The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.
Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.
Tendon disorders are common and highly prevalent among the aging, athletic, and active populations1,2,3. In the United States, 16.4 million connective tissue injuries are reported each year4 and account for 30% of all injury-related physician office visits3,5,6,7,8. The most commonly affected sites include the rotator cuff, Achilles tendon, and patellar tendon9. Although a variety of non-operative and operative treatments have been explored, including anti-inflammatory drugs, rehabilitation, and surgical repair, outcomes remain poor, with limited return to function and high rates of failure5,6. These poor clinical outcomes have motivated basic and translational studies seeking to understand tendinopathy and to develop novel treatment approaches.
Tensile biomechanical properties are the primary quantitative outcomes defining tendon function. Therefore, laboratory characterization of tendinopathy and treatment efficacy must include a rigorous testing of tendon tensile properties. Numerous studies have described methods to determine the biomechanical properties of tendons from animal models such as rats, sheep, dogs, and rabbits10,11,12. However, few studies have tested the biomechanical properties of murine tendons, primarily due to the difficulties in gripping the small tissues for tensile testing. As murine models have numerous advantages for mechanistically studying tendinopathy, including genetic manipulation, extensive reagent options, and low cost, development of accurate and efficient methods to biomechanically test murine tissues is needed.
In order to properly test the mechanical properties of tendons, the tissue must be gripped effectively, without slipping or artifactual tearing at the grip interface or fracturing of the growth plate. In many cases, particularly for short tendons, the bone is gripped on one end and the tendon is gripped on the other end. Bones are typically secured by embedding them in materials such as epoxy resin13 and polymethylmethacrylate14,15. Tendons are often placed between two layers of sandpaper, glued with cyanoacrylate, and secured using compression clamps (if the cross section is flat) or in a frozen medium (if the cross section is large)15,16,17. These methods have been applied to biomechanically test murine tendons, but challenges arise due to the small size of the specimens and the compliance of the growth plate, which never ossifies18. For example, the diameter of the murine humeral head is only a few millimeters, thus making gripping of the bone difficult. Specifically, tensile testing of murine supraspinatus tendon-to-bone samples often results in failure at the growth plate rather than in the tendon or at the tendon enthesis. Similarly, biomechanical testing of the Achilles tendon is challenging. Although the Achilles tendon is larger than other murine tendons, the calcaneus is small, making gripping of this bone difficult. The bone can be removed, followed by gripping the two tendon ends; however, this precludes the testing of the tendon-to-bone attachment. Other groups report gripping the calcaneus bone using custom-made fixtures19,20, anchoring by clamps21, fixing in self curing plastic cement22 or using a conical shape slot22, yet these prior methods remain limited by low reproducibility, high gripping failure rates, and tedious preparation requirements.
The objective of the current study was to develop an accurate and efficient method for tensile biomechanical testing of murine tendons, focusing on the supraspinatus and Achilles tendons as examples. Using a combination of 3D reconstructions from native bone anatomy, solid modeling, and additive manufacturing, a novel method was developed to grip the bones. These fixtures effectively secured the bones, prevented growth plate failure, decreased specimen preparation time, and increased testing reproducibility. The new method is readily adaptable to test other murine tendons as well as tendons in rats and other animals.
Animal studies were approved by Columbia University Institutional Animal Care and Use Committee. Mice used in this study were of a C57BL/6J background and were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). They were housed in pathogen-free barrier conditions and were provided food and water ad libitum.
1. Development of custom-fit 3D printed fixtures for gripping bone
2. Biomechanical testing of murine tendons
3D-printed fixtures were used to test 8-week old murine supraspinatus and Achilles tendons. All mechanically tested samples failed at the enthesis, as characterized by microCT scans, visual inspection, and video analysis after tensile tests. A one-to-one comparison of the previous and current methods for supraspinatus tendon testing in our laboratory is shown in Figure 3. In the previous method28,29,30, the humerus bone was embedded in epoxy and a paper clip was placed over the humeral head in an effort to prevent growth plate fracture. 4-6 hours were necessary to allow for the epoxy to fully cure (Figure 3), allowing for only 6-8 specimens to be tested in a typical day. A further limitation of the approach was the user-dependent effectiveness of the paper clip placement for preventing growth plate fracture. The testing results using these prior methods were highly variable, with coefficients of variation on the order of 30% for most parameters and growth plate failure rates of approximately 10%–20%. As summarized in Figure 3, specimen preparation time using the new methods was decreased to 5–10 minutes, making it practical to test 16–20 samples per day. Furthermore, growth plate failures were eliminated.
