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OPEN

Received: 8 May 2018
Accepted: 13 July 2018
Published: xx xx xxxx

Utilization of 3D printing
technology to facilitate andstandardize soft tissue testing
Mario Scholze1,2, Aqeeda Singh 1, Pamela F. Lozano1, Benjamin Ondruschka3,
Maziar Ramezani4, Michael Werner5 & Niels Hammer1,5,6
Three-dimensional (3D) printing has become broadly available and can be utilized to customize
clamping mechanisms in biomechanical experiments. This report will describe our experience using 3D
printed clamps to mount soft tissues from different anatomical regions. The feasibility and potential
limitations of the technology will be discussed. Tissues were sourced in a fresh condition, including
human skin, ligaments and tendons. Standardized clamps and fixtures were 3D printed and used tomount specimens. In quasi-static tensile tests combined with digital image correlation and fatigue trials
we characterized the applicability of the clamping technique. Scanning electron microscopy was utilized
to evaluate the specimens to assess the integrity of the extracellular matrix following the mechanicaltests. 3D printed clamps showed no signs of clamping-related failure during the quasi-static tests, andintact extracellular matrix was found in the clamping area, at the transition clamping area and thecentral area from where the strain data was obtained. In the fatigue tests, material slippage was low,
allowing for cyclic tests beyond 105 cycles. Comparison to other clamping techniques yields that 3D
printed clamps ease and expedite specimen handling, are highly adaptable to specimen geometries andideal for high-standardization and high-throughput experiments in soft tissue biomechanics.
Standardization of mechanical experiments involving biological soft tissues when tested under strain remains an
ongoing issue with negative impact on the accuracy and validity. Soft tissues are characterized by a number of
special characteristics including anisotropy, heterogeneity, viscoelasticity and inter-individual variation. Thesevariables are further potentiated particularly in human samples by the post-mortem delay and therefore autolysis,
alterations in water content, and underlying traumatic or internal pathology, complicating the standardizationof the mechanical tests. Consequently, to obtain robust results, large sample sizes are necessary to outweigh thestatistical scatter which is likely to occur.
To overcome issues such as material slippage in uniaxial tensile tests, a number of adjustments and fixtures
have been introduced1–3, including steel clamps with roughed surface areas4,5 and high friction areas6–9, cryogenic clamps10,11 or pneumatic clamps1–3,9,12–14. However, these existing methods have limitations regarding theirapplicability to the soft tissues, especially regarding the formation damage due to avulsion at the clamping site or
the risk of temperature-induced changes in the mechanical behavior. In recent years, we have developed a technique called partial plastination to address material slippage15–17. Partial plastination helps to minimize material
slippage by using ceramic-reinforced polyurethane resins at the mounting sites of the tissue and leaving behindan area of native tissue for mechanical testing with a sharp boundary to the plastinated area15,16. This technique
however comes with certain limitations including the time-consuming preparation, the need for highly specialized plastination equipment such as vacuum pumps and casting fixtures, and the use of further chemicals such asacetone with unknown effects on tissue mechanics and potential hazard for the users. Furthermore, the difficultyof positioning soft tissues in a vertical test setup (involving the effects of gravity) can introduce errors during theclamping by deviations from the axial position which could also lead to variances in strain and force recordings.
1

Department of Anatomy, University of Otago, New Zealand Department of Anatomy, Dunedin, New Zealand.
Institute of Materials Science and Engineering, Chemnitz University of Technology, Chemnitz, Germany. 3Instituteof Legal Medicine, Medical Faculty University of Leipzig, Leipzig, Germany. 4Department of Mechanical Engineering,
Auckland University of Technology, Auckland, New Zealand. 5Department of Trauma, Orthopedic and Plastic
Surgery, University Hospital of Leipzig, Leipzig, Germany. 6Fraunhofer Institute for Machine Tools and Forming
Technology, Dresden, Germany. Correspondence and requests for materials should be addressed to M.S. (email:mario.scholze@mb.tu-chemnitz.de) or N.H. (email: nlshammer@googlemail.com)
2

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Figure 1. Standardization in material testing and test setup. Focus of this study will be the boxes highlighted inred. The figure is numbered in italics according to the steps described in Materials and Methods.

