The Influence of In Vitro Test Conditions on the Biomechanical Properties of Degenerated Lateral Menisci - A Ring Study

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Weiske, Oihana Piquet, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6870107/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Aug, 2025 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted 13 You are reading this latest preprint version Abstract The biomechanical properties of degenerated meniscal tissue are increasingly being studied in the context of osteoarthritis research. Spatial indentation testing using a multiaxial testing machine allows non-destructive characterization of viscoelastic properties. However, in vitro testing conditions can significantly influence the results. The purpose of this round robin study was to evaluate the effects of different fixation methods and laboratory environments on the viscoelastic properties of degenerated lateral menisci. Spatial normal indentation tests were performed on nine degenerated human lateral menisci in two laboratories using a multiaxial testing machine. Key parameters, including the maximum applied force (P max ), instantaneous modulus (IM), and elastic modulus (E t10 ), were analyzed across different meniscus regions. Significant differences in the IM, E t10 , and P max were observed between the laboratories, highlighting the influence of testing conditions on biomechanical results. The results indicated that variations in fixation methods, environmental conditions, and freeze-thaw cycles significantly affect the elastic and viscoelastic properties of meniscal tissue. Unphysiological strains in the inner region of the menisci suggested that strain-controlled indentation may be preferable to distance-controlled testing. These results underscore the importance of standardizing in vitro conditions for meaningful comparisons with the existing literature. meniscus osteoarthritis mechanical examination instantaneous modulus elastic modulus different fixation methods sGAG collagen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Knee osteoarthritis (OA) is a prevalent condition affecting millions globally [ 1 ], including 37% of persons aged 60 years and older [ 2 ], with multiple knee joint tissues implicated in its development. Furthermore, it is more common in women than in men [ 3 ]. The prevalence of OA is expected to increase with the aging populations in the U.S. [ 4 ] and Europe [ 5 ]. The meniscus plays a crucial role in load distribution and shock absorption, and its biomechanical dysfunction may accelerate OA progression [ 6 , 7 ]. Meniscal impairment results from traumatic injuries and degeneration driven by microdamage accumulation, inflammation, and aging. Recently, increasing attention has been given to tissue alterations and the molecular mechanisms underlying meniscal injury and degeneration [ 8 ]. The biomechanical properties of degenerated meniscal tissue are of increasing interest in the context of OA research [ 9 – 11 ]. In contrast to biomechanical testing of extracted samples under confined or unconfined conditions, spatial indentation mapping allows for non-destructive mechanical characterization of the complex, wedge-shaped meniscus [ 10 , 12 ]. However, when testing viscoelastic, biological tissues, the in-vitro test conditions are very likely to affect the outcome measures [ 11 , 13 ]. Neither the U.S. Food and Drug Administration nor the American Society for Testing and Materials has published guidelines for the mechanical characterization of meniscal tissue. In their systematic review, Schwer et al. derived recommendations for mechanical testing based on a quantitative analysis of the reproducibility and reliability of the reviewed studies [ 14 ]. These can be used as a guide for future investigations and provide a basis for standardization and reporting guidelines. Moreover, the C4Bio initiative [ 15 ] was recently launched to achieve a common consensus on test protocols for material characterization of biological tissues, which could facilitate the development of standards for meniscus testing and reporting recommendations. On the basis of these prior works [ 14 , 15 ], we derived the hypothesis that the in vitro testing conditions significantly influence the biomechanical parameters of lateral human menisci. Therefore, the aim of this study was to investigate the influence of different fixation methods and related laboratory environments on the elastic and viscoelastic properties of the same degenerated lateral menisci [ 10 , 11 ]. Moreover, we aimed to assess the structure-function relationship in these menisci by adding biochemical quantifications. 