www.nature.com/scientificreports

OPEN

An ossifying bridge– on the structural continuity
between the Achilles tendon
and the plantar fascia
Johann Zwirner1,2*, Ming Zhang1, Benjamin Ondruschka3, Keichi Akita2 &Niels Hammer4,5,6*
Highly regular aligned trabeculae are found in the superficial posterior and inferior calcaneus
appearing to connect the Achilles tendon (AT) to the plantar fascia (PF) in a bridge-like manner. This
provides a morphological basis for the stretching-based heel pain treatment. However, the continuityof collagen fibres between the AT and the PF remains debated controversially to date. The given
study morphologically investigated the AT-calcaneus-PF complex using histology and plastination.
Moreover, the AT-calcaneus-PF complex was biomechanically mapped based on 13 sub-regionswith a total of 76 tested samples. Regular calcaneal trabeculae were surrounded by tendon-likecollagen fibre bundles and adipocytes. The orientation of calcaneal trabeculae was further closely
related to the course of the PF collagen fibre bundles. The pooled biomechanical analysis revealed
low elastic moduli (minimum = 4 MPa) and ultimate tensile strengths of the decalcified calcaneal
samples (minimum = 0.4 MPa) and the calcaneal periostea (minimum = 2 MPa) and high respective
values (elastic modulus maximum of 144 MPa; ultimate tensile strength maximum of 29 MPa) for
the PF samples compared to the other sub-regions. This study provides structural evidence for amorphological connection between the AT and PF via the highly aligned calcaneal trabeculae ofthe posterior calcaneus. The AT-calcaneus-PF complex was biomechanically mapped to allow for anassessment of its site-dependent mechanical characteristics.
Plantar fasciitis is the most frequent cause of inferior heel pain and affects approximately one percent of the adult­population1. A variety of stretching techniques including both the plantar fascia (PF) and Achilles tendon (AT)have been established as effective non-surgical treatment options to alleviate the plantar fasciitis pain patients
describe as piercing, searing or t­ hrobbing2. Night splints, which keep the foot in a neutral or slightly dorsiflexed
position while the patient rests are another stretching-related option, involving the concept of an AT-PF connection to mitigate the PF-related pain. Moreover, it was shown that high-load strength training including repetitive
heel rises out of a dorsiflexed position positively influenced the therapy outcome of plantar fasciitis pain p­ atients3.
A shortened or tight AT, however, is an established risk factor for plantar fasciitis, once more underlining a closefunctional relationship of the two collagenous s­ tructures4,5. From a morphological perspective, highly regularaligned trabeculae were noted in the calcaneus that seemed to anatomically connect AT and PF in a bridge-like­manner6. These published findings raised the question whether the AT and PF are two structurally different
tissues, inserting separately into the calcaneus from two ends, or if the AT and PF resemble one continuing collagenous structure, which is secondarily divided by the calcaneal ossification during l­ ife6. Supporting the latter,it was shown that AT and PF are connected by thick collagen fibres in neonates, which quantitatively diminishedduring life and could not be detected in late(r) adulthood as they were likely integrated into the bony c­ alcaneus7.
Integration of the formerly continuous tendon into the posterior calcaneus, which then further subdivides into1
Department of Anatomy, University of Otago, Dunedin, New Zealand. 2Department of Clinical Anatomy,
Tokyo Medical and Dental University, Tokyo, Japan. 3Institute of Legal Medicine, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany. 4Department of Macroscopic and Clinical Anatomy, Medical
University of Graz, Graz, Austria. 5Department of Orthopaedic and Trauma Surgery, University of Leipzig, Leipzig,
Germany. 6Fraunhofer IWU, Dresden, Germany. *email: medijo@gmx.de; niels.hammer@medunigraz.at

Scientific Reports |(2020) 10:14523| https://doi.org/10.1038/s41598-020-71316-z

1Vol.:(0123456789)

www.nature.com/scientificreports/
the distal PF and the proximal AT likely allows for a more efficient energy use during push-off movements of thefoot and shows an adaption to b
­ ipedalism8. The calcaneal periosteum, which receives fibres from both AT and
PF is only vaguely accepted as a continuous morphological connection between the two ­structures7,9–11, certainlywith insignificant load-bearing characteristics. Also, it was noted that the calcaneal periosteum in the area ofthe highly regular trabeculae that possibly connect AT and PF differs from the remaining calcaneal periosteum,indicating the potential previous continuity of the two ­tendons9. If the AT and PF form one continuous structure
which becomes separated throughout development, it can be hypothesized that the AT represents the proximal
part, the regular calcaneal trabeculae including the calcaneal periosteum the intermediate part and the PF the
distal part of one united tendon of the gastrocnemius and soleus muscle. To shed light on the structural connection between the AT and the PF via the calcaneus this study followed two aims: Firstly, the proximal–distaltransition of the AT-calcaneus-PF complex should be morphologically investigated continuously between theformation of the AT starting at the distal calf and ending at the insertion of the PF at the heads of the metatarsal
bones. Secondly, it aimed at establishing a biomechanical testing setup for this heterogenous complex, whichallows for a uniform investigation of the structural organization by means of a materials testing approach tosubsequently enable the structural analysis of the same from a materials testing perspective.

