwater streams
(stream frogs) and waterfalls (rock and torrent frogs) that also possess adhesive
toe pads [1]. The purpose of this study is to investigate the morphology of the
toe pads of one such rock frog, the Lesser rock frog, Staurois parvus Inger and
Haile, found in and around waterfalls within the rainforests of Borneo. Is the
pad morphology very similar to those of tree frogs, or can we find differences
that might relate to its rather different habitat?
The foundations of our understanding of the structure and function of tree
frog toe pads dates back 40 years, but has received a significant boost in recent
years by the realization of the biomimetic significance of mechanisms of animal
adhesion (e.g. [2,3]). Many of these biomimetic studies have been aimed at
replicating the dry adhesive mechanisms of geckos (e.g. [4–7]), but tree frog
adhesion has also been highlighted as having potential for the development
of new smart adhesives able to adhere under wet conditions [8–11].
The morphology of the adhesive toe pads of tree frogs has been extensively
investigated by both transmission and electron microscopy [12–21]. There is
also a recent study by Barnes et al. [22] that combined atomic force microscopy
(AFM) and cryo-scanning electron microscopy (including freeze-fracture).
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These investigations show that the cells that make up the
stratified columnar epithelium of the toe pads differ from
those of the skin on the dorsal surface of the toes or indeed
any other epidermal cell type [1], being specialized for their
adhesive function. Indeed, circumferal and, in many species,
proximal margin grooves separate the toe pad epithelium
from neighbouring, less specialized skin. Accessory adhesive
tubercles, found more proximally on the digits as well as on
the main body of the feet, may be similarly specialized, but
their precise role in adhesion and/or friction remains to be
confirmed. The outer layer of cells, largely hexagonal in sur-
face view, appears flat-topped at the microscale, the apical
portions of individual cells being separated from each other
by deep, fluid-filled channels, giving an aspect ratio of
ca 0.5 for the outer parts of the epithelial cells. The fluid
within the channels is secreted by mucous glands that have
ducts ending in these channels, the channels serving to
spread the mucus over the surface of the pad. Additionally,
on surfaces wetted by rain, the channels may act to disperse
excess fluid to the pad edge. Finally, the epithelial cells are
covered by a dense array of nanopillars [23] that originate
from desmosomes [12]. Within the epithelial cells, there are
large numbers of keratin fibrils (tonofilaments), arising from
the nanopillars, that are oriented approximately normal to
the surface [12,20]. They are the main cytoskeletal elements
of the toe pad epithelium and will confer a degree of rigidity
to the cells [15]. Additionally, by analogy with compara-
ble research on insects [24–26], it is probable that they are
important for both toe pad adhesion and detachment.
Arboreal frogs (tree frogs) are found in at least seven dif-
ferent families including the Hylidae and Rhacophoridae,
whose members are almost exclusively arboreal in the New
and Old Worlds, respectively. The morphological features
described above show little variation between the different
families of tree frog and, as common ancestors with toe pads
are lacking, clearly demonstrate convergent evolution [1].
Frog pads employ wet adhesion [27,28] that functions
optimally when the fluid layer under the pad is very thin
(�1 mm) and there are no air pockets [29,30]. Given the pres-
ence of this fluid joint, the fact that toe pads also generate
significant friction forces is surprising. Federle et al. [29]
have proposed that this is achieved through direct contact
between the tops of the nanopillars and the substrate in
adhering frogs, and their experimental data demonstrate
static as well as dynamic friction. Toe pads detach by peeling
from the rear (i.e. in a proximal to distal direction) [28]. This is
a natural consequence of the forward and upward movement
of the distal part of the limb at the beginning of the locomotor
swing phase, but does mean that, on slopes, downward-
facing pads would have a tendency to peel spontaneously.
As a consequence, tree frogs on slopes usually place their
pads so that they face uphill [30]. Frictional forces prevent
sliding on sloping surfaces, produce the close contact upon
which good adhesion depends and, on overhanging surfaces
where the limbs are spread out sideways [31], prevent peeling
of the pads from the surface [32].
Much less is known about adhesion and toe pad mor-
phology in rock or torrent frogs. Ohler [33] carried out a
preliminary study of toe pad morphology of torrent-living
ranid frogs, demonstrating that the toe pads of Amolops spp.
had distinct anatomical differences from the typical pattern
seen in tree frogs. The toe pad epithelial cells were elongated,
providing much straighter channels between the centre of the
pad and the periphery. Experimental evidence is lacking, but
an obvious function for these shorter pathways from centre to
edge of pad would be to promote rapid drainage of excess
fluid from underneath the pad. Such elongated channels were
also found in the torrent frog, Odorana hosii, but only around
the edges of the toe pads in the rock frog, Staurois guttatus [34].
A biomechanical study has been carried out on the Trinidadian
stream frog, Mannophryne trinitatis, by Barnes et al. [35], while
Endlein et al. [34] made a detailed comparison between the
adhesive abilities of the rock frog S. guttatus and a rhacophorid
tree frog of comparable size. In both studies, the stream/rock
frog was good at adhering to rough wet surfaces, but very
poor at sticking to dry rough ones. They also slipped on
smooth wet surfaces. These data suggest that torrent/stream
frogs can cope with running water so long as the surfaces are
rough. This study searched for morphological features that
could underlie these abilities and discusses the implications of
these findings for the development of new smart adhesives.
2. Material and methods2.1. AnimalsThe frog species of our study was the Lesser rock frog, S. parvus(family Ranidae), a native of Borneo. The frogs (N ¼ 5) were sub-
adult with a snout–vent length of 30 mm. They were donated by
Schonbrunn Zoo (Vienna, Austria).
2.2. Sample preparation for scanning electronmicroscopy
Frog toes were cut off with a scalpel and fixed in 2.5% glutaral-
dehyde in 0.1 M phosphate buffer for 2 h at room temperature.