2 Materials and Methods 2.1 Study design After IRB approval (IRB 305/10 Albert-Ludwigs University Freiburg) spatial indentation stress relaxation tests of nine degenerated lateral human menisci (70 ± 9 years) were performed in two independent laboratories using a multiaxial testing machine (Mach-1 v500css; Biomomentum Inc.). For this purpose, indentation stress relaxation tests were performed with thickness measurements at the lateral menisci to calculate the instantaneous modulus (IM), the modulus after a relaxation time of 10 s (E t10 ), and the maximum applied load (P max ). The degeneration state of the menisci was assessed by determining the water, sulfated glycosaminoglycan (sGAG), and collagen contents in both laboratories and correlated with the biomechanical data. Nonparametric statistical and correlation analyses were performed to evaluate the results. 2.2 Biomechanical tests 2.2.1 Stress relaxation tests The lateral menisci were harvested from nine patients who were initially indicated for a total knee replacement surgery. The tissues were tested in a fresh condition at Laboratory A (Lab A, Freiburg) using a multiaxial testing machine equipped with a multiaxial load cell (70N; MA23X, ATI Industrial Automation, Apex, NC, USA)). For tissue fixation, the menisci were directly glued on the specimen holder using fully removable adhesive (Fig. 1 A) [ 9 ]. The non-residue removability was confirmed in pretests utilizing histological analyses. To ensure repeatability of the spatial mappings, both, the measurement points were marked directly on the meniscus surface using a tissue marker (Fig. 1 A) and the indentation maps of each meniscus were exported by Lab A and re-imported in the software of the testing machine by Lab B (Ulm). The indentation parameters for both test runs were as follows: indenter diameter = 1 mm, indentation depth = 0.2 mm, indentation velocity = 0.2 mm/s, relaxation time = 10 s [ 9 , 11 ]. After the first test, the menisci were frozen and shipped at − 30°C to Lab B. After thawing the menisci were embedded in a custom-made polymethylmethacrylate (PMMA) cast to confine the meniscus along its circumference (Fig. 1 B) as previously described [ 11 ]. During all tests, the samples were kept moist with saline solution. P max , as well as the IM representing the initial elastic response and the E t10 representing the initial viscous response of the meniscus tissue were determined. The IM was evaluated according to Hayes et al. [ 16 ] using the following equation for normal indentation: $$\:IM=\:\frac{P}{H}\:\bullet\:\:\frac{1-\:\vartheta\:²}{2ak\bullet\:\left(\frac{a}{h}\:\vartheta\:\right)}$$ Where IM is the instantaneous modulus; P is the load; H is the depth of indentation; a is the radius of the contact region; ϑ is the Poisson´s ratio (assumed 0.5 for soft tissues); k is the correction factor dependent on a/h and ϑ; and h is the sample thickness. 2.2.2 Thickness measurements The thickness of the menisci was determined after the second test run using a needle technique, as described by Jurvelin et al. [ 17 ]. For this measurement, the indenter was replaced with a cannula (Sterican 18G; B. Braun, Melsungen, Germany). To prevent bending and potential inaccuracies in thickness measurements, the needle was replaced after each measurement point. The testing parameters were set as follows: Contact criterion: 0.1 N (indenter–sample) Stage velocity: 0.5 mm/s Stage repositioning: 2× load resolution Termination criterion: 5 N (to protect the load cell) The same scan grid as used in the indentation test was applied. In this method, the needle’s displacement between the initial contact (0.1 N) with the meniscus tissue and the break-off contact (5 N) with the metallic base was recorded and analyzed using standard software of the multiaxial testing machine. The thickness measurement was adjusted using the cosine of the indentation angle, following the approach by Sim et al. [ 12 ] and Veronesi et al. [ 18 ]. The correction angle was automatically determined for all normal indentation positions. 