Materials and methods

Harvesting and sample preparation for mechanical testing. A total of nine complexes comprising of the human AT, adjacent calcaneus and PF (subsequently referred to as AT-calcaneus-PF complex) wereretrieved post mortem (mean age 74, age range 28 to 93). One of the complexes was used for histological assessment (28-year-old male), two for plastination (87-year-old female, corresponding to one left and one right complex) and six for mechanical testing (four phenoxyethanol-based embalmed c­ adavers12, left and right complex
was used of two cadavers, two right complexes of two other cadavers, mean age 82 years, 3 males, 1 female). The
histologically processed AT-calcaneus-PF complex was retrieved at the Institute of Legal Medicine, Universityof Leipzig, Germany during leg preparation and the remaining ones were acquired from bequeathed cadavers
for medical education and research purposes at the Department of Anatomy of the University of Otago in Dunedin, New Zealand. The University of Otago Ethics Committee approved this study (approval number H17/20),
in conjunction with Maori consultation being sought from the Ngai Tāhu Research Consultation Committee
and the Ethics committee of the University of Leipzig, Germany Study approved the tissue sampling (protocol
number 486/16-ek).
Dissection, decalcification and sample retrieval.

Following the retrieval, the six AT-calcaneusPF complexes of the mechanical testing group were prepared for decalcification as follows: First, the AT was
exposed carefully until the calcaneus was reached distally, consecutively removing all surrounding tissues except
for the periosteum. Second, the AT was proximally transsected, distally to the macroscopically last visible muscle
fibres of the soleus muscle. Thereafter, the PF was exposed and mobilized from the heads of the metatarsal bones
distally to the calcaneus proximally with a sharp scalpel. Following this, the lower half of the calcaneus was cut
with an ultrasound knife (PIEZOSURGERY®, Mectron, Saline, MI, USA) with subsequent blunt removal of trabecular bone parts by means of the handle of a scalpel to facilitate the following decalcification of the superficial
calcaneal trabecular layer, delineating from the surrounding trabeculae. Decalcification of the samples removed
the hydroxyapatite leaving behind the collagen scaffold that connects the AT and the PF. This allowed to investigate the mechanical properties of the collagenous scaffold that connects the AT and the PF and compare it to
the two structures. Following the hydroxyapatite removal, the collagen scaffold mechanically behaves in a soft
tissue-like manner. For the decalcification of calcaneal samples, the resulting complexes of AT and PF connectedby the most superficial inferior and posterior calcaneal trabeculae were submerged into a 10wt% ethylenediamine-tetra-acetate (EDTA) solution for four weeks with a weekly renewal. Then, dog bone-shaped samples
adapted from an ISO-527–2 ­template13 for biomechanical testing were cut from the AT-calcaneus-PF complex
according to Fig. 1A. Samples for biomechanical testing were retrieved along the AT, calcaneus and PF along a
medial and a lateral line based on the following morphological observations: the median fibrous septum of thesoleus muscle (separating the AT fibre bundles into a medial and lateral line), median fibrous septae proximal
to the most proximal insertion of the AT into the calcaneus (symmetrically separating the AT into medial andlateral bundles shown in histology sections) and finally the division of the PF into two larger bundles (central
and lateral, the medial bundle was neglected here due to its smaller size). Along the medial and lateral lines, thefollowing corresponding pairs were retrieved: MPAT (medial proximal AT) and LPAT (lateral proximal AT),MDAT (medial distal AT) and LDAT (lateral distal AT); MB (medial bone) and LB (lateral bone); MCP (medialcalcaneal periosteum) and LCP (lateral calcaneal periosteum); MPPF (medial proximal PF) and LPPF (lateralproximal PF) and MIPF (medial intermediate PF) and LDPF (lateral distal PF). The MDPF (medial distal PF) of
the medial line did not have a lateral line counterpart.

Uniaxial quasi‑static mechanical testing. The samples’ cross-sectional areas were determined by creating polysiloxane (medium-bodied, Exahiflex; GC Corporation, Tokyo, Japan) cross section casts, which were
subsequently scanned (Perfection 7V750Pro; Seiko Epson Corporation, Suwa, Japan) and computed by meansof the Measure 2.1d software (DatInf, Tübingen, Germany). Self-printed clamps with sharp pyramids to prevent
specimen slippage during tensile testing were used (Fig. 1B)14. Before tensile testing was conducted, the samples
were speckled using a black pencil to create a randomly distributed pattern, necessary for the digital image correlation (DIC) (Fig. 1C). A uniaxial testing machine (Allround Table Top Z020; Zwick Roell, Ulm, Germany)equipped with an Xforce P load cell (2.5 kN; Zwick Roell, Ulm, Germany) was used to conduct the tensile tests
at room temperature. The testControl II software (Zwick Roell, Ulm, Germany) was used. Twenty load-unload
Scientific Reports |Vol:.(1234567890)(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

2

www.nature.com/scientificreports/

Figure 1.  The images display the preparation steps of samples for tensile testing and the subsequent uniaxial
load application at different testing stages. (A) A lateral calcaneal periosteum sample is depicted after it has
been cut into a “dog bone” shape (view from inside). (B) The sample from A was mounted into 3D-printed
squeezing clamps (view on the sample from outside). (C) Speckled and mounted sample in the material testingmachine, upper (UC) and lower (LC) clamp. (D) A load is applied to the sample, indicated by the UC crossheaddisplacing cranially. The sample elongates (represented by the dotted arrow) and narrows in the ‘shaft’ regionof the dog bone, indicated by the yellow arrows. (E) Continuous load application causes tearing of the sample(indicated by the white arrow).