As a consequence, we aimed to explore alternative techniques which may facilitate tissue clamping, and aid instandardizing the clamping of soft tissues for biomechanical testing in a less time-consuming manner. 3D printing has meanwhile become broadly available, and such professional extrusion solutions can be utilized for customizing and printing fixtures and adjustments for biomechanical testing using commercially-available filaments.
Furthermore, it can be utilized to provide affordable add-ons to existing testing devices all over the world, going
beyond just soft-tissue biomechanics. The possibility of sharing existing digital models enables a broad availability
and exchange of research and knowledge. 3D printing may also be used for clamping mechanisms, and variations in clamping design appear to be eased by the rapid-prototyping approach with the ubiquitously-available
software.
This report will describe our preliminary experience using 3D printed clamps to mount human soft tissues
from three different anatomical regions with different properties, including skin, ligaments, and tendons, andcompare this method to our previous experience with partial plastination15,17–20. The feasibility and potential
limitations of the technology will be discussed, and two scenarios will be shown for quasi-static and cyclic (fatigue)
tests.

Materials and Methods

A general overview of the workflow for the experiments and evaluation of this study is given in Fig. 1. All experiments were performed in accordance with relevant guidelines and regulations.

Tissue sampling and water content adaptation. Human tissues were sourced from different anatomical regions for these experiments in a fresh and unfixed condition. Scalp skin samples (n = 196 from 49 cadavers,
age range 6–94 years), and iliotibial band tissues (ligaments; n = 58 from 15 cadavers, age range 6–97 years) wereobtained from human cadavers in forensic medicine (ethical approvals 486/16 EK and 156-10-12072010 fromthe University of Leipzig, Germany). Flexor digitorum superficialis and profundus tendons (n = 79, age range
77–91 years) were harvested from the arms of another five human cadavers (ethical approval H17/20 from the
University of Otago, New Zealand). The different age range of the tissues is explained by the different cohorts inanatomy environments as opposed to forensic medicine. A protocol was applied to adapt the tissues’ water content. For human skin and ligament samples, the water content was adapted to the average of the native conditionin the sample population20,21. These samples were later tested in quasistatic tensile tests with a short test duration(1–2 min), where the adaptation minimized the influences related to age and the post-mortem interval. Human
tendons were pre-treated in 0.9% tris-buffered saline, because of the long test duration (up to 12 hours) and thepossibility to moisten the samples during the tests in predefined intervals with the same liquid (every 20 minutes).
3D printed clamping systems for preparation and mounting.

The clamping systems, including
clamps and fixtures were developed by computer-aided design (CAD) in Creo 4.0 (PTC software, Needham,
MA, USA). 3D printing was conducted using the Ultimaker 3 Extended (Ultimaker B.V., Geldermalsen, The
Netherlands), applying the Fused Deposition Modelling (FDM) technique. All models were processed with the
Ultimaker Cura 3 slicing application prior to printing. Commercially-available filaments of acrylonitrile butadiene styrene (ABS), nylon (polyamide), polylactic acid (PLA) and thermoplastic polyurethane (TPU) were trialed
at printing parameters according to the distributor (Ultimaker B.V.). For all 3D prints, the parts were produced

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Failure type

Absolute number and relative %Transverse edge

13

8%

Transverse middle

00%Longitudinal edge

47

30%

Longitudinal middle

1

1%

Total failures

60

38%

Total Number

158

Table 1. Number and type of clamp failures in the cyclic (fatigue) setup for the 158 clamps used for 79 tendons.
Note: One clamp showing both modes of longitudinal failure.

with 100-micron layers using a 0.4-mm brass nozzle. The clamps were printed in an upright position for a lateralorientation of the pyramids, allowing a very detailed quality of these surface structures.
Tables and templates for standardizing tissue dimensions. In the first step, specimen tables suitable for tissue
preparation prior to testing were designed to include cutting into a ‘dog-bone’ shape and casting (determinationof the cross-sectional area). The tables were constructed so that different sample dimensions could be mounted,
varying between 5.0 and 80.0 mm to account for different tissue geometries and dimensions. The tables in the
final design introduced here primarily have the function of simplifying tissue preparation and offer an approachto standardize the dimension and geometry of soft biological samples. They were printed of PLA and consisted of
the following setup and parts:•••

Base with a central area to place the parallel testing area of the sample, one central and two peripheral openings. The central opening serves for determining the samples’ cross section, the peripheral opening for theclamps.
Cutting template, according to the ISO 527-2 (International Standard Organization, 1996) to cut samples intoa ‘dog bone’ shape.
Molding device: These clips were used as micro versions of impression trays commonly used in dentistry.
Clips were attached to the devices, to align them in parallel and to minimize the use of impression material
required for the cast (determination of specimen cross section). The resulting impressions served to determine the samples’ cross-sectional areas.