They were then rinsed three times (5 min per rinse) in 0.1 M
phosphate buffer containing 2% sucrose. Toes were post-fixed
with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h,
then washed three times in distilled water (15 min per wash),
before en bloc staining with 0.5% aqueous uranyl acetate for
1 h in the dark (as this is a light-sensitive stain). Following two
further washes in distilled water, samples were dehydrated
through an acetone series (30, 50, 70 and 90%, 15 min in each),
then four changes of absolute acetone (15 min each) and four
changes of dried absolute acetone (3A molecular sieve). Toe
samples were placed in critical point drying (CPD) specimen pro-
cessing capsules (type C211, TAAB Laboratories Equipment Ltd,
Aldermaston, Berkshire, UK) under dried absolute acetone and then
critical point dried using a Polaron Critical Point Drying Apparatus
E3000 (Quorum Technologies, Lewes, East Sussex, UK). Dried
frog toes were orientated on aluminium pin scanning electron
microscopy (SEM) stubs using double sided copper conductive
tape and quick drying silver DAG paint, all from Agar Scientific
(Stansted, Essex, UK). Mounted toes were gold/palladium coated
(approx. 20 nm thickness) using a Polaron SC515 SEM coating
system (Quorum Technologies) and then viewed on a JEOL6400
SEM (JEOL, Tokyo, Japan) running at 6–10 kV. The above describes
procedures used in Glasgow. Procedures carried out in Kiel were
similar, with specimens being viewed on a Hitachi S-4800 SEM
(Hitachi High-Technologies Corp., Tokyo, Japan).
2.3. Sample preparation for transmission electronmicroscopy
Frog toes were cut off as described earlier, with the initial sample
preparation procedures following those described above for SEM.
Following fixation in glutaraldehyde, post-fixation in osmium
1 mm
Figure 1. SEM image of forelimb of S. parvus; ventral view showing toe padsand subarticular tubercles (arrows). The ‘thumb’ (digit 1) is the small pad onthe right hand side.
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tetroxide, en bloc staining with uranyl acetate and washing in dis-
tilled water, the toes were processed through a dehydration series
of ethanol starting with 30%, then 50, 70 and 90% (15 min in each
solution), before four changes in absolute ethanol (for 15 min each)
and drying in absolute ethanol (3A molecular sieve; four changes
of 15 min each). The absolute ethanol was then replaced with 100%
propylene oxide for three 15 min changes, and then into a 50 : 50
propylene oxide/Araldite 502/812 resin mix (TAAB Laboratories
Equipment Ltd) minus accelerator overnight. The toes were
given several changes of pure resin minus accelerator over the
next day; then placed in pure resin plus accelerator overnight
and fresh resin embedded in flat-bed moulds the next morning.
They were then left to polymerize at 608C for 48 h. Semi-thin
0.35 mm light microscope sections were cut using a Leica Ultracut
UTC Ultratome (Leica Microsystems, Milton Keynes, UK) and a
Drukker histomicrotome knife with trough (Drukker International,
Cuijk, The Netherlands). Sections were dried on a glass slide on a
hotplate (808C) and then stained with 1% toluidine blue/1% borax
in distilled water. Ultrathin sections (60–70 nm) were also cut using
the Leica Ultracut UTC Ultratome and a Drukker Ultramicrotome
knife. Sections were picked up on slot or 100 mesh formvar-
coated copper grids and contrast-stained with 2% methanolic
uranyl acetate for 5 min and Reynolds lead citrate for 5 min. Toe
samples were viewed on a LEO912AB transmission electron micro-
scope (Carl Zeiss Ltd, Cambridge, UK), running at 120 kV. Images
were captured using an Olympus iTEM soft imaging system.
2.4. Sample preparation for cryo-SEMFrog toes were tied with a synthetic fibre to prevent blood loss, cut
off with a scalpel and plunge-frozen in liquid propane cooled by
liquid nitrogen. This procedure avoided formation of ice crystals
and prevented damage of pad structure. The specimens were
stored in liquid nitrogen. Cryo-SEM analysis was performed with
a Hitachi S-4800 SEM (Hitachi High-Technologies Corp.) using a
cryo-preparation chamber Gatan ALTO-2500 (Gatan Inc., Abing-
don, UK). The frozen frog toes were fixed with Tissue-Tek O.C.T
(Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) to
the specimen holder and immediately plunged in liquid nitrogen
to avoid thawing. They were then placed in the cooled (21408C)
cryo-preparation chamber (using liquid nitrogen). The frozen speci-
mens were sputter-coated with a gold–palladium layer (1 : 9, layer
thickness 10–20 nm) and transferred to the microscope. The
samples were examined at 21208C at an accelerating voltage of
2–5 kV.
2.5. Sample preparation for atomic force microscopyThe AFM studies were performed with a NanoWizard I AFM (JPK
Instruments AG, Berlin, Germany). The frog toe was tied with a
synthetic fibre and cut off with a scalpel. The toe was fixed with
superglue to a glass slide in an AFM BioCell chamber (JPK
Instruments AG, Berlin, Germany) and submerged in frog Ringer
solution. The measurements were conducted with a pyrex-nitride-
probe cantilever (silicon nitride SPM-Sensor, NanoWorld AG,
Neuchatel, Switzerland) in intermittent (tapping) mode. The resol-
ution of the scans was 1024 � 1024 pixels. The images were
processed with the SPIP software (Scanning probe image processing
software, v. 5.1.2, Image Metrology A/S, Hørsholm, Denmark).
3. Results3.1. Toe morphology (SEM and cryo-SEM)3.1.1. Toe padsThe toe pads are located on the expanded tips of each digit, of
which there are four in each forelimb (figure 1) and five in each
hindlimb. Additionally, there are accessory adhesive structures
called subarticular tubercles located more proximally on
the digits (figure 1, arrows). The toe pads are surrounded by
circumferal and proximal grooves (figures 1 and 2). The prox-
imal groove, which delineates the proximal margin of the
pad, is shallow as seen in the longitudinal section of the pad
(figure 2b, small arrow), while the circumferal groove that
delineates the distal and lateral pad margins is a large deep
channel, especially at the distal tip of the pad (figure 2b, large
arrow). Interestingly, as this is not a common feature of tree
frogs, the larger pads extend distally around the front of the
toe (figure 2a,b). The following features were visible.