Determination of biomechanical parameters
UTS

Stress [MPa]

0.6
0.4

SFmax

Emod
0.2
0.0

MCP sample

0

5

10

Strain [%]15

Figure 2.  The determination of the biomechanical parameters in this study is shown representatively on a
medial calcaneal periosteum (MCP) sample of this study. The elastic modulus (­ Emod) was determined by a linear
regression analysis between the zero-point and the point that equals 70% of the maximum stress. The ultimate
tensile strength (UTS) equals the maximum stress ­(Fmax) divided by the cross section value before the tissue
failed when being stretched. The strain at maximum force (­ SFmax) reflects how much the sample was strained at
the point of the UTS compared to its initial length.

preconditioning cycles with a force range of 0.5 to 2.0 N were applied before the tissues were stretched until
failure (Fig. 1C–E). All tissues were strained in the longitudinal axis according to the samples’ predominantcollagen orientation. The displacement rate was 20 mm/min and the sample reading rate was 100 Hz. A singlecharge coupled camera with a resolution of 2.8 Megapixels (Q400; Limess, Krefeld, Germany) and the ISTRA
4D software (VRS 4.4.1.354; Dantec Dynamics, Ulm, Germany) were used for strain data evaluation of themechanical tests.

Data processing and statistical analysis. Synchronized force readings by MATLAB R2017b software(Mathworks, Natick, USA) and DIC data were used to calculate mechanical properties. Elastic modulus (­ Emod),strain at maximum force ­(SFmax) and ultimate tensile strength (UTS) were evaluated (Fig. 2). For statisticalevaluation, Excel version 16.15 (Microsoft Corporation, Redmond, USA) was chosen. Firstly, the biomechanicalparameters of the subsamples were averaged according to their respective location (e.g., all MB values of the different tested samples were averaged) within the AT-calcaneus-PF complex. Following this, the averaged valuesof the subsamples were compared among the 13 different sites (e.g., MB vs. LB) and classified into the following
groups according to being part of the percentile fractions shown in parentheses: low (0 to 24th percentile), below
median (25th to 49th percentile), median (50th percentile), above median (51 to 74th percentile) and high (75th
to 100th percentile).
Scientific Reports |(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

3Vol.:(0123456789)

www.nature.com/scientificreports/

Figure 3.  A graphical overview of the obtained biomechanical parameters and the retrieval site of the ‘dogbone’ samples from the Achilles tendon-calcaneus-plantar fascia complex is depicted. The colour code reflects
the respective fraction of percentiles that the individual sample belongs to in comparison to all the othersamples. (A) Samples of the decalcified human Achilles tendon-calcaneus-plantar fascia complex were retrieved
according to a medial (blueish ‘dog bones’) and lateral (yellow ‘dog bones’) line, view from inside. MPAT, medial
proximal Achilles tendon; MDAT, medial distal Achilles tendon; MPPF, medial proximal plantar fascia; MIPF,
medial intermediate plantar fascia and MDPF, medial distal plantar fascia. The following samples were retrieved
from the lateral line from proximal to distal: LPAT, lateral proximal Achilles tendon; LDAT, lateral distal Achilles
tendon; LPPF, lateral proximal plantar fascia and LDPF, lateral distal plantar fascia. (B) A medial bone (MB)and medial calcaneal periosteum (MCP) sample were retrieved. Equally, a lateral bone (LB) and lateral calcaneal
periosteum (LCP) sample were retrieved. d, distal; ­Emod, elastic modulus; l, lateral; m, medial; p, posterior; S­ Fmax,
strain at maximum force; UTS, ultimate tensile strength.

E12‑sheet plastination and histology.

The two E12-plastinated AT-calcaneus-PF complexes used inthis study were taken from one entirely plastinated cadaver. The cadaver was embalmed according to Xu et al.15.
The plastinates were scanned at 1200 dpi (Epson Perfection V750 Pro Scanner, Epson, Jakarta, Indonesia). For
histological analyses, one fresh AT-calcaneus-PF complex was embedded into paraffin and horizontally sectioned with a section thickness of 20 µm using the same orientation as for the plastinates. Subsequently, theselected sections that corresponded to the plastinated slices were stained with hematoxylin eosin (H&E) and
Masson–Goldner as trichromic staining (all consumables by Dr. Hollborn GmbH & Co KG, Leipzig, Germany)and photographed using a Zeiss Axioskop 40 (Carl Zeiss AG, Oberkochen, Germany) combined with an Olympus DP22 camera system (Olympus K.K., Shinjuku, Japan).