Figure 2A represents an exploded assembly drawing of the complete preparation table with a tissue sample and molding devices as well as the flexible supporting arms. The stepwise preparation of tissues with thisapproach is shown in Fig. 3.
Clamping design and supporting arms for tissue mounting. Clamping devices of varying geometry and surface
characteristics were trialed using different materials. The clamping area itself was built to have included pyramid
structures on both sides, which interlock with the sample by form and force closure when being compressed. For
the reusable design, the walls were constructed to interlock and to have a self-locking mechanism (2-part design,
Fig. 2A). Additionally, a breakable single-use design based on the same structures and clamping mechanisms wasmade especially for small clamp opening widths, where the use of the 2-part design is impossible. These clamps
were designed as tubes with two openings as 1-part design and later refined especially for fatigue testing purposes, given that such machines have much smaller clamping widths, only allowing for relatively small mounts.
Preliminary experiments were conducted for both sharp- and smooth-edged pyramid tips (Fig. 2B). Sharp pyramids were found out to be more suitable for quasi-static experiments and/or when testing thicker samples, wherea greater interlock with the samples is necessary, therefore forming the basis for the application with full thickness
skin samples. Smooth pyramids were found out in preliminary tests to offer suitable interlock with thinner samples like tendons or ligaments. They were especially used in the breakable design for fatigue testing of the tendons.
As part of this design, flexible supporting arms were constructed out of TPU to facilitate the mounting of the tissues in the material testing machine at a standardized clamping length. Prior to the tests, the clamps were soakedwith isotonic saline to circumvent dehydrating effects of the tissue samples from the dry plastics.

Biomechanical tests. Tensile mechanical experiments were conducted with all tissues at 21° Celsius andfollowing osmotic adaptation of the water content (skin and ligaments) or moistening (tendons), according to theflow chart given in Fig. 1.
Following the tapering, mounting and clamping as shown in Figs 3 and 4, quasi-static experiments were conducted for the skin and ligament samples in a universal testing machine (Zwick Roell 20 kN Allround Table Top
Z020 and 2.5-kN Xforce P load cell, Ulm, Germany). A pair of pneumatic grips Zwick type 8297 was used tomount the 3D printed clamps in the machine and to facilitate a constant compressive gripping force (6 bar operative pressure which equals 6500 N gripping force and 10 MPa pressure at the 3D printed clamps). The samples
tested quasi-static were preconditioned to 20 load-unload cycles conducted at 30% of the assumed maximum
stress before the tissues underwent a final cycle reaching until material failure, indicated by a >30%-decrease
from the previous maximum force. Crosshead displacement was 20 mm/min and sampling rate was 100 Hz. A
strain measurement by contacted extensometers was impossible when testing soft tissues given the high risk of
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Figure 2. (A) Mounted sample in the self-locking clamps (top right), and schematic representation of the
preparation table (center of image) including a (enlarged) molding tool for the determination of cross sectionsof the samples prior to testing (right bottom). (B) Clamp designs with 4-sided pyramid structures in crosssectional view. Pyramid dimensions (d = diagonal, h = height) are enlarged. Additionally, corresponding
scanning electron microscopy images of clamped ligament tissues after testing at 25x magnification are
represented at the right. Note the elongated impressions in the ligament tissues clamped with the sharp
pyramids (scale bar 100 μm).

sample damage. Therefore, a contactless optical (digital) image correlation system (DIC; Q400, Limess, Krefeld,
Germany and Istra4D, Dantec Dynamics, Skovlunde, Denmark) was synchronized with the testing device forstrain measurements (image sampling rate 10 Hz). This technique offers a precise evaluation of strain directly at
the surface of the sample during deformation by tracking of speckles having a stochastic pattern with high contrast. A schematic overview of this approach is given in Fig. 5.
The untapered flexor tendons underwent cyclic fatigue testing using an MTS Landmark Servohydraulic Test
System (Eden Prairie, MN, USA) with hydraulic grips. The samples were mounted in a similar manner at aslightly lower clamp pressure than in quasi-static tests (6 MPa) to avoid clamping related micro-damages andresulting issues in fatigue tests. Following a preload of 10 N, cyclic loading was conducted with a minimum loadof 10 N and a maximum load ranging from 200 N to 500 N. Mounting of the samples was conducted as shownin Fig. 6. The tests were effectuated at a frequency of 4 Hz, and the samples remained in tension throughout the
tests. For all experiments, the type and location of failure of the biological tissues were recorded. Additionally, thesamples were inspected for material slippage and for potential failure of the clamps after testing.