3.1.2. Toe pad surface micropattern—epithelial cellsAs shown in figure 3a,b, the toe pad surface is made up of
flat-topped epithelial cells of ca 13 mm diameter separated by
channels with a width of ca 2 mm in fixed material, but prob-
ably narrower in living cells where channel width varies with
the amount of fluid under the pad. Mucus glands are rare in
toe pads (less than five per pad), less common than in other
skin areas. The duct of one such mucus gland is shown in
figure 3b, opening into an intercellular channel. The majority
of epithelial cells are irregular hexagons, but other shapes are
also seen. In a total number of 122 epithelial cells, the hexago-
nal shape was predominant (56%), followed by pentagonal
(33%) and heptagonal (11%). A single octagonal epithelial
cell was also recorded. The white lines on figure 3a show the
existence of relatively straight channels crossing the pad in an
approximatelyanterior–posterior direction. We have quantified
the lengths of such channels in two pads (10 channels in each
pad), including those in figure 3a. Relative to a straight line
(length ¼ 1), such channels have a length of 1.13+0.04
(mean+ s.d., N ¼ 20). This is significantly better than a pattern
of regular hexagons, which have a relative channel length
of 1.33. Interestingly, channels going across the pad also have
a relative channel length of less than 1.33, but are not as straight
as the anterior–posterior channels ( p , 0.001, Wilcoxan signed-
rank test). Such channels should promote good drainage of
excess water in the rock frogs’ stream habitat, those running in
an anterior–posterior direction being of greater significance as
pads normally face uphill. For full details of this analysis, see
electronic supplementary material, table S1 and figure S1.
Figure 3c shows such a channel at a point where three epithe-
lial cells meet. The channel walls can be seen to be ridged, the
200 mm 100 mm
ventral
(a) (b)
dorsal
Figure 2. (a) SEM image of digit 2 of forelimb of S. parvus, showing toe pad epithelium, circumferal and proximal grooves. (b) Longitudinal section of toe pad(light microscope image) showing the same features. Note relatively large size of circumferal groove (large arrow) compared with proximal groove (small arrow).
(a)
100 mm
1 mm 500 nm
10 mm
(b)
(c) (d )
Figure 3. SEM images of toe pad epithelium of S. parvus. (a) White lines show relatively straight channels crossing the pad. (b) Polygonal epithelial cells (mainlyhexagonal in shape) surrounded by deep channels with a single mucous pore (centre). (c) Edge of a pad epithelial cell showing dense array of nanopillars coveringthe pad surface. (d ) High-power view of nanopillars.
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ridges probably reflecting the presence of bundles of keratin
tonofilaments beneath the surface.
3.1.3. Toe pad surface nanopattern—nanopillarsAs in tree frogs, the surface of the epithelial cells is covered in a
dense array of nanopillars that form a surface nanopattern con-
sisting of regular polygons 200–300 nm in diameter (figure 3d).
Their height is not measurable from this image, partly because
they are so densely packed and partly because they merge with
the ridges that characterize the channel walls. However, trans-
mission electron microscopy (TEM) images show their height
to be 400–500 nm, giving them an aspect ratio of 1.4–2.0
(figure 10). Bundles of filaments, presumed to be keratin tono-
filaments from immunohistochemical studies on other frog
species (e.g. [36]), are visible through the walls of the nanopil-
lars in some of our cryo-SEM images (figure 4b). Such images
(figure 4a,b) also revealed that the nanopillars have a slightly
concave terminal and in, unspecialized ventral toe epithelium
at least, can have aspect ratios of up to 2.5. One of the
advantages of using cryo-SEM is that it allows one to view
the mucus covering of the pad. In figure 4a, the frozen mucus
can be seen surrounding but not covering the nanopillars and
also filling their dimpled tops.
3.1.4. Epithelium of toe tuberclesProximal to each toe pad are located subarticular tubercles
(figures 1 and 5a). Their epithelial surface possesses similar
features to those of the toe pads (figure 5c,d ). Polygonal
(a)
2 mm
1 mm
(b)
Figure 4. High-power images of nanopillars in S. parvus. Cryo-SEM images ofunspecialized ventral epithelium showing (a) the tops of the nanopillars sur-rounded by frozen mucus and (b) fibrils (bundles of tonofilaments) visiblethrough the walls of the nanopillars (arrows).
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epithelial cells (mostly 5 or 6-sided) are separated by channels
(figure 5c,d ), but the channel depth in tubercular epithelium
is significantly shallower (1–3 mm, figure 8b) than in the
pad epithelium (10 mm, figure 8a), and significantly shal-
lower and narrower in the peripheral region of the tubercles
(figure 5d ) than in the centre (figure 5c). Epithelial cells in
the tubercle are also covered by an array of nanopillars
with similar dimensions and arrangement as in toe pads,
but at a significantly lower density (figure 5b). By contrast,
the density of mucous pores (several of which are visible in
both figure 5c,d ) appears higher than in the toe pad epi-
thelium. These images also show marks on their surface
(actually gaps in the nanopillar array as figure 5b makes
clear) that clearly indicate the position of the layer of cells
that previously had lain above them (figure 5d, arrows).
Such marks are also present on the toe pad epithelium
(obvious in figure 11a, less so in figure 3b). The different
cell layers are thus displaced with respect to each other in
both x and y axes, as previously noted for tree frogs [22].
3.1.5. Ventral unspecialized toe epitheliumExamination of the ventral epithelium of the toes, excluding toe
pads and subarticular tubercles, shows a degree of adaptation for
adhesion and friction. As figure 6 shows, the epithelial cell sur-
face is covered by nanopillars, but at lower density than in the
more specialized epithelia of the toe pads and subarticular tuber-
cles. Such nanopillars are absent at the cell boundaries, giving
rise to narrow, shallow, intercellular channels. Mucous pores
are common. The cryo-SEM images of nanopillars discussed ear-
lier (figure 4a,b) were taken from this area, their lower density
making structural features easier to see and measure.
3.1.6. Dorsal toe epitheliumIn contrast to the ventral epithelium, the epithelium of the
dorsal surface of the toes has neither nanopillars nor intercellu-
lar channels; i.e. it shows no adaptations for either adhesion or
friction. At low power (figure 7a), the surface appears relatively
smooth, though large Y-shaped pores, possibly of mucous
glands (though the pore structure is very different than that
of the ventrally located mucous pores), are common. At
higher power (figure 7b), the dorsal epithelium is seen to be
covered by nanostructures (150 nm across) that are joined
end to end to form a network that covers the cell surface.