Results

Biomechanical mapping of the Achilles tendon‑calcaneus‑plantar fascia complex. All 76 tested
subsamples withstood the tensile tests conducted in this study. Low elastic moduli were noted in the decalcified
calcaneal samples (MB: 4 MPa; LB: 7 MPa) and the lateral calcaneal periosteum sample (LCP: 16 MPa). Highelastic moduli were found exclusively in the PF (MPPF: 118 MPa; LDPF: 142 MPa; MDPF: 144 MPa) with thehighest value observed in the lateral proximal PF (LPPF: 192 MPa). The UTS was low for the decalcified calcaneal samples (MB: 0.4 MPa; LB: 0.6 MPa) and the medial calcaneal periosteum sample (MCP: 2 MPa). As noted
for the elastic moduli, high tensile strengths were exclusively found in the PF samples (MDPF: 22 MPa; LDPF:
24 MPa; LPPF: 29 MPa) with the highest value observed in the medial proximal PF (MPPF: 29 MPa). The ­SFmax
was low in the calcaneal periosteum samples (MCP: 15%; LCP: 16%) as well as in the lateral decalcified bone
sample (LB: 16%). High ­SFmax values were noted in all tested AT samples (MPAT: 31%; LPAT: 35%; MDAT: 34%;
LDAT: 42%). The biomechanical parameters are graphically depicted in Fig. 3.
Macroscopic observations of the Achilles tendon insertion into the calcaneus. The AT weakly
attached to the upper posterior calcaneus between the proximal retrocalcaneal bursa and the distal calcaneal
tuberosity (lower calcaneus) (Fig. 4A). After the AT was detached, a smooth surface remained on both the AT
and calcaneus within the area of the former attachment (Fig. 4A). Continuing the separation of the two structures distally, the area of the calcaneal tuberosity revealed a stronger connection between the AT and the calcaneus, leaving a frayed surface on both the calcaneus and the opposite detached thin layer of calcaneal periosteum
Scientific Reports |Vol:.(1234567890)(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

4

www.nature.com/scientificreports/

Figure 4.  Macroscopic observations during the separation of the Achilles tendon-calcaneus-plantar fascia
complex (AT-calcaneus-PF complex). (A) The AT-calcaneus-PF complex can be observed looking fromproximal to distal after the AT was separated from its weak attachment on the calcaneus and reverted
backwards. (B) The calcaneal tuberosity becomes visible if the separation of the AT from the calcaneus
is continued. The white arrow points at the median fibrous septum originating from the calcaneus andcontributing to the calcaneal periosteum, which is depicted more proximally in the plastinate and histology
sections shown in Fig. 7. (C) The macroscopic transition of ligamentous structures between the AT and
the PF is shown after the calcaneus was removed (view from medial to lateral onto a sagittal plane after the
AT-calcaneus-PF complex had been longitudinally divided into two parts). AT, Achilles tendon; black arrows,
transverse insertion line; CAL, calcaneus; CP, calcaneal periosteum; PF, plantar fascia; RCB: retrocalcaneal
bursa, white asterisk, most proximal point of the calcaneal tuberosity; d, distal; m, medial; l, lateral; p, proximal.

(Fig. 4B). The proximal border of the calcaneal tuberosity ran in a wavy fashion with the most proximal pointin the mid of a mediolateral axis. After AT and PF are separated from the calcaneus the calcaneal periosteum
connects the two structures in a bridge-like manner (Fig. 4C).

Morphological observations of the Achilles tendon‑calcaneus‑plantar fascia complex based onplastinated slices. In the proximal AT, the median septum of the soleus muscle was identified separating

the AT into medial and lateral fibres (Fig. 5a, b). The AT-calcaneus-PF complex revealed a course change of the
fibrous septae within the AT from running perpendicular to the anterior AT border proximally (Fig. 5b), running oblique to it more distally (Fig. 5c), and parallel to the anterior AT border directly proximal of the proximal
AT insertion into the calcaneus (Fig. 5d). At the latter level, fibrous septae were depicted at the midpoint of amediolateral AT axis. Fibrous septae within the AT seemed to continue in the superficial calcaneus within asuperficial trabecular area that differed from the rest of the calcaneal trabeculae in terms of a darker appearance(Fig. 5e). These darker trabeculae changed from being a superficial band at the level of the proximal AT insertion (Fig. 5e) to being a demarcating line only with regular calcaneal trabeculae between the aforementioned
and the calcaneal surface distally (Fig. 5f–h). At the level of the calcaneal tuberosity, several finger-shaped bonyextensions were observed, yielding a regular trabecular architecture (Fig. 5f), compatible with the definition ofenthesophytes. These areas seemed to develop in the areas of the fibrous AT septae (Fig. 5f). The inspection of
the superficial AT indicated a distinct crossing of collagen fibres from proximal medial to distal lateral (Fig. 6A).
In the more distal medial AT, straight superficial fibres without a crossing of sides were observed (Fig. 6A).
Deeper calcaneal layers revealed a trabecular course, which is in line with the orientation of the more superficially observed collagen orientation of the medial and lateral bundle of the PF (Fig. 6B–D).

Morphological observations based on histology. Fibrous septae seen in plastinates (Fig. 7A, corresponding to the level of the lower part of the posterior calcaneal surface in Fig. 4 and illustrated as orientationline in Fig. 5) are running between the calcaneus and the AT paratenon perpendicularly to the course of theremaining AT fibre bundles as indicated in the Masson–Goldner stain in greater detail (Fig. 7B). The median
fibrous septum originating from a slight median calcaneal prominence symmetrically separates the most medial
running longitudinal AT fibre bundles (Fig. 7B, yellow dotted line). Based on H&E slices, the demarcating superficial trabecular layer within the calcaneus seen on plastinates consists of calcaneal trabeculae surrounded bycollagens and adipocytes (Fig. 7C).