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Figure 3. Steps of trimming, mounting and casting specimens. (1) The cutting template consists of a bottom
and a top plate (not shown), and a guide rail allowing for exact positioning. Radii and lengths can be adjusted
according to the ISO standard to get a ‘dog bone’ shape. Removed tissues can be used for further analyses,
e.g. on ultrastructure or water content. (2) Each clamp consists of four identical parts interlocking pairwise,
mounted to the end of the specimen pairwise in a fixed sample length given by the mounting Table. (3) Casts ofthe specimens’ cross-sectional areas can optionally be made using a designated opening at the mounting table.
Impression material may either be directly placed in this opening, or in a two-part impression device shown in
the picture, allowing for less use of the impression material.

Scanning electron microscopy. Following the experiments, scanning electron microscope analyses of rep-resentative samples were conducted using a JSM-6700F field emission microscope (JOEL Ltd., Tokyo, Japan) at
magnifications between 25x and 15,000x to examine the specimens’ failure sites. Random samples were chosen of
the skin (n = 48 regional samples) and ligaments (n = 12 regional samples).

Micro computed tomography (μCT) and sterilization.

Six clamps were scanned using a General
Electric phoenix v/tome/x s μCT (GE Measurement & Control Solutions, Wunstorf, Germany) with an isovoxel
size of 110 µm prior to and following steam sterilization. Three clamps underwent sterilization at 121 °C for
20 minutes at 2.048 bar with a drying time of 10 minutes. The remaining three clamps were steam sterilized at
134 °C for 10 minutes at 3.043 bar, followed by a drying time of 20 minutes. The resulting geometries were thenassessed using VGSTUDIO MAX 3.1 (Volume Graphics GmbH, Heidelberg, Germany).

Statistical evaluation.

All statistical comparisons were done using Microsoft Excel (Microsoft Corp.,
Redmond, WA, USA), SPSS version 24 (IBM, Armonk, VA, USA) and Graphpad Prism 7a (GraphPad SoftwareInc., La Jolla, CA, USA). Cramér’s ϕ correlations were determined following the Chi-square test to assess theassociation between extracellular matrix integrity and the location site of investigation as an indicator of proteinpreservation. This was done at the clamping site, the border between the clamping and the testing area, and thefailure site. P values of ≤ 0.05 were considered as statistically significant.

Results

Applicability and first experiences with the technique. The combination of tissue preparation, clamp-

ing and mounting with 3D printed parts and accessories allowed for soft tissues to be tested in a highly-standardized and high-throughput approach. PLA was primarily used for the tables, templates and clamps, and TPU
for the specimens´ mounting frame, which consisted of two supporting arms. With the given design, the arms
remained at the samples’ clamps when positioned in the testing machine and this led to the samples being compressed between the closing grips. Consequently, the supporting arms needed to be smaller than the gripping
width and elastic enough to be pulled out of the fully compressed clamps and inserted without excessive wear of
breakage, allowing for reuse. The framed design helped to maintain the sample at a parallel clamping position andat a predefined clamping length without alterations related to manual handling of the sample itself. Moreover, it
formed one of the prerequisites for rapid sequence/high-throughput testing of samples at high standardization.
In pilot trials, a sample throughput of up to 32 samples/day (4 specimens/hour) was achieved for thequasi-static uniaxial tensile tests with combined DIC, beginning with the freshly-harvested and untapered sample. Examples of load-deformation curves and the resulting load-deformation appearance of tissue under scanning electron microscopy (SEM) are given in Figs 7 and 8. In case of the fatigue testing setup, cycles beyond
1.6 × 105 were reached, allowing to obtain fatigue cycle stress-cycle (S-N) characteristics (Wöhler curves) for 50

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Figure 4. Specimen mounting and removal of positioning ring for quasi-static experiments.