2.3 Biochemical characterization The meniscus body was divided into three anatomical regions: anterior horn (AH), pars intermedia (PI), and posterior horn (PH), with a further subdivision in an inner, middle, and outer zone, leading in total to nine distinct regions (Fig. 1 C). Cylindrical samples with a diameter of 3.8 mm were collected from each region. Then, their wet weight was determined using a precision scale (AC120S, Sartorius AG, Göttingen, Germany). Consecutively the samples were lyophilized (Labconco Freezone 2.5, Labconco, Kansas City, MA, USA), dry weight and their water content calculated. Subsequently, the samples were liquefied with proteinase K and their sGAG and DNA contents were determined with the dimethyl-methylene blue (DMMB)-Assay [ 19 ]. The soluble collagen content was determined for the AH, PI and PH regions following an established protocol [ 20 ] for Lab B and on the determination of hydroxyproline in Lab A [ 21 ]. 2.4 Statistical Analyses Data were normally distributed; thus all results are presented as mean ± standard deviation. Measurements were analyzed using one-way ANOVA (Tukey posthoc test, non-parametric) at a significance level of p < 0.05. Differences in the P max , IM, and E t10 between Lab A and Lab B were analyzed for all regions using Wilcoxon testing. All statistical analyses were performed using Origin 2023 Professional SR1 (OriginLab, Northampton, MA, USA) and Graphpad Prizm 10.2.2 (Graphpad Software Inc., Boston, MA, USA). p ≤ 0.05 was considered statistically significant. 3 Results 3.1 Mechanical properties At the inner and middle meniscus zones, statistically lower values for the P max (p < 0.05) were assessed in Lab B when comparing to those of Lab A, except for the AH middle zone (Fig. 2 A). The P max of the outer zones at the AH, PI, and PH was by tendency higher in Lab B compared to Lab A. Analyzing the elastic IM, significantly lower values were found in the inner and middle PI zones and in the inner PH zone in Lab B (p < 0.05) (Fig. 2 B). In the outer PI and PH zones, the IM was statistically higher in Lab B (p < 0.05) compared to Lab A. No differences for the IM were found for the AH region. Statistically lower values for the viscoelastic E t10 were assessed in Lab B both, at the inner and middle zones of the AH, PI, and PH (p < 0.05) (Fig. 2 C). In both test environments (Lab A, Lab B) the highest P max , IM, and E t10 values were assessed in the PH region of the lateral menisci. All outer zones displayed higher E t10 values in Lab B. The measured values for P max were in a similar range (0–0.7 N) in both laboratories (Fig. 3 ). However, the samples measured by Lab A displayed higher values particularly in the inner area. In Lab A the inner zones were more loaded while in Lab B the outer zones experienced relatively more loading. These distinct differences were further analyzed by subtracting each according measurements to determine a qualitative map (Fig. 4 ). Starting from the yellow area, mainly all values related to the outer regions of the meniscus (in purple) were higher for Lab B. The middle area (dark blue) was in a similar range in both laboratories, being slightly higher for Lab A, while in the inner area (green to red) the values were clearly higher for Lab A. 3.2 Biochemical properties 3.2.1 Water content The water content was determined in both laboratories, with a mean value of 82.0 ± 2.3% in Lab A and 76.7 ± 2.9% in Lab B (Table 1 ). Table 1 Mean values of the water content in the three meniscus regions, determined in both laboratories. Meniscus compartment Mean water content [%] Lab A Lab B AH 83.0 ± 4.1 78.1 ± 1.8 PI 79.4 ± 2.8 77.4 ± 2.2 PH 83.6 ± 4.2 74.5 ± 1.8 3.2.2 sGAG The sGAG dry weight was determined in both laboratories and indicated comparable results: In detail, at the AH sGAG, determined in Lab B was 18% higher than in Lab A. The sGAG levels for the PI did not differ between the two laboratories, whereas for the PH a 42% higher value was measured in Lab B than in Lab A (Fig. 5A). No significant difference in the sGAG dry weight between the meniscal compartments was observed (p > 0.05). 