Discussion

Successful stretching exercises involving the AT when treating various forms of heel pain including plantar fasciitis have highlighted the functional connection between AT and ­PF2. Beyond that, highly regular interindividualtrabecular arrangements in the superficial posterior and inferior calcaneus, which seem to form an osseous

Scientific Reports |(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

5Vol.:(0123456789)

www.nature.com/scientificreports/

Figure 5.  Plastinated sections of the Achilles tendon (AT) and its insertion into the calcaneus are depictedin the transverse plane. Section levels (a-h) are illustrated on a dissected AT shown from dorsal on the left.
(a) Section level: upper posterior subtalar joint, (b) Section level: posterior subtalar joint, (c) Section level:
uppermost part of the posterior calcaneal surface, (d) Section level: transition between the upper and middle
part of the posterior calcaneal surface, (e) Section level: middle part of the posterior calcaneal surface, (f)Section level: lower part of the posterior calcaneal surface (calcaneal tuberosity), (g) Section level: lower
posterior calcaneal surface, (h) Section level: proximal end of the heel fad pad; Black arrows, trabecular bonelayer appearing to be denser compared to the surrounding tissue; black dotted lines, thin fibrous AT septae;
CAL, calcaneus; red arrows, thin AT layer covering the calcaneus; white arrows, median septum of soleus
muscle; black asterisks, enthesophytes; yellow arrow, thick fibrous septae; a, anterior; i, inferior; l, lateral; m,medial; p, posterior; s, superior; Scale bars: grey, 24 mm; orange, 6 mm.

Figure 6.  The course of both Achilles tendon (AT) and plantar fascia (PF) collagen fascicles is depicted. (A) A
dissected AT is shown from dorsally. The black arrows indicate a collagen bundle running from proximal medial
to distal lateral in the superficial AT layer. In contrast, the white arrows indicate that the most medial superficial
fascicles of the proximal AT stay medial in the distal part of the AT. (B) A plantar view on the most inferior tipof the calcaneus (CAL) is displayed with the inserting lateral (LB) and central bundle (CB) of the PF. The whitearrows point on collagen fibres that originate from the medial CAL taking a course towards the LB. Inversely, anumber of fibres can be observed taking a medially directed course (indicated by the black arrows). The whiteasterisk indicates a bony artefact. (C) A plantar view on the CAL is displayed at a slightly more proximal levelcompared to B. Focusing on the trabecular architecture, the black arrows indicate representative trabeculae thattake a lateromedial course, similar to the more distal fibres of the CB shown in B. Similarly, the white arrows
highlight representative trabeculae that are in line with the orientation of the LB. (D) An overview of the sliceshown in B is depicted, showing the CB and LB originating from the CAL taking a distal course (view fromplantar). Directions A-C: indicated by a cross; d, distal; l, lateral; m, medial; p, proximal.

Scientific Reports |Vol:.(1234567890)(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

6

www.nature.com/scientificreports/

Figure 7.  The insertion of the Achilles tendon (AT) into the calcaneus (CAL) is depicted on plastinates and inhistology. (A) Plastinated slice. Black arrows: demarcated trabeculae; dotted box: corresponds to an area similar
to the dotted box in B; 1–3: fibrous septae, similar to the ones in B. Scale bar: 4 mm, (B) Masson–Goldner stain.
Black box, median fibrous septum; PT, paratenon; 1–3: fibrous septae, similar to the ones in A; white arrow:
median calcaneal prominence; yellow dotted line: demarcation between AT and CAL. Scale bar: 1 mm, (C)H&E stain. AC, adipocytes (located in bone marrow); black arrows: border between AT and CAL in greater
detail compared to B; blue arrows: collagen bundles; white asterisk: folding artefact; yellow arrows: calcaneal
trabeculae. Scale bar: 200 μm; a, anterior; l, lateral; m, medial; p, posterior.