Their appearance is similar to that of vermicular (worm-like)
structures seen on lizard scales [37].
3.2. Toe pad anatomy (light microscopy and TEM)3.2.1. Epithelial cell layersExamination of longitudinal sections of the toes under the light
microscope reveals that the epithelium of the toes of S. parvus is
stratified. In the toe pads, it has a thickness of ca 50 mm, consist-
ing of four to five cell layers, with deep channels separating the
outer portions of the outer cell layer (figure 8a). This layer has
dark pyknotic nuclei and consists of dying or dead cells. Per-
ipheral portions of the cytoplasm of the two outmost layers
are stained darkly, owing to the presence of keratin. The
large vesicles in the second layer will give rise to the intercellu-
lar channels when the second layer becomes the outer layer,
through the shedding of the outer layer. By contrast, unspecia-
lized ventral epithelium and the dorsal epithelium of the toe
are only 15–20 mm thick (figures 2b and 8b). The dorsal epi-
thelium has no channels, while the channels separating the
outer portions of the outer layer of cells in unspecialized ven-
tral toe epithelium are only about 1 mm deep. The extent of
the keratinization is much reduced. The epithelium of the sub-
articular tubercles is broadly similar to that of the toe pads,
though its thickness is reduced (data not shown).
3.2.2. Transmission electron microscopyThe electron microscope reveals a lot more detail of the ultra-
structure of the different layers. As, with each sloughing of
the outer layer, layer IV will progressively become layer III,
then II and finally I (the outer layer), examination of the differ-
ent layers also provides insights into how the specialized outer
layer with its deep channels, nanopillar arrays and keratin
fibrils develops from unspecialized beginnings. The outer
layer, seen in figure 9a, shows the cells to be filled with dark-
staining keratin tonofilaments that have their origin in the
nanopillars, three of which are shown in enlarged view in
figure 10. Completely filling the nanopillars (figure 10), they
initially run normal to the cell surface (figure 9a), but gradually
bend proximally before stopping about halfway across the cell.
Even the peripheral cytoplasm appears dark, owing to keratin
deposition. Figure 9b shows the cell border between layers I
and II. Here, the outer cell layer is devoid of both tonofilaments
and keratin, though both are present in the peripheral part of
layer II. The outermost part of layer II also shows how the
nanopillars arise, as bundles of tonofilaments are becoming
separated from each other by clear cytoplasm and there are
some invaginations of the cell boundary into these clear areas
(figure 9b, arrows). Figure 9d shown the same layer I/layer II
boundary, but now including one of the large vesicles (seen
in figure 8a) that will give rise to the deep channels between
the outer parts of the epithelial cells in layer I. Figure 9c,
(a)
100 mm 500 nm
(b)
(c)
10 mm 10 mm
(d )
Figure 5. SEM images of subarticular tubercle epithelium of S. parvus. (a) Basal tubercle of digit 4 of forelimb. (b) High-power view of nanopillars, intercellularchannel (black arrow) and gap in the nanopillar array at the position of a cell boundary of the previous outer cell layer (white arrow). (c) Epithelium of centre oftubercle with larger intercellular channels. (d ) Epithelium near edge of tubercle with smaller intercellular channels (black arrows indicate channels; white arrows,gaps in the nanopillar array as described in b).
(a)
5 mm 500 nm
(b)
Figure 6. SEM images of nanopillar array and intercellular channels of unspecialized ventral epithelium of S. parvus digits. (a) Low power; arrow indicates openingof mucous gland. (b) High power.
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which illustrates the boundary between layers II and III, shows
an even earlier stage in the development of the structure of the
outer layer. Both cell layers are clearly living tissue, with typical
nuclei visible in the cells shown. The cell boundary is invagi-
nated, but to a lesser extent than the layer I/II boundary. In
some cases, short tonofilaments can be seen to be going both
inwards and outwards from desmosomes joining the two
surfaces together (figure 9c, arrows). Electron-dense particles,
probably corneous (horny) material containing keratin [38],
are visible in both layers.
3.3. Atomic force microscopic studies of the surfaceof the toe pads
3.3.1. Cell surface probed by the atomic force microscopyThe AFM studies provide further insight into the structure
and components of toe pad epithelium. Unlike SEM, TEM
and light microscopy, the tissue does not require to be
fixed, so problems of shrinkage and distortion are avoided.
However, as an AFM cantilever is not able to penetrate
narrow clefts, it cannot provide data on the depths of the
channels between either epithelial cells or nanopillars. AFM
scans showed the polygonal epithelial cells separated by
channels that did not exceed 1 mm in width (figure 11a),
considerably narrower than they appear in SEM images
(figure 3b). They also show the gaps in the nanopillar array
that reflect the position of the previous cell layer, a feature
not obvious in figure 3b. The narrowness of the channels
did not allow the cantilever tip to penetrate them and, there-
fore, the depth of the channels could not be estimated.
Figure 11b shows the nanostructures on the epithelial cell sur-
faces. The height profile (figure 11c) quantifies the extent to
which the tops of the nanopillars are not flat-terminated
but slightly concave. Precise quantification is difficult because
it is necessary for the profile to run precisely through the
(a)
10 mm 2 mm
(b)
Figure 7. SEM images of dorsal epithelium of S. parvus digits. (a) Low-power image of several epithelial cells. (b) High-power image of cell surface and the pore ofa ( presumed) mucous gland.
(a)
10 mm 10 mm
(b)
Figure 8. Light microscope sections of ventral epithelium of toes of S. parvus (�100 objective). (a) Toe pad epithelium showing four cell layers with deep channelsbetween the outer portions of the outer cell layer (arrows). (b) Unspecialized ventral epithelium (near proximal groove) showing small shallow grooves (arrows) betweenthe outer portions of the outer cell layer.
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centre of the nanopillar. Our best data give a figure for the
depth of the dimple in the range 6–8 nm.
4. DiscussionThe surface of toe pads of tree, rock and torrent frogs shows a
particular topography with distinct features at the micro- and
nanoscales [20–23,29,33,34,39–41]. The role that this surface
structure plays in reversible attachment and locomotion of
the animals in wet and even flooded environments is a cur-
rent focus of research for zoologists and material scientists
eager to transfer new ‘wet’ adhesive principles from natural
to artificial systems.