Figure 5. Principle of digital image correlation used for the strain evaluation in the quasi-static experiments: A
calibrated camera recorded the tapered testing area of the samples during tensile deformation. By the evaluationof randomized speckles at the surface, the system can track facets and calculate an in-plane surface strain field.
The nominal strain for the evaluation of the tests was further calculated by a perpendicular virtual extensometer
between two points defined in the parallel testing length of each sample.

flexor tendons (the remaining 29 samples were used for pilot trials). Depending on the used force, the testing timereached intervals up to 11 hours. A scatter plot comparing the number of cycles to failure and used stress is given
in Fig. 9. Sharp pyramid geometries in the clamping areas gave more accurate results in the higher strains of thequasi-static material tests reaching to material failure, whereas flatter pyramid geometries were more useful forthe fatigue tests, allowing for higher cycles before material failure occurred. Clamps with sharp pyramids (fourper specimen, two interlocking per side, see Fig. 2) were primarily used for thicker samples and static tests, which
involved the skin specimens. Clamps with flatter pyramids (2 per specimen, one per side) were primarily used forthe fatigue tests and thinner samples. A total of 200 clamps with sharp pyramids were used for the skin tests, andanother batch of 60 clamps with flat pyramids for the ligament tests, resulting in an average reusability of 4 times
for both designs, ranging between 3x and 5x reuse.
In contrast, the partial plastination technique requires a two-day setting17–21. Preparing the samples to thestep of the embedding basically follows the same steps as above, succeeded by surface coating of the central
specimen part to be evaluated with gelatin for protection purposes and mounting before the samples’ ends are
dehydrated in acetone15. In the following step using acetone, the specimens were prepared for embedding in the
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Extra-cellularmatrix type (n = 48)•Area of sample

Extracellular matrix integrity
Frequency(existence)

Correlation

fully
intactpartially
disrupted

fully
disrupted

Correlation

Collagen• Clamp81%

Cramér’s

23%

54%

23%

Cramér’s• Edge56%

ϕ = 0.37

11%

56%

33%

ϕ = 0.40• Failure100%

p = 0.033

6%

88%

6%

p < 0.001

Keratin• Clamp100%

Cramér’s

19%

69%

13%

Cramér’s• Edge100%

ϕ = 0.86

38%

50%

13%

ϕ = 0.68• Failure13%

p < 0.001

0%

0%

100%

p < 0.001

Table 2. Evaluation from scanning electron microscopy regarding collagen and keratin existence and integrityin three different areas of human skin after testing: Clamped area, transition/edge between clamping andtesting area and in the area of failure. All specimens were tested in uniaxial quasi-static tensile tests until failureusing 3D printed clamps made of polylactic acid with pyramid structures. Note: Percentages rounded to whole
numbers.

Figure 6. Specimen mounting for cyclic tests (left and middle), and photographs showing fatigue experimentsof human flexor tendons.

ceramic-polyurethane resin, ideally applying a mild vacuum for forced impregnation. Subsequently following thecuring, the removal of the templates, protruding resin and of the gelatin covering the testing area of the samples
was necessary and time-consuming. The steps and chemicals used makes the entire process necessary to adjust
the water content following the plastination. Preparation time averages 20–30 minutes per sample, and a full
two-day time frame is necessary to test specimens in a standardized manner, resulting in a potential throughput
of 16 samples in 2 days (1 sample per hour), beginning with a freshly-harvested sample. Sand paper clamping
at first step appears to be easier to carry out but brings significant issues with slippage or avulsion and lacks inopportunities for standardization.

Material failure, slippage and clamping failure. Using the approach with 3D printed accessories, failure

occurred in the central area in 189/196 human skin samples (96%) in the quasi-static experiments and in 48/58
of the ligament samples (83%). In case of the fatigue tests with the untapered flexor tendons, failure occurred
predominantly in the transition of the clamping area in 29/50 samples (58%) being used for the fatigue testing. Of
the total of 79 flexor tendons used, material wear and failure of the clamps occurred as given in Table 1 (2 clamps
per tendon, 158 clamps in total, single use).

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Figure 7. (A) Stress-strain curve of human calvarian skin and the corresponding strain fields from digital
image correlation in three steps before the specimen fails. A schematic sample with squares indicates the area
where tissues were removed for electron microcopy. (B–E) Scanning electron microscopy of human calvarianskin. Left side 500x magnification (scale bar 40 μm), right side 5,000x magnification (scale bar 4 μm), red arrows
indicate the area of magnification for the right picture. (B) Clamping area. Images correspond to skin obtained
from the upper parietal bone area of an infant male. Partially ruptured keratin sheets in low magnification.
Partial collagen fiber rupture can furthermore be seen in higher magnifications. (C) Edge area. Images
correspond to skin obtained from the upper occipital bone area of a geriatric male. Partially ruptured keratins
can be seen in low magnification. In higher magnifications, ruptured collagen bundles are found surrounded bykeratin sheets. (D) Failure area. Images obtained from upper parietal bone area of a geriatric male. In this area,
mainly collagen bundles were found, with both retracted and intact areas, building a dense network. (E) Failurearea. Obtained from the area upper superficial to the occipital bone of an infant male. At lower magnifications,a similar pattern can be seen as in the geriatric sample. At higher magnifications, it can be seen that a similararea is composed of lesser collagen at a lower density but higher quantity, which may be partially responsible forhigher elasticity and lower ultimate stress features.