bridge between AT and PF suggested that this connection might exist even m­ orphologically7. The given study
provides evidence in favour of a morphological entity of the AT, the superficial posterior and inferior calcaneus
and PF based on plastination and histology. The sheet plastination sections assessed in this study revealed that thediminution of the AT when inserting into the calcaneus from proximal to distal is associated with the occurrenceof a delimitable highly regular trabecular band in the superficial posterior and inferior calcaneus, which differs
in colour intensity from the surrounding hard tissue and seems to continue the course of the AT towards the
PF. The histological observations revealed that this band consists of calcaneal trabeculae, which are surroundedby collagens and adipocytes. This is compatible with the concept of a formerly continuous tendon, that ossified
in the area of the calcaneus resulting in two separated tendons, namely the AT and PF. Fibrous septae within
the AT seemed to continue their course in the aforementioned area of regular trabecular arrangement. In moredistal plastinated slices, a trabecular orientation within the calcaneus was noted, which matched the orientationof the adjacent PF fibres. It is conceivable that predominantly during movements the mechanical stimuli fromthe AT proximally and the PF distally contribute to the ossification of the tendon into the bone at the interface
between the AT/PF and the calcaneus. The observed enthesophytes might be an example that in certain cases
this metaplasia might happen to a greater extent than ‘normally’, again being closely connected to the structuralorganization of the tendons (in the given study the AT). Supporting this, the prevalence of calcaneal spurs wasshown to increase with age with no sex-dependency16,17. Reciprocally, the observed differences in colour intensity within the superficial calcaneal regular trabeculae itself might be an indication that the load distribution
is irregular when mechanically strained. Consequently, the relation of trabeculae, tendinous collagens and fat
might vary in different regions of these highly regular trabeculae ranging from a dense dark greyish line to an‘inconspicuous’ trabecular architecture in other plastinated sections as observed in this study.
This is the first study to map the load-deformation characteristics of the entire AT-calcaneus-PF complex
using a uniaxial tensile testing setup. Decalcification of the calcaneal samples for the mechanical tests was conducted, to remove all hydroxyapatite components, leaving behind a collagen scaffold, which allowed to use an
identical tensile testing protocol for hard and soft tissues (Scholze, et al. 2018). Thereby, it is possible to focus
on the bone component that might have been part of the continuous AT-PF tendon before it ossified in thearea of the calcaneus. The pooled UTS of all samples, describing the applicable maximum stress a material canwithstand before breaking when stretched, showed that the decalcified bone samples were at the lower end of
the obtained values. This can be interpreted from different perspectives. If it is assumed that the highly regulartrabeculae of the AT represent a formerly continuous tendon, which subsequently ossified, the organic matrix
might have dismounted a substantial amount of the longitudinally oriented collagens while bricking the same.
Consequently, this resulted in a low tensile strength when tested in the given setup. The group of Snow et al.
stated that in neonates thick continuous fibres were observed between AT and PF, which decreased during life
to ultimately, apart from the calcaneal periosteum, completely disappear in late a­ dulthood7. However, the given
study revealed oppositional results as the decalcified calcaneal samples originating from an old adulthood groupwith a mean age of 74 years without a single failing specimen withstood the uniaxial tensile tests conducted in thisstudy. This evidences a certain amount of structural integrity of the collagenous scaffold, which is independentof the calcaneal ossification. Overall, the morphological results of the given study favour an initial continuous
tendon between AT and PF that becomes integrated into the superficial posterior and inferior calcaneus during
Scientific Reports |(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

7Vol.:(0123456789)

www.nature.com/scientificreports/
life. In the given study the ‘bridging’ was detectable histologically in a sample retrieved from a cadaver at the endof the second decade of life indicating that the subsequent ossification at the calcaneal level should have been
finished in the younger adulthood already. There seems to be a continuous ossification of the AT as a proximal
movement of the AT insertion with age was shown based on MRI ­datasets18. Another possibility is that the high
UTS of the tendinous structures observed in the given study compared to the decalcified bone samples is theresult of a collagen increase in the AT and PF in relation to the collagenous structure that becomes integrated into
the calcaneus early in life. This could be caused by the repetitive mechanical stimuli generated during walking
or jumping, consequently strengthening both AT and ­PF19,20. On the contrary, based on the observed low UTS
values of the decalcified bone samples in this study it could be argued that a tendinous connection between the
AT and PF, being integrated into the calcaneus later in life, was never existing and the observed longitudinallytrabecular structure that seems to connect the AT and the PF only represents a structural adaptation of thecalcaneal bone to the pulling forces of the AT and the PF into oppositional directions. The E­ mod of the decalcified calcaneal samples quantitatively reached roughly a quarter of the values stated for comparable soft tissue
scaffolds in an identical testing ­setup21,22. The higher elasticity (meaning lower elastic moduli) of the decalcified
bone samples compared to all other soft tissues in this study resembles the free spaces that occurred after theremoval of the organic matrix, allowing the remaining collagen scaffold to expand into the same. In contrast,
AT and PF samples revealed higher values, most likely caused by their dense collagenous organization resulting
in both higher elastic moduli and tensile strengths.
Several authors indicated a twist of the AT-forming fascicles on their course from the origin at the medial/
lateral gastrocnemius and soleus muscle to the insertion at the c­ alcaneus23–27. The macroscopic observationsin the given study support the concept of a twisting AT based on the changing arrangement of AT septae fromproximal to distal noted in plastinated sections and the mediolateral course of superficial posterior AT fibres
seen in the dissected samples. Also, an AT fibre twist might explain that the most proximal point of the calcaneal
tuberosity ended up at the midpoint of a mediolateral calcaneal axis in this study, similar to the bulking middle
part when twisting a sheet of paper. According to Szaro et al.26 the medial gastrocnemius fibres are located posteriorly (superficially), the lateral gastrocnemius fibres anteriorly (deeply) and the soleus muscle fibres medially/
centrally in the AT. None of the aforementioned groups followed the course of AT fibres beyond the calcaneal
insertion as it was done here for the first time to our knowledge. This study indicated the feasibility to subdivide
the AT-calcaneus-PF complex into numerous subsamples, allowing to use a uniform uniaxial quasi-static testing protocol for both soft and hard tissues at the same time. Due to the limited amount of available tissues forthe given study the authors refrained from performing correlation analyses, which in a larger sample size mightreveal information about the structural organization of the AT-calcaneus-PF complex from a material testing
point of view. Thus, applying the given setup investigating the AT-calcaneus-PF complex using a medial andlateral line could shed light on the complex reorganization of collagen bundles from proximal (calf) to distal(sole). Also, the possible impact of the collagen bundle rearrangement on the mechanical properties of the ATcalcaneus-PF complex in a larger sample size might enable to draw conclusions on the structural organizationof this complex. Further investigations with regards to age, sex and bodyweight deploying a larger sample size
might provide further insights into the here stated site-dependent biomechanical mapping of the human ATcalcaneus-PF complex. Using larger sample sizes the here stated site-dependent biomechanical mapping of thehuman AT-calcaneus-PF complex can be applied to investigate the biomechanical properties across different
species. This might provide further insights into the evolutionary anatomy of these ­structures8. This given study
provides evidence for a structural continuity of the AT and the PF via the posterior calcaneal trabeculae. It should
be noted that the plantaris longus muscle has been shown to be de-coupled from the PF in humans and, therefore,
lost its potential to strengthen the f­ ormer8. This has been achieved by a firm attachment of the plantaris muscle
tendon to the calcaneal p­ eriosteum8. The former concept of immobilizing the spatially closely related plantaris
muscle tendon appears to be similar for the AT-calcaneus-PF complex discussed in this given study.