The outermost cell layer of the toe pad consists of epithelial
cells forming polygonal pillars of 10–15 mm width. As in hylid
and rhacophorid tree frogs [22,39], the majority are hexagonal
but pentagonal and heptagonal geometries also occur. The
occurrence of such geometries in a predominantly hexagon pat-
tern has recently been studied in the corneal nipple arrays that
cover the ommatidia (facets) of the compound eyes of the
butterfly, Nymphalis antiopa [42]. Nipples are occasionally sur-
rounded by either seven or five neighbours and five/seven
pairs are relatively common, a feature shared by the toe pads
of the tree frog, Rhacophorus prominanus, where pentagonal epi-
thelial cells are surrounded by more heptagons than would be
expected by chance and vice versa [22]. Space filling due to the
domed surface of each ommatidium is considered by Lee and
Erb to be one reason for the existence of such irregularities.
Indeed, this mechanism of introducing curvature into a close-
packed structure may be quite common in nature. For example,
the honeycombs of bees and social wasps use similar interrup-
tions of the hexagonal pattern to account for curvature,
confined boundary conditions and transition zones from smal-
ler to larger cells [43,44]. Tree and rock frog epithelial cells are
much less regular in shape than either corneal nipple arrays
or honeycombs and the presence of relatively straight channels
crossing the pad (see below) also necessarily affects cell shape,
changing regular hexagons into rather squarer cells. However,
as in butterfly corneal nipple arrays, the coexistence of heptago-
nal and pentagonal shapes with the predominantly hexagonal
one is probably required to fill the pad space and accommodate
irregularities caused by the pad curvature and the mucus pores.
The polygonal epithelial cells are separated at their tips by
narrow (ca 1 mm width) channels of around 10 mm depth.
This is a characteristic feature of the toe pad epithelia of
tree frogs, suggesting an important functional role for the
channels. In ranid frogs, this feature has been observed in tor-
rent frogs such as Amolops spp. [33], in rock frogs of the genus
Staurois ([34] and this study) and also, somewhat surpris-
ingly, in the unexpanded toe pads of the amphibious
leopard frog, Rana pipiens [45]. In S. parvus, the channels
are restricted to the ventral epithelium of the toes; i.e. pads
(deep channels), subarticular tubercles (shallower channels)
and unspecialized ventral epithelium (channels are hardly
more than gaps in the nanopillar array) as illustrated in
figures 3b, 5, 6 and 8. They are absent from dorsal toe epi-
thelium, where nanopillars are also absent (figure 7). The
role of the channels in reversible ‘wet’ adhesion has been
recently investigated in artificial tree frog toe pad replicas
(a)
1 mm
1 mm 2 mm*
*
1 mm
(b)
(c) (d )
Figure 9. TEM images of toe pad epithelium of S. parvus digits. (a) Outer cell layer showing nanopillars and dense bundles of keratin tonofilaments (arrows).(b) Border between outer cell layer on left and the second layer of cells; arrows show invaginations of cell membrane. (c) Border between cell layers II (left) and III;arrows indicate desmosomes. (d ) Lower-power image of border between layers I and II showing large vacuoles (stars) that will form the channels between theepithelial cells when the outer layer is shed.
200 nm
Figure 10. High-power TEM image of nanopillars in S. parvus. Section of toepad epithelium showing nanopillars filled with tonofilaments lying at rightangles to the pad surface.
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[11]. The channel structure allows draining of a wetting fluid
out of the contact surface and establishment of direct (dry)
contact in the presence of shear forces (i.e. friction). This con-
clusion is supported by data from live frogs studied using
interference reflection microscopy, which allows direct
measurement of the fluid layer thickness under the pad
[29]. This draining role would be expected to be more impor-
tant in frogs that live in wet environments, such as rock and
torrent frogs. Indeed, Ohler [33] observed elongated epi-
thelial cells in the toe pads of torrent frogs (Amolops spp.)
which, she hypothesized, provided shorter and straighter
channels for the removal of excess fluid from under the
pad. Elongated epithelial cells do not appear to be a
common feature of rock frog pads in spite of their habitat
in the vicinity of waterfalls. However, as shown by Endlein
et al. [34] in S. guttatus and here in S. parvus, there are chan-
nels crossing the pad (particularly in a distal/proximal
direction as would be appropriate for water drainage) that,
while not straight, are significantly shorter than would be
found in an array of regular hexagons (figure 3a). A role in drai-
nage of excess fluid thus seems probable. Although the
presence of a deep circumferal groove (figure 2b) is not
unique to rock frogs (WJP Barnes 2012, unpublished data on
Z range: 1.591 mm
(a) (b)
(c)
25
2
1
0
15
20
25
30
35
0
0.4 1.2 2.0position (mm)
heig
ht (
nm)
2.8 3.6 4.4
2.5(mm)
(mm
)
5.0
1900
2000
2100
2200
2300
0 25
1.2
0.8
0.4
(mm)
(mm
)
Figure 11. AFM images of toe pad epithelium cells of S. parvus. (a) Topographical image at the microscale. (b) Topographical image of the toe pad surfacenanostructures. The horizontal line indicates the position of the height profile shown in (c). (Online version in colour.)
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rhacophorid tree frogs), this groove, which surrounds the distal
and lateral parts of the pads, is ideally placed to channel fluid
around rather than under the pad.
Studies on toe pad mimics have also investigated the
importance of aspect ratio [11]. Microstructures with low
aspect ratio features are more resistant to bend and collapse
during shear and allow more effective establishment of
direct contact; i.e. higher friction forces are achieved. It is
thus possible that the lower aspect ratio subarticular tubercles
(epithelial cells are surrounded by shallow channels as
described above) have evolved to generate high friction
forces, but this remains to be tested experimentally.