Compared to methodology reported by others for testing murine tendons14,15,17,25,28,29,30,31,32,33, the new methods were more efficient and reproducible. For supraspinatus tendons, structural properties such as maximum load (3.8 ± 0.6 N) and stiffness (12.7 ± 1.8 N/mm), as well as normalized material properties such as maximum stress (8.7 ± 3.0 MPa), and modulus (51.7 ± 13.5 MPa) had considerably lower coefficients of variations compared to results from the literature (Table 1). For the Achilles tendon, mechanical properties such as maximum load (7.8 ± 1.1 N) and stiffness (13.2 ± 1.9 N/mm) had lower coefficients of variations compared to results from the literature19,21,22,32,33,34,35,36,37,38, whereas maximum stress (24.2 ± 5.4 MPa) and modulus (73.2 ± 22.1 MPa) had coefficients of variations similar to those reported in the literature (Table 2).
Animal sex had a significant effect on the mechanical properties of the supraspinatus and Achilles tendons (Figure 4). When comparing male and female supraspinatus tendons, there were significant increases in maximum force (p = 0.002) and work to yield (p = 0.008). There were trends between the two groups for stiffness (p = 0.057), stress (p = 0.068), modulus (p = 0.061) and resilience (p = 0.078). When comparing male and female Achilles tendons, there were significant increases in maximum stress (p = 0.0006) and resilience (p = 0.0019). There were trends between the two groups for work to yield (p = 0.079), and modulus (p = 0.074) and no difference for maximum force (p = 0.1880) and stiffness (p = 0.6759).
Figure 1: Representative 3D models of fixtures for the humerus (top row) and the calcaneus (bottom row). (A) 3D models of the bones. (B) Disassembled models of the fixtures. (C) Assembled models of the fixtures. Please click here to view a larger version of this figure.
Figure 2: Representative 3D printed fixtures. (A) Fixture for biomechanical testing of supraspinatus tendons of 8-week old mice at an angle of 180° between the humerus and supraspinatus tendon. (B) Fixture for biomechanical testing of supraspinatus tendons of 8-week old mice at an angle of 135° between the humerus and supraspinatus tendon. (C) Fixture for biomechanical testing of murine Achilles tendons at an angle of 120° between the calcaneus and Achilles tendon. (D) Fixture for biomechanical testing of supraspinatus tendons of adult Sprague Dawley rats at an angle of 180° between the humerus and supraspinatus tendon. Scale bar: 5 mm. Please click here to view a larger version of this figure.
Figure 3: Comparison of previous and current methods for mechanical testing of murine supraspinatus tendons. (A) Previous specimen preparation methods used in our laboratory prior to mechanical testing: the humerus was potted in epoxy up to the humeral head to stabilize the bone, a paper clip was placed over the humeral head to prevent growth plate fracture, and, for the epoxy to cure, the specimens were left in room temperature for 4-6 hours prior to mechanical testing. (B) Specimen preparation methods used in the current study (Steps 1.2 and 2.1.4): Top left shows a 3D representation of the fixtures as produced by a solid modeling program. The 3D printed fixtures are reusable and easily assembled and disassembled. The bone end of the specimen is inserted into the fixtures, securing the growth plate and exposing the tendon for gripping and testing. The tendon end is glued between a folded thin tissue paper and inserted into the grips. Preparation time for each specimen is 10–15 minutes. (C) Representative load-deformation curves for tensile testing of supraspinatus tendon using current methods. (D) Representative load-deformation curve for tensile testing of supraspinatus tendon showing a growth plate failure. Please click here to view a larger version of this figure.