3.2.3 Collagen At Laboratory A, the collagen dry weight varies between 75 and 83 µg/mg, while at Laboratory B, they ranged between 3 and 4 µg/mg. When percentages are calculated, both laboratories are in the same range, 3–5%. For the AH, Lab B determined a value of 5.2 ± 3.6% (p = 0.0001) of the collagen content as that determined by Lab A. For the PI, the collagen content determined by Lab B was 3.1 ± 2.7% (p < 0.001) of the value as that determined by laboratory A, and for the PH, 1.7 ± 1.2% (p < 0.001) of the value determined by Lab A (Fig. 5B). No significant difference was found for the collagen values determined by Lab A. The collagen content determined by Lab B indicated a significant difference between the AH and PH (p = 0.029). 4 Discussion The aim of this study was to investigate the influence of different fixation methods and related laboratory environments on the elastic and viscoelastic properties of the same degenerated lateral menisci. Moreover, the degeneration state of the meniscus samples was identified by means of biochemical quantifications. The fixation method in Lab A was based on directly gluing the menisci to the specimen holder, while in Lab B a confining cast, mad of PMMA was utilized. The results displayed higher IM values in the inner zones of the glued meniscus fixation, while the PMMA embedded menisci indicated higher IM values in the outer zones, thus corroborating our hypothesis. In addition, differences in biochemical parameters were observed which could be attributed to the additional freezing/thawing cycle as well as to different measurement techniques for the collagen content. 4.1 Mechanical properties This study indicated that both, the initial elastic and viscoelastic properties are influenced by the meniscus fixation method during indentation stress relaxation testing. At the inner zone, where the meniscus is thin, the defined indentation depth of 0.2 mm could have led to very high, unphysiologic strains in the meniscus tissue which led to higher P max and IM values (particularly for the fixation method applied in Lab A) compared to the outer zones. A similar result is seen by Pordzik et al. [ 10 ] – there, the thinner outer areas of the meniscus were rated above average in terms of IM as well. Seitz et al. [ 11 ] came to similar conclusions regarding mildly degenerated menisci. They used the same fixation method as Lab B. Fixation B confined the circumference without the need to glue the sample to the older, explaining the lower P max , IM, and E t10 values in the inner zones and slightly higher values in the outer zones. A cast would be preferable to gluing to the test holder, but it should be much less rigid than the PMMA cast used in the present study. In our opinion, a silicone cast, mimicking the confinement ability of the joint capsule might be the best choice. Furthermore, strain-controlled indentation testing would be superior for spatial indentation testing of viscoelastic tissues compared to distance-controlled testing. In this way, unphysiologically high strains could also be avoided, particularly in the case of anatomically thin structures such as the inner zone of the lateral meniscus. Furthermore, each meniscus underwent a freeze-thaw cycle and was tested twice, which is known to decrease the intrinsic compressive resistance of meniscal tissue [ 4 ], potentially impacting the biomechanical outcome parameters. Ekiert et al. [ 22 ] demonstrated a correlation between the number of freeze-thaw (F/T) cycles and the tensile properties of subcutaneous deep frontalis tendon fascicle bundles, and that as the number of F/T cycles increase, the tensile properties decrease. Following one F/T cycle, the measured values were already 8.5% less, after two cycles 14% smaller, and increasing to 19% after three cycles. This influence can be expected to be transferred to other soft tissues in a similar manner as described above, but with