Ventral surfaces are also characterized by the presence
of nanopillar arrays that cover the surface. These arrays are
particularly dense on the toe pads, but less so on the sub-
articular tubercles and unspecialized ventral toe epithelium
(figures 3c,d; 4; 5b and 6b). They are unlikely to play a role in
capillarity as such forces are generated by the meniscus (air–
water interface) around the edge of the pad [9], but a role in
friction and/or viscous flow between adhering surfaces
seems likely. Indeed, the thickness of the fluid layer under
the pad is such that the tops of the nanopillars are likely to
make direct dry contact with the substrate. This is shown
both by cryo-SEM images such as figure 4a and by calculations
of the thickness of the fluid layer thickness using interference
reflection microscopy [29]. Also, as noted previously in a tree
frog [23], the tops of the nanopillars are clearly concave
(figure 4b, a cryo-SEM image and figure 11c, the AFM height pro-
file), with a dimple depth in the range 6–8 nm, similar to those
measured in tree frogs [22,23]. The reason for this concavity is
unclear, but it is noteworthy that the shape (flat, T-shaped, con-
cave) of the tops of fibrillar microstructures can significantly
affect both adhesion and friction [11,46]. Varenberg and Gorb
propose that, under appropriate circumstances, each element
can act as a passive suction device, but this is disputed by
Drotlef et al., who favour a combination of capillary and direct
contact forces.
Interestingly, nanopillar arrays have also been repor-
ted in clingfish, where they occur around the margin of the
ventrally placed suction disc. Clingfish use their adhesive
disc to stick under water to many different kinds of surfaces
and, surprisingly as they adhere by suction, adhesion is not
hindered by surface roughness [47]. The array of nanopillars
(called microvilli by the authors), aided by secreted mucus, is
able to maintain a good seal and resist frictional forces, even
on the roughest of surfaces. As S. parvus lives in and around
fast-flowing water, it is unsurprising that the nanopillar
arrays in these frogs are present not only on the toe pads
and subarticular tubercles but also on unspecialized ventral
epithelium in the toe region. Indeed, as Endlein et al. [34]
have shown for S. guttatus, most of the ventral body surface
is used when they are clinging onto rough surfaces under
high rates of water flow.
Material properties are also critical for adhesion and fric-
tion. This paper does not address this matter directly, but the
TEM results indicate that the distal regions of the pads are ker-
atinized and that keratin tonofilaments fill the nanopillars and
the distal parts of the two outer cell layers (figures 9 and 10).
Their dense packing resembles the structure of many compo-
site materials and would confer higher mechanical strength
and flexibility to the epithelial cells. As frog toe pads have a
low elastic modulus [23,41], such keratinization probably
serves to increase the wear resistance of the outermost part of
the toe pads. Whether the keratin tonofilaments have a particu-
lar role in adhesion, as they do in insects [26], remains to be
investigated. However, their orientation (initially normal to
the surface they become angled to the surface in a proximal
direction as shown in figure 9a) could provide the pad with
directional adhesion, so that pads pulled towards the body
increase their contact with the substrate, while movements in
the other direction cause detachment.
The TEM images of the different cell layers within the toe
pad epithelium (figure 9a–d ) are broadly similar to those of
equivalent studies on hylid and rhacophorid tree frogs
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[12,14,20,21]. For example, in all groups, it appears that the
outer cell layer consists of dying or dead cells and is highly
keratinized, that nanopillars originate from desmosomes,
and that the keratin filaments are linked to desmosomes.
Our images also indicate that the specialized outer layer
develops in a similar way to the tree frogs. The degree of
evolutionary convergence is thus remarkable, though it is
probable that most of the underlying mechanisms (e.g. mech-
anisms of keratinization) will have been present in common
ancestors of these groups. Thus, in our view, it is the detailed
structure of the toe pad that arises by convergence in the
different frog families and that it does so predominantly
using pre-existing cellular mechanisms.
The adhesive mechanisms of climbing animals such as
geckos, anoles and skinks among reptiles, tree, torrent and rock
frogs among amphibians, and a wide variety of insects have
many features that are of interest to materials scientists. These
include the combination of good adhesion with easy detachment,
the fact that the adhesive can be used repeatedly without dete-
riorating, the ability to self-clean (e.g. [48]) and thus quickly
and efficiently remove attached dirt particles, and the property
of only sticking when required [2,4]. The amphibians additio-
nally have the ability to adhere under wet conditions. Thus,
understanding the adhesion principles behind the structural fea-
tures described in this paper will enhance the development of
novel adhesion strategies for reversible attachment in artificial
systems. The initial work on the tree/rock/torrent frog system
has already begun with theoretical studies of capillary adhesion
of soft, deformable surfaces [49,50] and nanofabrication and
testing of hexagonal patterns of cells surrounded by channels
(Varenberg & Gorb [46,51], whose work was insect-inspired,
and Drotlef et al. [11], who systematically examined the adhesive
and frictional properties of tree frog toe pad analogues under a
variety of conditions). More generally, studies on bioinspired
adhesive materials are increasingly combining materials with
very different properties in the development of new hybrid
adhesive surfaces (e.g. [52,53]). In respect of the frog adhesion
research, possible applications include improved design in wet
weather tyres [35,54,55], non-slip footwear, plasters for surgery
able to adhere to tissue, holding devices for surgery and the
assembly of functional elements in miniature electronic devices
(microelectromechanical systems). As both Barnes et al. [22] and
this paper make clear, both nanopillars and fibrils will also
need to be considered if the full biomimetic potential of the
rock frog adhesive system is to be realized.
Ethics statement. The frogs were sacrificed for these studies by submer-sion in water containing a lethal dose of benzocaine, in accordancewith current laws on animal experimentation in the UK.
Acknowledgements. We thank Margaret Mullin of Glasgow University’sLife Sciences Electron Microscopy Facility for her expert assistancewith both the scanning and transmission electron microscopy.
Funding statement. This work was supported by grants from theDeutsche Forschungsgemeinschaft (SPP1420 ‘Biomimetic materialsresearch: functionality by hierarchical structuring of materials’) toA.d.C. and W.J.P.B. and by an STMS grant from COST ActionTD0906 on ‘Biological adhesives: from biology to biomimetics’ toD.M.D.