Limitations

A number of limitations have to be addressed for this study. Firstly, the minute samples size was limited by the
number of available tissues. The mechanically tested samples in this study were embalmed using a phenoxyethanol-based ­fixative12 and subsequently decalcified by means of a 10wt% EDTA solution, which might haveinfluenced the biomechanical properties. However, the biomechanical purpose of the given study was to compare the biomechanical properties of several sub-structures of the AT-calcaneus-PF complex to each other withspecial interest of the change of the individual parameters from proximal to distal rather than giving a detailed
lifelike material description of the aforementioned. The bias introduced by the embalming and decalcification
likely influenced all samples to a comparable amount as a systemic error and should, therefore, not influence therelative relationship between the biomechanical properties of the tested subsamples. The study intended to test
the calcaneal samples within the area of the regular trabecular arrangement, which possibly reflects the former
tendinous part. In this regard, for the preparation of calcaneal samples for mechanical testing, the cancellous
bone outside the area of the highly regular trabecular arrangement might not have been removed entirely in all
samples, which potentially influenced the acquired material parameters. Furthermore, the elastic modulus washere evaluated in the linear part of each nominal stress–strain curve, using a regression analysis. Human soft
tissues, however, naturally present a non-linearity concerning their load-deformation behaviour. Therefore, thepresented values are suitable for the given purpose but likely represent an oversimplification when these parameters are applied for, e.g., lifelike computer simulations of human soft tissues. The post mortem interval of thetissues in this study is unknown. However, no obvious signs of tissue degradation such as skin discolouration
were noted, which would indicate a prolonged post mortem interval. It remains unknown at this stage whether

Scientific Reports |Vol:.(1234567890)(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

8

www.nature.com/scientificreports/
the EDTA affected the biomechanical properties of the extracellular matrix components of the scaffold, resulting
from hydroxyapatite removal, e.g., by destruction of glycosaminoglycans or collagens.

Conclusions

Morphological observations support the concept of a collagen fascicle continuity between the AT and the PF via
the superficial posterior and inferior calcaneus according to a functional and morphological ‘AT-calcaneus-PF
complex’. A biomechanical testing setup was established that allows to uniformly test the involved hard and soft
tissues of the AT-calcaneus-PF complex in a larger sample size to reveal structural information of this complex
from a material testing point of view in the future.
Received: 21 May 2020; Accepted: 13 August 2020