Material slippage using the 3D printed clamps for the quasi-static experiments varied and was qualitatively
dependent on sample thickness; yet with no statistically significant level of correlation (p > 0.05). Figure 10 shows
the ligament example with the most pronounced slippage (2.0 mm) and a representative comparison with minute slippage. As these quasi-static experiments were synchronized with DIC data of the specimen deformation,
the error in strain recording potentially resulting from the slippage could be ruled out effectively. During thequasi-static experiments, no clamping failure was observed. However, as a result of repeated opening and closing
and in particular for thick samples (skin tests), some of the clamps showed signs of weakening and were consequently excluded from further use.
In the fatigue tests, material slippage was generally low, likely as a consequence of the lower stresses
compared to the quasi-static experiments. In most samples, slippage was minimal and below the level ofmacroscopically-visible detection. Also, given the nature of these tests with partial failure as a consequence ofrepeated loading, it was impossible to separate slippage from preconditioning phenomena (stretching and parallelization of collagens). As the primary objective of the fatigue tests was, however, to obtain load-dependent life
cycles ranging beyond 105 to 106; speckle-based image correlation would have been impossible as the repetitive
moistening of the samples would have washed off surface marking.

Scanning electron microscopy. Of the representative samples taken for SEM, site-immanent characteristics were found for both skin and ligament samples. In the clamping area of the skin and ligament samples,
imprints of the pyramid patterns were seen, causing contact patterns in the epidermal and ligament surface
regions, especially in the keratins and collagens (Figs. 2B and 7; Table 2). In higher magnifications, partial collagendamage was observed. The edge regions between the unclamped and clamped parts showed similar findings withSCIENTIFIC ReportS | (2018) 8:11340 | DOI:10.1038/s41598-018-29583-4

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Figure 8. (A) Stress-strain curve of human ligament and the corresponding strain fields from digital image
correlation in three steps before the specimen fails. A schematic sample with squares indicates the area where
tissues were removed for electron microcopy. (B–D) Scanning electron microscopy of ligament samples. Leftside 1,000x magnification (scale bar 20 μm), middle 5,000x magnification (scale bar 2.5 μm), right side 15,000x
magnification (scale bar 1 μm), red arrows indicate the area of magnification for the next picture. (B) Clamping
area. Images correspond to the area under the pyramids of the clamps. Collagen fibers remained partially to
fully intact in all magnifications. (C) Edge area. Intact and partially ruptured collagen fibers are seen, indicatedby disruption of fibers and ball-shaped collagens in higher magnifications. (D) Failure area. Lumpy collagenappearance, collagen disarrangement and collagen balls indicate multiple sites of rupturing in the failure areas.

Figure 9. Stress-cycle (S-N) curve (Wöhler curve) for human flexor tendon fatigue behavior tests.
a decreased presence of collagen bundles, which tended to be more disrupted than in the clamping area. At the
failure site, the existence of keratins was minimal and frequently disrupted; collagens were present throughout thisregion, showing partial disruption with curling, elongation and retraction of the collagen proteins. Both existence(p ≤ 0.033) and integrity (p < 0.001) of collagens and keratins were significantly site-dependent, with a strong site
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Extra-cellularmatrix type (n = 12)•Area of sample

Extracellular matrix integrity

Frequency(existence)

Correlation

fully intact

partially disrupted

fully disrupted

n/a

50%

50%

0%

Cramér’s

0%

100%

0%

ϕ = 0.816

0%

0%

100%

p = 0.003

Correlation

Collagen• Clamp100%• Edge100%• Failure

100%

n/a

Table 3. Evaluation from scanning electron microscopy regarding collagen existence and integrity in threedifferent areas of human ligaments after testing: Clamped area, transition/edge between clamping and testingarea and in the area of failure. All specimens were tested in uniaxial quasi-static tensile tests until failure using
3D printed clamps made of polylactic acid with pyramid structures.