Figure 4: Sex effect on the mechanical properties of supraspinatus (SST) and Achilles (ACHT) tendons. There was a significant effect of sex on many of the mechanical properties based on unpaired t-tests (*sex effect, p < 0.05). Data shown as mean ± standard deviation. Please click here to view a larger version of this figure.
Figure 5: Cross-sectional area measurement from microCT. (A) Minimum cross-sectional area measurement along the length of supraspinatus tendon. (B) Minimum cross-sectional area measurement along the length of Achilles tendon. Only the tendon proper should be selected for measurement. Please click here to view a larger version of this figure.
Structural Properties | Material Properties | |||||||||||
Animals | Max Force (N) | Stiffness (N/mm) | Max Stress (Mpa) | Modulus (MPa) | ||||||||
著者 | N | Background | Mean ± SD | COV(%) | Mean ± SD | COV (%) | Mean ± SD | COV (%) | Mean ± SD | COV (%) | ||
Beason et al. Journal of Shoulder and Elbow Surgery (2013)15 | 10 | C57Bl/6 | 0.93±0.34 | 36.56 | 95.1±39.8† | 41.85 | 3.40±1.56 | 45.88 | 312.8±127.0 | 40.60 | ||
Bell et al. Journal of Orthopaedic Research (2014)31 | 6 | C57Bl/6 | 1.22 ± 0.52 | 42.62 | 2.37 ± 1.6 | 67.51 | NR | NR | ||||
Cong et al. Journal of Orthopaedic Research (2018)17 | 8 | C57Bl/6 | 5.38 ± 2.404# | 44.68 | 4.25 ± 1.67# | 39.29 | NR | NR | ||||
Connizzo et al. Annals of Biomedical Engineering (2014)32 | 10 | NR (db/+) | NR | 84.44 ± 27.23*† | 32.25 | NR | 476 ± 186.27* | 39.13 | ||||
Connizzo et al. Journal of Biomedical Engineering (2013)14 | NR | C57/BL6 | NR | NR | NR | 297 ± 148.90* | 50.13 | |||||
Deymier et al. Acta Biomaterialia (2019)28 | 12 | CD-1 IGS Mouse (WT) | 5.0 ± 0.7 | 14 | 9.2 ± 2.9 | 31.52 | 33 ± 35 | 106.06 | NR | |||
Eekhoff et al. Journal of Biomedical Engineering (2017)33 | 13 | Eln +/+ | NR | 8.50 ± 2.95 | 34.71 | 5.96 ± 3.23 | 54.19 | 101.2 ± 50.8 | 50.20 | |||
Killian et al. FASEB Journal (2016)29 | 8 | C57BL/6 | NR | NR | 7.79 ± 2.61* | 33.50 | 58.32 ± 31.73* | 54.41 | ||||
Schwartz et al. Bone (2014)25 | 20 | CD-1 IGS Mouse (WT) | 4.11 ± 0.79* | 19.22 | 8.58 ± 3.78* | 44.06 | 12.29 ± 5.95* | 48.41 | 133.80 ± 59.41* | 44.40 | ||
Schwartz et al. Development (2015)30 | 12 | (Rosa-DTA (DTA) x Gli1-CreERT2 ) ScxCre;Smofl/fl (WT) | 4.16 ± 0.29* | 6.97 | 11.04 ± 1.98* | 17.93 | 26.24 ± 5.81 | 22.14 | 121.89 ± 44.18 | 36.25 | ||
Average COV | 27.34 | Average COV | 38.64 | Average COV | 51.70 | Average COV | 45.02 | |||||
New Method | 10 | C57BL/6J | 3.79 ± 0.62 | 16.41 | 12.73 ± 1.81 | 14.20 | 8.71 ± 3.04 | 34.91 | 51.67 ± 13.54 | 26.20 |
Table 1: Mechanical properties of supraspinatus tendons. Mean ± SD and coefficient of variation (COV) for structural and material properties estimated using new methods compared to ones reported in the literature. [NR: not reported, * estimated from figure(s), # standard deviation calculated from reported standard error, † measured deformation using optical stain lines].