References
1. Duellman W, Trueb L. 1997 Biology of amphibians,2nd edn. Baltimore, MA: Johns Hopkins UniversityPress.
2. Barnes WJP. 2007 Biomimetic solutions to stickyproblems. Science 318, 203 – 204. (doi:10.1126/science.1149994)
3. Creton C, Gorb S. 1997 Sticky feet: from animals tomaterials. MRS Bull. 32, 466 – 468. (doi:10.1557/mrs2007.79)
4. Autumn K. 2007 Gecko adhesion: structure, functionand applications. MRS Bull. 32, 473 – 478. (doi:10.1557/mrs2007.80)
5. Bhushan B. 2007 Adhesion of multi-levelhierarchical attachment systems in gecko feet.J. Adhes. Sci. Technol. 21, 1213 – 1258. (doi:10.1163/156856107782328353)
6. del Campo A, Greiner C, Arzt E. 2007 Contactshape controls adhesion of bioinspired fibrillarsurfaces. Langmuir 23, 10235 – 10243. (doi:10.1021/la7010502)
7. Zhao B, Pesika N, Zeng H, Wei Z, Chen Y, Autumn K,Turner K, Israelachvili J. 2009 Role of tilted adhesionfibrils (setae) in the adhesion and locomotion ofgecko-like systems. J. Phys. Chem. B 113,3615 – 3621. (doi:10.1021/jp806079d)
8. Barnes WJP. 2007 Functional morphologyand design constraints of smooth adhesivepads. MRS Bull. 32, 479 – 485. (doi:10.1557/mrs2007.81)
9. Barnes WJP. 2012 Adhesion in wet environments—frogs. In Encyclopedia of nanotechnology, part 2 (ed.B Bhushan), pp. 70 – 83. Berlin, Germany: Springer.
10. Gupta R, Frechette J. 2012 Measurement andscaling of hydrodynamic interactions in the presenceof draining channels. Langmuir 28, 14 703 – 14 712.(doi:10.1021/la303508x)
11. Drotlef D-M, Stepien L, Kappl M, Barnes WJP, ButtH-J, del Campo A. 2013 Insights into the adhesivemechanisms of tree-frogs using artificial mimics.Adv. Funct. Mater. 23, 1137 – 1146. (doi:10.1002/adfm.201202024)
12. Ernst V. 1973 The digital pads of the tree frog, Hylacinerea. I. The epidermis. Tissue Cell 5, 83 – 96.(doi:10.1016/S0040-8166(73)80007-2)
13. Ernst V. 1973b The digital pads of the tree frog,Hyla cinerea. II. The mucous glands. Tissue Cell 5,97 – 104. (doi:10.1016/S0040-8166(73)80008-4)
14. Welsch U, Storch V, Fuchs W. 1974 The finestructure of the digital pads of rhacophorid treefrogs. Cell Tiss. Res. 148, 407 – 416. (doi:10.1007/BF00224267)
15. Green DM. 1979 Treefrog toe pads: comparativesurface morphology using scanning electronmicroscopy. Can. J. Zool. 57, 2033 – 2046. (doi:10.1139/z79-268)
16. McAllister A, Channing C. 1983 Comparisons oftoepads of some Southern African climbing frogs.S. Afr. Zool. 18, 110 – 114.
17. Green DM, Simon P. 1986 Digital microstructure inecologically diverse microhylid frogs generaCophixalus and Sphenophryne (Amphibia: Anura)from Papua New Guinea. Aust. J. Zool. 34,135 – 145. (doi:10.1071/ZO9860135)
18. Hertwig I, Sinsch U. 1995 Comparative toe padmorphology in marsupial frogs (genus Gastrotheca):arboreal versus ground-dwelling species. Copeia 1,38 – 47. (doi:10.2307/1446798)
19. Ba-Omar TA, Downie JR, Barnes WJP. 2000Development of adhesive toe-pads in the tree frog(Phyllomedusa trinitatis). J. Zool. Lond. 250,267 – 282. (doi:10.1111/j.1469-7998.2000.tb01077.x)
20. Mizuhira V. 2004 The digital pads of rhacophoridtree-frogs. J. Electron Microsc. 53, 63 – 78. (doi:10.1093/jmicro/53.1.63)
21. Chakraborti S, Das D, De SK, Nag TC. 2014 Structuralorganization of the toe pads in the amphibianPhilautus annadalii (Boulenger, 1906). Acta Zool.(Stockholm) 95, 63 – 72. (doi:10.1111/azo.12008)
22. Barnes WJP, Baum M, Peisker H, Gorb SN. 2013Comparative cryo-SEM and AFM studies of hylid andrhacophorid tree frog toe pads. J. Morphol. 274,1384 – 1396. (doi:10.1002/jmor.20186)
23. Scholz I, Barnes WJP, Smith JM, Baumgartner W.2009 Ultrastructure and physical properties of anadhesive surface, the toe pad epithelium of the treefrog, Litoria caerulea White. J. Exp. Biol. 212,155 – 162. (doi:10.1242/jeb.019232)
rsfs.royalsocietypublishing.orgInterface
Focus5:20140036
11
on May 21, 2018http://rsfs.royalsocietypublishing.org/Downloaded from
24. Gorb SN, Jiao Y, Scherge M. 2000 Ultrastructuralarchitecture and mechanical properties ofattachment pads in Tettigonia viridissima(Orthoptera Tettigoniidae). J. Comp. Physiol. A 186,821 – 831. (doi:10.1007/s003590000135)
25. Perez-Goodwyn PJ, Peressadko A, Schwarz H,Kastner V, Gorb SN. 2006 Material structure,stiffness, and adhesion: why attachment pads of thegrasshopper (Tettigonia viridissima) adhere morestrongly than those of the locust (Locustamigratoria) (Insecta: Orthoptera). J. Comp. Physiol. A192, 1233 – 1243. (doi:10.1007/s00359-006-0156-z)
26. Dirks J-H, Li M, Kabla A, Federle W. 2012 In vivodynamics of the internal fibrous structure in smoothadhesive pads of insects. Acta Biomater. 8,2730 – 2736. (doi:10.1016/j.actbio.2012.04.008)
27. Emerson SB, Diehl D. 1980 Toe pad morphology andmechanisms of sticking in frogs. Biol. J. Linn. Soc. 13,199 – 216. (doi:10.1111/j.1095-8312.1980.tb00082.x)
28. Hanna G, Barnes WJP. 1991 Adhesion anddetachment of the toe pads of tree frogs. J. Exp.Biol. 155, 103 – 125.
29. Federle W, Barnes WJP, Baumgartner W, DreschlerP, Smith JM. 2006 Wet but not slippery: boundaryfriction in tree frog adhesive toe pads. J. R. Soc.Interface 3, 589 – 601. (doi:10.1098/rsif.2006.0135)
30. Barnes WJP, Oines C, Smith JM. 2006 Whole animalmeasurements of shear and adhesive forces in adulttree frogs: insights into underlying mechanisms ofadhesion obtained from studying the effects of sizeand scale. J. Comp. Physiol. A 192, 1179 – 1191.(doi:10.1007/s00359-006-0146-1)
31. Barnes WJP, Pearman J, Platter J. 2008 Applicationof peeling theory to tree frog adhesion, a biologicalsystem with biomimetic implications. Eur. Acad. Sci.E-Newsletter Sci. Technol. 1, 1 – 2.