References

1. Nahin, R. L. Prevalence and pharmaceutical treatment of plantar fasciitis in United States Adults. J. Pain. 19, 885–896. https​://doi.
org/10.1016/j.jpain​.2018.03.003 (2018).
2. Cole, C., Seto, C. & Gazewood, J. Plantar fasciitis: evidence-based review of diagnosis and therapy. Am. Fam. Physician 72,
2237–2242 (2005).
3. Rathleff, M. S. et al. High-load strength training improves outcome in patients with plantar fasciitis: a randomized controlled trial
with 12-month follow-up. Scand. J. Med. Sci. Sports 25, e292-300. https​://doi.org/10.1111/sms.12313​ (2015).
4. Riddle, D. L., Pulisic, M., Pidcoe, P. & Johnson, R. E. Risk factors for Plantar fasciitis: a matched case-control study. J. Bone Joint
Surg. Am. 85, 872–877. https​://doi.org/10.2106/00004​623-20030​5000-00015​ (2003).
5. Bolivar, Y. A., Munuera, P. V. & Padillo, J. P. Relationship between tightness of the posterior muscles of the lower limb and plantar
fasciitis. Foot Ankle Int. 34, 42–48. https​://doi.org/10.1177/10711​00712​45917​3 (2013).
6. Milz, S. et al. Three-dimensional reconstructions of the Achilles tendon insertion in man. J. Anat. 200, 145–152. https​://doi.org/
10.1046/j.0021-8782.2001.00016​.x (2002).
7. Snow, S. W., Bohne, W. H., DiCarlo, E. & Chang, V. K. Anatomy of the Achilles tendon and plantar fascia in relation to the calcaneus
in various age groups. Foot. Ankle Int. 16, 418–421. https​://doi.org/10.1177/10711​00795​01600​707 (1995).
8. Sichting, F., Holowka, N. B., Ebrecht, F. & Lieberman, D. E. Evolutionary anatomy of the plantar aponeurosis in primates, including
humans. J. Anat. 237, 85–104. https​://doi.org/10.1111/joa.13173​ (2020).
9. Kim, P. J., Richey, J. M., Wissman, L. R. & Steinberg, J. S. The variability of the Achilles tendon insertion: a cadaveric examination.
J. Foot Ankle Surg. 49, 417–420. https​://doi.org/10.1053/j.jfas.2010.05.002 (2010).
10. Stecco, C. et al. Plantar fascia anatomy and its relationship with Achilles tendon and paratenon. J. Anat. 223, 665–676. https​://doi.
org/10.1111/joa.12111​(2013).
11. Benjamin, M., Theobald, P., Suzuki, D. & Toumi, H. in The Achilles Tendon (eds N. Maffulli & C. Almekinders) (Springer, Berlin,2007).
12. Crosado, B. et al. Phenoxyethanol-based embalming for anatomy teaching: an 18 Years’ experience with crosado embalming at
the University of Otago in New Zealand. Anat. Sci. Educ. https​://doi.org/10.1002/ase.1933 (2019).
13. International Organization for Standardization. Plastics-determination of tensile properties (ISO Standard No. 527–2). https:​ //www.
iso.org/stand​ard/56046​.html (1996).
14. Scholze, M. et al. Utilization of 3D printing technology to facilitate and standardize soft tissue testing. Sci. Rep. 8, 11340. https​://doi.org/10.1038/s4159​8-018-29583​-4 (2018).
15. Xu, Z., Chapuis, P. H., Bokey, L. & Zhang, M. Denonvilliers’ fascia in men: a sheet plastination and confocal microscopy studyof the prerectal space and the presence of an optimal anterior plane when mobilizing the rectum for cancer. Colorectal Dis 20,
236–242. https​://doi.org/10.1111/codi.13906​ (2018).
16. Beytemur, O. & Oncu, M. The age dependent change in the incidence of calcaneal spur. Acta. Orthop. Traumatol. Turc. 52, 367–371.
https​://doi.org/10.1016/j.aott.2018.06.013 (2018).
17. Benjamin, M., Rufai, A. & Ralphs, J. R. The mechanism of formation of bony spurs (enthesophytes) in the achilles tendon. Arthritis
Rheum 43, 576–583. https​://doi.org/10.1002/1529-0131(20000​3)43:3%3c576​::AID-ANR14​%3e3.0.CO;2-A (2000).
18. Kim, P. J., Martin, E., Ballehr, L., Richey, J. M. & Steinberg, J. S. Variability of insertion of the Achilles tendon on the calcaneus: an
MRI study of younger subjects. J. Foot Ankle Surg. 50, 41–43. https​://doi.org/10.1053/j.jfas.2010.10.007 (2011).
19. Nirmalanandhan, V. S. et al. Improving linear stiffness of the cell-seeded collagen sponge constructs by varying the componentsof the mechanical stimulus. Tissue Eng. Part A 14, 1883–1891. https​://doi.org/10.1089/ten.tea.2007.0125 (2008).
20. Krahl, H., Pieper, H. G. & Quack, G. Bone hypertrophy as a results of training. Orthopade 24, 441–445 (1995).
21. Zwirner, J., Scholze, M., Waddell, J. N., Ondruschka, B. & Hammer, N. Mechanical properties of human dura mater in tension – ananalysis at an age range of 2 to 94 years. Sci. Rep. 9, 16655. https​://doi.org/10.1038/s4159​8-019-52836​-9 (2019).
22. Hammer, N. et al. Do cells contribute to tendon and ligament biomechanics?. PLoS ONE 9, e105037. https:​ //doi.org/10.1371/journ​al.pone.01050​37 (2014).
23. Cummins, E. J., Anson, B. J. & et al. The structure of the calcaneal tendon (of Achilles) in relation to orthopedic surgery, withadditional observations on the plantaris muscle. Surg. Gynecol. Obstet 83, 107–116 (1946).
24. Edama, M. et al. The twisted structure of the human Achilles tendon. Scand. J. Med. Sci. Sports 25, e497–503. https​://doi.
org/10.1111/sms.12342​(2015).
25. Parsons, F. G. On the morphology of the tendo-achillis. J. Anat. Physiol. 28, 414–418 (1894).
26. Szaro, P., Witkowski, G., Smigielski, R., Krajewski, P. & Ciszek, B. Fascicles of the adult human Achilles tendon - an anatomical
study. Ann. Anat. 191, 586–593. https​://doi.org/10.1016/j.aanat​.2009.07.006 (2009).
27. Shim, V. B., Handsfield, G. G., Fernandez, J. W., Lloyd, D. G. & Besier, T. F. Combining in silico and in vitro experiments to characterize the role of fascicle twist in the Achilles tendon. Sci. Rep. 8, 13856. https​://doi.org/10.1038/s4159​8-018-31587​-z (2018).

Author contributions

All authors were involved in the study design. J.Z. performed the tensile testing and drafted the first version of
the manuscript. N.H, M.Z. and K.A. gave technical advice on the conducted experiments. B.O. conducted thehistology. All authors reviewed, edited and approved the manuscript for submission.

Funding

The funding was provided to J.Z. by the Yokochi Fund of the Ichiro Kanehara Foundation.

Scientific Reports |(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

9Vol.:(0123456789)

www.nature.com/scientificreports/

Competing interests

The authors declare no competing interests.

Additional information

Correspondence and requests for materials should be addressed to J.Z. or N.H.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps andinstitutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly fromthe copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/.
© The Author(s) 2020

Scientific Reports |Vol:.(1234567890)(2020) 10:14523 |https://doi.org/10.1038/s41598-020-71316-z

10