Figure 10. Examples of low to minute material slippage in 3D printed clamps for quasi-static (A,B) and fatigue
testing (C,D) indicated by red distance signs at the lower parts of each picture.

association for keratin and a moderate association for collagen, indicating that both keratins and collagens are
more likely to remain intact at the clamps.
In the ligament samples, collagens were observed at all areas, including the clamps, the edge between theclamped and unclamped regions. At the clamps, fully intact and partially disrupted collagens were predominantly
found, at the edge exclusively partially ruptured collagens, and at the failure site exclusively fully ruptured collagens (Table 3). Significant correlations were found for the different areas and collagen integrity, indicating thatcollagens are more likely to remain intact in the clamping area and consequently less damaged as a consequenceof the clamping (p = 0.003). Figure 8 summarizes the ultrastructural findings from SEM for the three different
areas of the clamp.

Assessment of sterilization-induced deformations. Following the sterilization, the optically visible
deformation was minute. However, quantifiable deformations were observed by μCT for the clamps at both temperatures. Using the 121 °C protocol, 73.9 ± 3.5% of the clamp surface matched with the original shape within
0.2 mm deviation, and 88.34 ± 2.5% within 0.3 mm. These values were similar to the 134 °C protocol, where thesurfaced of the sterilized clamps matched with the original shape in 66.2 ± 12.1% within 0.2 mm and 76.3 ± 9.1%
within 0.3 mm, respectively. Using the 121 °C protocol, 95% of the surface varied less than 0.38 ± 0.02 mm, and0.50 ± 0.14 mm for the 134 °C protocol. The deformations observed are summarized in Fig. 11 and were locatedespecially at the margins and edges of the clamps, not allowing to continue using the clamps reliably including
the locking mechanism.
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Figure 11. Graph summarizing the accuracy of the 3D printed clamps following sterilization using two
different protocols. Percentage indicates the amount surface identical prior to and following sterilization within
the given range of accuracy. The 95% value indicates the maximum deviation observed for 95% of the clamp
surface. The illustrated an overlay between the initial shape and the shape of the clamps following sterilization.

Discussion

In summary, 3D printing of clamps and fixtures provides a number of advantages over other techniques, making
it particularly suitable if a relatively high throughput is required under a standardized testing environment forinvestigation of sufficient high numbers of test samples in biomedical research. It moreover, in quasi-static testing,allows for pre-determining the area of failure in a central specimen region, as indicated by SEM. This feature isof particular importance if one may hypothesize that failure might primarily occur at the clamps due to avulsion.
For fatigue tests, the given design will need further refinement if a similar pre-determination of the rupture area
is desired or necessary. This may potentially involve tapering of these samples, likely with negative impact on thenumber of load cycles to failure.
A variety of soft tissue clamping methods exist, which are often adapted from engineering materials such as
plastic, carbon or glass fiber composites. As an example, European DIN EN ISO 527 T5 (combined with probe
geometries according to the DIN EN ISO 527 T4) allows for sufficient testing with fiber orientations deviating up
to 45 degrees from the direction of load application but uses strong adhesives to mount the probe to the fixture. It
is however unclear to which extent the chemicals and solvents in the adhesives will alter the areas of the clamped
tissues, and this may also impact on the areas outside of the clamping. This applies also to the partial plastination technique15,16. Roughened steel or sand paper-reinforced clamps form further soft tissue clamping alternatives1–7 but have disadvantages regarding the repeatability of the mounting procedure and the re-use of the clamps
themselves. To overcome material slippage, a number of adjustments and fixtures have been introduced1–3,6–9.
Cryogenic clamping brings about further temperature (or freeze breaking) induced changes in material properties. Other adjustments in clamping have attempted to address the issue of stress concentrations at standard
friction clamps, e.g. roller fixtures22. Such adjustments however have the disadvantage that a relatively long sample length is required, which is usually difficult to achieve with most soft tissues, especially when testing humansamples. Moreover, bone-ligament-bone complexes13,23 are difficult to be obtained and standardized especially
if samples are obtained as part of a biopsy only, and they do not exclusively reflect the mechanical properties ofthe ligament of interest. As shown in this given study, the clamping using 3D printed PLA clamps appears not tohave a negative influence on the integrity of the extracellular matrix in the testing area and at the transition to theclamps to the end that the clamping is a major source of material failure.
The given method with 3D printing therefore addressed successfully the primary aim at improving standardization, while simultaneously allowing for high-volume sample testing in biomechanical setups. The method has
rendered reliable results for human soft tissues, though further refinements will be necessary depending on thenature of the proteins composing the samples and the type of experiment running. Table 4 summarizes in brief
the features of sand paper clamping, partial plastination, cryogenic clamping and 3D printed clamps as comparison between well-known and new clamping techniques. It can be summarized that the given clamping design
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Level of standardization(e.g. specimen alignment,
clamping length)

Sand paper clamping

Partial plastination

Cryogenic clamping

3D printed clamping system

Low

Moderate

Moderate

High

Ease of use

Easy

ComplexModerate

Easy

Use of potentially
dangerous goods?