32. Endlein T, Ji A, Samuel D, Yao N, Wang Z, Barnes WJP,Federle W, Kappl M, Dai Z. 2013 Sticking like stickytape: tree frogs utilise friction forces to enhanceattachment to overhanging surfaces. J. R. Soc.Interface 10, 20120838. (doi:10.1098/rsif.2012.0838)
33. Ohler A. 1995 Digital pad morphology in torrent-living Ranid frogs. Asiatic Herpetol. Res. 6, 85 – 96.
34. Endlein T, Barnes WJP, Samuel D, Crawford N, BiawAE, Grafe U. 2013 Sticking under wet conditions: theremarkable attachment abilities of the torrent frog,Staurois guttatus. PLoS ONE 8, e73810. (doi:10.1371/journal.pone.0073810)
35. Barnes WJP, Smith J, Oines C, Mundl R. 2002 Bionicsand wet grip. In Tire Technol. Int. December ‘02, pp.56 – 60. Dorking, UK: UK & International Press.
36. Alibardi L. 2001 Keratinization in the epidermis ofamphibians and the lungfish: comparison withamniote keratinization. Tissue Cell 33, 439 – 449.(doi:10.1054/tice.2001.0198)
37. Spinner M, Westhoff G, Gorb SN. 2013 Subdigitaland subcaudal microornamentation inchamaeleonidae—a comparative study. J. Morphol.274, 713 – 723. (doi:10.1002/jmor.20137)
38. Alibardi L. 2010 Cornification of the beak ofRana dalmatina tadpoles suggests the presenceof basic keratin-associated proteins. Zool. Stud. 49,51 – 63.
39. Smith JM. 2003 Effects of allometric growth and toepad morphology on adhesion in hylid tree frogs. PhDthesis, University of Glasgow, Glasgow, UK.
40. Smith JM, Barnes WJP, Downie JR, Ruxton GD. 2006Structural correlates of increased adhesive efficiencywith adult size in the toe pads of hylid tree frogs.J. Comp. Physiol. A 192, 1193 – 1204. (doi:10.1007/s00359-006-0151-4)
41. Barnes WJP, Perez Goodwyn PJ, NokhbatolfoghahaiM, Gorb SN. 2011 Elastic modulus of tree frogadhesive pads. J. Comp. Physiol. A 197, 969 – 978.(doi:10.1007/s00359-011-0658-1)
42. Lee KC, Erb U. 2013 Grain boundaries andcoincidence site lattices in the corneal nanonipplestructure of the mourning cloak butterfly.Beilstein J. Nanotechnol. 4, 292 – 299. (doi:10.3762/bjnano.4.32)
43. von Frisch E. 1974 Animal architecture. New York,NY: Harcourt Brace Jovanovitch.
44. Capinera JL. 2008 Encyclopedia of entomology, 2ndedn. Heidelberg, Germany: Springer.
45. Fahrenbach WH, Knutson DD. 1975 Surfaceadaptations of the vertebrate epidermis to friction.
J. Invest. Dermatol. 65, 39 – 44. (doi:10.1111/1523-1747.ep12598036)
46. Varenberg M, Gorb SN. 2008 A beetle-inspiredsolution for underwater adhesion. J. R. Soc. Interface5, 383 – 385. (doi:10.1098/rsif.2007.1171)
47. Wainwright DK, Kleinteich T, Kleinteich A, Gorb SN,Summers AP. 2013 Stick tight: suction adhesion onirregular surfaces in the northern clingfish. Biol.Lett. 9, 20130234. (doi:10.1098/rsbl.2013.0234)
48. Crawford N, Endlein T, Barnes WJP. 2012 Self-cleaning in tree frog toe pads; a mechanism forrecovering from contamination without the need forgrooming. J. Exp. Biol. 215, 3965 – 3972. (doi:10.1242/jeb.073809)
49. Butt H-J, Barnes WJP, del Campo A, Kappl M,Schoenfeld F. 2010 Capillary forces between soft,elastic spheres. Soft Matter 6, 5930 – 5936. (doi:10.1039/c0sm00455c)
50. Zakerin M, Kappl M, Backus EHG, Butt H-J,Schonfeld F. 2013 Capillary forces between rigidspheres and elastic supports: the role of Young’smodulus and equilibrium vapour adsorption. SoftMatter 9, 4534 – 4543. (doi:10.1039/c3sm27952a)
51. Varenberg M, Gorb SN. 2009 Hexagonal surfacemicropattern for dry and wet friction. Adv. Mater.21, 483 – 486. (doi:10.1002/adma.200802734)
52. Ruffato III D, Parness A, Spenko M. 2014 Improvingcontrollable adhesion on both rough and smoothsurfaces with a hybrid electrostatic/gecko-likeadhesive. J. R. Soc. Interface 11, 20131089. (doi:10.1098/rsif.2013.1089)
53. Shahsavan H, Zhao B. 2014 Bioinspired functionallygraded adhesive materials: synergetic interplay oftop-viscous-elastic layers with base micropillars.Macromolecules 47, 353 – 364. (doi:10.1021/ma4018718)
54. Barnes WJP. 1999 Tree frogs and tire technology. InTire Technol. Int. March ‘99, pp. 42 – 47. Dorking,UK: UK & International Press.
55. Persson BNJ. 2007 Wet adhesion with application totree frog adhesive toe pads and tires. J. Phys. Cond.Matter 19, 376110. (doi:10.1088/0953-8984/19/37/376110)