Structural Properties | Material Properties | |||||||||
Animals | Max Force (N) | Stiffness (N/mm) | Max Stress (Mpa) | Young's modulus (MPa) | ||||||
著者 | N | Background | Mean ± SD | COV(%) | Mean ± SD | COV (%) | Mean ± SD | COV (%) | Mean ± SD | COV (%) |
Boivin et al. Muscles, Ligaments and Tendons Journal (2014)19 | 6 | Non-diabetic lean control mice | 8.1 ± 0.6 | 7.41 | 3.9 ± 0.7 | 17.95 | NR | 16 ± 3.7 | 23.13 | |
Connizzo et al. Annals of Biomedical Engineering (2014)32 | 10 | db/+ | NR | 20.39 ± 2.43* | 11.92 | NR | 152.94 ± 44.12* | 28.85 | ||
Eekhoff et al. Journal of Biomechanical Engineering (2017)33 | 8 | Eln +/+ | NR | 18.86 ± 3.37 | 17.87 | 10.55 ± 2.97 | 28.15 | 443.8 ± 131.7 | 29.68 | |
Mikic et al. Journal of Orthopaedic Research (2006)34 | 20 | C57BL/6-J x 129SV/J | NR | NR | 18 ± 5 | 27.78 | 61 ± 20 | 32.79 | ||
Probst et al. Journal of Investigative Surgery (2000)22 | 20 | BALB/c | 8.4 ± 1.1 | 13.10 | 6.3 ± 1.2 | 19.05 | NR | NR | ||
Shu et al. Peer J (2018)21 | 9 | C57BL/6 | 9.6 ± 3.84 | 39.96 | 8.19 ± 3.63 | 44.32 | 27.55 ± 10.54 | 38.26 | NR | |
Sikes et al. Journal of Orthopaedic Research (2018)35 | 7 | C57BL/6 | NR | NR | 19.53 ± 7.03 | 0.36 | 62.82 ± 20.20 | 32.16 | ||
Wang et al. Journal of Orthopaedic Research (2006)36 | 9 | A/J | 8.4 ± 1.2 | 14.29 | 12.2 ± 2.8 | 22.95 | 78.2 ± 8.6 | 11.00 | 713.9 ± 203.7 | 28.53 |
Wang et al. Journal of Orthopaedic Research (2006)36 | 8 | C57BL/6J | 10.2 ± 1.4 | 13.73 | 13.1 ± 2.5 | 19.08 | 97.4 ± 11.4 | 11.70 | 765.1 ± 179.6 | 23.47 |
Wang et al. Journal of Orthopaedic Research (2006)36 | 7 | C3H/HeJ | 12.5 ± 1.7 | 13.60 | 14.1 ± 3.2 | 22.70 | 97.5 ± 10.9 | 11.18 | 708.6 ± 127.8 | 18.04 |
Wang et al. Journal of Orthopaedic Research (2011)37 | 7 | C57BL/6 | 6.6 ± 1.7 | 25.76 | 8.2 ± 1.4 | 17.07 | 13.4 ± 3.7 | 27.61 | 86.8 ± 15.5 | 17.86 |
Zhang et al. Matrix Biology (2016)38 | NR | CD-1 and C57BL/6J | 6.73 ± 3.74* | 55.57 | 12.03 ± 3.34* | 27.76 | 25.4 ± 15.14* | 59.61 | 632.31 ± 113.79* | 18.00 |
Average COV | 22.93 | Average COV | 22.07 | Average COV | 23.96 | Average COV | 25.25 | |||
New Method | 12 | C57BL/6J | 7.8 ± 1.08 | 13.91 | 13.19 ± 1.86 | 14.08 | 24.16 ± 5.42 | 22.45 | 73.17 ± 16.14 | 22.06 |
Table 2: Mechanical properties of Achilles tendons. Mean ± SD and COV for structural and material properties estimated using new methods compared to ones reported in the literature. [NR: not reported, * estimated from figure(s), # standard deviation calculated from reported standard error].
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Murine animal models are commonly used to study tendon disorders, but characterization of their mechanical properties is challenging and uncommon in the literature. The purpose of this protocol is to describe a time efficient and reproducible method for tensile testing of murine tendons. The new methods reduced the time required to test a sample from hours to minutes and eliminated a major gripping artifact that was a common problem in previous methods.