No

Yes (solvents & resin)

Yes (liquid N2)

No

Adaptability to various
tissues

None

None

ModerateHigh

Modularity

None

None

None

High

Material slippageHigh

MinuteLow

Low

Work time involved for
preparation excluding
testing

15 mins/sample

30 mins/sample

15 mins/sample

10–15 mins/sample

Applicability for quasistatic testing

Suitable

Suitable

Suitable

Suitable

Applicability for cyclic and Unsuitable (material
fatigue testingslippage)

Unsuitable (avulsion at the clamping
interface)

Unsuitable (continuous
cooling necessary,
temperature decrease oftissues)

Suitable for ≥105 cycles

Throughput

high

low

moderate

highType & price ofconsumables andmounting materials per
specimen

Sandpaperca. 0.30 NZ$(0.18 US$/€)

Acetone, resin
5–7 NZ$(3–4 US$/€)

Liquid nitrogen
1 NZ$/l(0.60 US$/l)

Printing materials
ca. 0.50 NZ$(0.30 US$/€)

Reusability of
consumables

not given

not given

not given

estimated 3–5×*

Desinfectability

poor

poor

good

good

Sterilizability

poor

poor

good

poor

Suitable field of
application

Preliminary tests

Highly-specialized applications

Small sample sizes, hightensile forces

High-standardization-high-throughput experiments

Table 4. Comparison of four clamping techniques and their suitability for biomechanical testing. *Applied to
aseptic conditions using 50% ethanol or Viraclean .

®

adds a novel tool for specimen mounting to the field of soft tissue biomechanics, providing a standardized andeasy to manufacture clamping method that is useful for a variety of adjustments in customized tests.
The key advantages of the 3D printed clamps, supporting arms, tables and templates are the ease of use, adaptability, reusability, modularity and the fact that they can be manufactured easily and at local research environments. Furthermore, recent developments in prices, usability and variety of desktop FDM-printers (which wasused in this study) allow an easy integration into laboratory environments. Without the maintenance time of theprinter, which is often reduced to some minutes (when changing a filament spool or cleaning the build-plate),no additional working time is required for the manufacturing of the parts for testing. Conventional 3D printing
materials like PLA and TPU can be printed easily without any hazard of air contamination and are available assingle filaments spools for moderate prices (e.g. PLA: from 30 NZ$/kg), which can be used for printing e.g. a
couple of hundred clamps. Geometries and print settings can be shared easily, forming a basis for affordable
add-ons to existing testing devices for tissue biomechanics. Given that no specialized equipment or procedures
are required for the preparation of the samples, the entire pre-testing procedure can be conducted immediately
before to the actual experiments in the testing environment.
A limitation of the 3D printing techniques utilizing PLA or TPU is formed by the relatively low melting
temperatures, which limit the possibility of PLA- and TPU-based plastics to be sterilized. This is especially thecase with autoclaving or dry heat as the widest spread sterilization techniques24,25, which has been confirmed byour results in μCT based visualization. Ethylene oxide and gamma irradiation may provide a solution here24,25.
However, it needs to be acknowledged that for most testing conditions, aseptic conditions provide a sufficientenvironment and that a sterile environment is difficult to be achieved with most mechanical testing machines.
If sterile conditions must apply, customized clamps printed of heat and chemically resistant metal powders or
milled fixtures may be preferential. Furthermore, when using metals, it is unclear if the pyramid structures or thesnapping mechanism may then need further adjustments to minimize avulsion between the sharp geometries.

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Acknowledgements

The authors would like to thank Jean-Baptiste Le Joncour and Corentin Rouquette for their help with the fatigue
experiments. Elisabeth Girvan helped with the electron microscopy samples. The publication costs of this article
were funded by the German Research Foundation/DFG and the Chemnitz University of Technology in thefunding program Open Access Publishing.

Author Contributions

M.S., B.O., M.R., M.W. and N.H. planned the research and designed the experiments. M.S., A.S., P.F.L., B.O. and
M.W. conducted the experiments and obtained the data. M.S. and N.H. drafted the manuscript, M.S., A.S., M.R.,
M.W. and N.H. critically revised the manuscript prior to publication.

Additional Information

Competing Interests: The authors declare no competing interests.
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