Several steps described in this protocol are critical to produce effective fixtures mechanically testing murine supraspinatus and Achilles tendons. First, step 1.1.4 is necessary to create a 3D model of the desired bone; however, due to the typically high resolution used for this scan, the file size may be too large to use with solid modeling programs. The software used in this protocol successfully reduced the size of the file (step 1.1.6) and preserved object geometry, although other softwares may also be effective to achieve this. Second, each anatomic site has specific design criteria to consider for effective gripping. For the design of the supraspinatus tendon fixture, it is critical to: (i) secure the humeral head to prevent growth plate failure (step 1.2.1.12), (ii) define a clearance fit that avoids disengaging of the humerus bone from the mold during testing (step 1.2.1.12.1) and (iii) orient the humerus bone to form a 180° angle with the long axis of the tendon (step 1.2.1.7). For the Achilles tendon fixture design, it is critical to: (i) define a clearance fit that grips the small calcaneus bone without slipping out from the fixture during testing and (ii) orient the calcaneus bone to form a 120° angle (30° plantar flexion) with the long axis of the tendon. Third, accurate measurement of the tendon cross-sectional area (step 2.1.2) is critical to properly calculate engineering stress for determination of material properties. To measure the cross-sectional area of the supraspinatus tendon, we recommend microcomputed tomography scans of the bone-tendon-muscle specimen suspended in a cryotube with a flat bottom, with the bone held upside down in the tube with agarose. Only the humerus bone should be inserted into the agarose gel, while the humeral head with the tendon and muscle attached should be scanned in air. As the supraspinatus tendon has a splayed geometry as it inserts into the bone, the most consistent way to measure the cross-sectional area is to determine the minimum cross-sectional area along the length of the tendon. A similar procedure should be followed to measure the cross-sectional area of the Achilles tendon. For the Achilles tendon, high resolution microcomputed tomography scans reveal two distinct tissues: the tendon proper and the surrounding sheath, which appears as a lighter shade. To consistently estimate the minimal cross-sectional area for the Achilles tendon, only the tendon proper should be selected for measurement (Figure 5). Lastly, the grips are reusable and small variations from sample to sample do not affect their effectiveness. Each bone should be scanned once (e.g., for the current study, left humerus, right humerus, and calcaneus) and one 3D model should be created for each bone. In addition, for animals of the same age, the bone geometry is nearly identical, thus the same fixture can be used for testing of all specimens. In this manuscript, 3D printed fixtures specific to 8-week old mice (skeletally mature adult mice) were used to test tendons. It was not necessary to create separate male and female fixtures. For other age groups (e.g., 4-week old mice) or mice with unique bone phenotypes, it is recommended that fixtures that fit the particular geometries of the bones are manufactured.
After design and 3D printing of the fixtures, to ensure reproducibility and efficiency of the approach, 10 tendon samples from mice of the same background and age of the planned study should typically be tested (the exact sample size may vary depending on the tissue and animal model). The mechanical properties of these tendons should be determined to ensure that coefficients of variation for structural and material properties are within the expected range, as described in Table 1 and Table 2. These pilot tests should also confirm that artifactual failures (e.g., growth plate failure) do not occur. Multiple cycles of design, prototyping, and validation may be needed to achieve the desired results for tendons other than the supraspinatus and Achilles tendons described in the current paper.
A number of groups have reported the mechanical properties of murine tendons. The coefficient of variations in these studies are typically high, often making it difficult to pick up differences among the comparison groups. Furthermore, methodological differences in tissue gripping among the various studies makes it difficult to determine whether failure properties are relevant to tendon or due to artifactual grip failures. To compare the new testing methods with existing methodologies, a literature review was performed and the results from 20 studies were summarized (Table 1 and Table 2). In the literature, for supraspinatus tendon mechanical testing, the average coefficients of variation for maximum force, stiffness, maximum stress, and modulus were 27%, 39%, 52%, and 45%, respectively. For Achilles tendon mechanical testing, the average coefficients of variation for maximum force, stiffness, maximum stress, and modulus were 23%, 22%, 24%, and 25%, respectively. In the current study, the new method for testing murine tendons resulted in a 32%–63% reduction of supraspinatus tendon coefficients of variation and 6%–39% reduction in Achilles tendon coefficients of variation.
There is no current standard methodology for gripping bones, thus it is unclear to what extent artifactual gripping issues has affected reported mechanical properties of murine tendons. Most groups report gripping the humerus bone by using epoxy resin13, polymethylmethacrylate (PMMA)14,15, or cyanoacrylate16 and securing the humeral head by applying a second coating of PMMA14, using custom fixture39 and/or inserting a paper clip25,28,30. Similarly, other groups report gripping of the much smaller calcaneus bone using custom-made fixtures19,20, anchoring by clamps21, fixing in self curing plastic cement22 or using a conical shape slot22. However, these methods remain limited by low reproducibility, high artifactual failure rates, and time-consuming preparation requirements. The new methods presented in this study have eliminated artifactual grip failures and have tripled the number of specimens that can be tested in a day. Furthermore, these methods are not limited to the supraspinatus and Achilles tendons, as they are easily adapted to testing other murine tendons and tendons from larger animal models. To test tendons from larger animals, however, the modulus of the 3D printed fixture material must be high enough that it is not compliant relative to the strength of the tendon being tested.
Several studies have shown sex-based differences in tendon disorders indicating that women have reduced function following treatment after tendon injury40,41,42. In the current study, sex had a significant effect on the mechanical properties of murine tendons. As guided by the National Institutes of Health (NIH), we recommend accounting for sex as a biological variable in the research design of animal models where tendon mechanical properties will be measured.
The authors have nothing to disclose.
The study was supported by the NIH / NIAMS (R01 AR055580, R01 AR057836).
Agarose | Fisher Scientific | BP160-100 | Dissovle 1g in 100 ml ultrapure water to make 1% agarose |
Bruker microCT | Bruker BioSpin Corp | Skyscan 1272 | Used by authors |
ElectroForce | TA Instruments | 3200 | Testing platform |
Ethanol 200 Proof | Fisher Scientific | A4094 | Dilute to 70% and use as suggested in protocol |
Fixture to attach grips | Custom made | Used by authors | |
Kimwipes | Kimberly-Clark | S-8115 | As suggested in protocol |
MicroCT CT-Analyser (Ctan) | Bruker BioSpin Corp | Used by authors for visualizing and analyzing micro-CT scans | |
MilliQ water (Ultrapure water) | Millipore Sigma | QGARD00R1 (or related purifier) | 100 ml |
Meshmixer | Autodesk | http://www.meshmixer.com/ | Free engineering software used by authors to refine mesh |
Objet EDEN 260VS | Stratasys LTD | Precision Prototyping | |
Objet Studio | Stratasys LTD | Used by authors with 3D printer | |
PBS – Phosphate-Buffered Saline | ThermoFisher Scientific | 10010031 | 2.5 L of 10% PBS |
S&T Forceps | Fine Science Tools | 00108-11 | Used by authors |
Scalpel Blade – #11 | Fine Science Tools | 10011-00 | Used by authors |
Scalpel Handle – #3 | Fine Science Tools | 10003-12 | Used by authors |
SkyScan 1272 | Bruker BioSpin Corp | Used by authors for visualizing and analyzing micro-CT scans | |
Skyscan CT-Vox | Bruker BioSpin Corp | Used by authors for visualizing and analyzing micro-CT scans | |
SkyScan NRecon | Bruker BioSpin Corp | Used by authors for visualizing and analyzing micro-CT scans | |
SolidWorks CAD | Dassault Systèmes | SolidWorks Research Subsription | Solid modeling computer-aided design used by authors |
SuperGlue | Loctite | 234790 | As suggested in protocol |
Testing bath | Custom made | Used by authors | |
Thin film grips | Custom made | Used by authors | |
VeroWhitePlus | Stratasys LTD | NA | 3D printing material used by authors |
WinTest | WinTest Software | Used by authors to collect data |