different characteristics, which would explain the differences in the E t10 readings between Labs A and B. 4.2 Biochemical properties The water content tended to decrease slightly after freezing in the measurements of Lab B. Other authors similarly showed that the water content may depend on the degree of degeneration [ 23 , 24 ]. Son et al. [ 25 ] determined the water content of OA menisci to be similar to ours at 79.6%, as did Morejon et al. [ 26 ] with 76.8%. Finally, while there was a slight difference in the water content between the two laboratories, no significant difference was found. Table 2 Mean water content as a percentage of the menisci of human knee joints; values from the literature in comparison with the results of the present study. Author Water content (%) Comments n total lat/med Lateral Medial Herwig et al. [ 24 ] 17/0 77.1 Increased water content as degeneration progressed Bursac et al. [ 27 ] 33/25 75.4 77.9 Healthy allograft group Son et al. [ 25 ] 13/7 79.6 78.0 OA menisci Danso et al. [ 28 ] 13/13 79 79 Seitz el al. [ 29 ] 25/25 72.2 73.3 Lewis et al. [ 13 ] 9/9 59.1 58.1 Warnecke et al. [ 23 ] 24/0 76.3 Increased water content as degeneration progressed Morejon et al. [ 26 ] 3/5 76.8 Katsuragawa et al.[ 30 ] 76.5 75.2 Nishimuta [ 31 ] Seitz et al. [ 11 ] 36/36 75.0 76.4 Severely degenerated Present study 9/0 82.0 76.7 Fresh (Lab A) Fresh frozen (Lab B) Like a previous study on lateral degenerated menisci [ 5 ], no zone-specific dependencies were found for the collagen or sGAG content. For both laboratories, the measured values for sGAG were in the same measurement range with no significant difference, since similar assays based on the DMMB assay were used (Table 3 ). The sGAG content was determined by Aggad et al. [ 32 ] also using a DMMB assay. Unfortunately, the meniscus was not divided into compartments, but is given as a total value of 3.2 ± 0.48 µg/mg. These values differ by a factor of 16–20 from our measurements (Labs A and B). However, this may be due to the fact that Aggad et al. [ 32 ] used a slightly different method. Instead of freeze-drying the samples, they digested them with papain at 65°C for 6 hours and subsequently incubated them with chondroitin lyase for 30 minutes before adding them to the DMMB complex. Rothrauff et al. [ 33 ] obtained slightly lower dry matter values for sGAG than ours, but they were still in the same range. They also performed a DMMB assay. Sanchez-Adams et al. [ 34 ] obtained a similar range as those in the present study with slightly lower values, which can be explained by using a chondroitinase assay to determine the sGAG content. Table 3 Overview of mean sGAG content in the present study compared to other studies Author sGAG content [µg/mg] Comments AH Pi PH Aggad et al. [ 32 ] 3.2 3.2 3.2 Menisci not subdivided Otani et al. [ 8 ] 18 25 30 Blyscan Glycosaminoglycan Assay Kit Rothrauff et al. [ 33 ] 31.7 31.7 31.7 Menisci not subdivided into AH/PI/PH Sanchez-Adams et al. [ 34 ] 9.1 12.0 38.8 Nishimuta et al. [ 35 ] 7 7 7 Menisci not subdivided Morejon et al. [ 26 ] 8.8 8.8 8.8 Menisci not subdivided Nishimuta et al. [ 31 ] 161 161 161 Menisci not subdivided Present study 58.8 69.3 52.0 50.5 48.6 69.3 Fresh (Lab A) Fresh frozen (Lab B) When measuring collagen content, a difference was observed between the values obtained by the two laboratories in the present study (Table 4 ). This can be attributed since Lab A used the hydroxyproline content to determine the total collagen content, whereas Lab B used a Sircol collagen assay (SCA). Lareu et al. [ 36 ] demonstrated that the SCA can overestimate collagen content by a factor of 3–24 compared to the hydroxyproline method of determining collagen content. Aggad et al. [ 32 ] also determined the amount of hydroxyproline in their study and obtained a similar order of magnitude as Lab A. However, they distinguished between males and females as well as between lateral and medial menisci, finding 84.4 ± 7.2 µg/mg in lateral menisci of males and 79.3 ± 6.7 µg/mg in females. Table 4 Overview of mean collagen content in the present study compared to other studies Author Collagen content [µg/mg] Comments AH Pi PH Aggad et al. [ 32 ] 84.4 79.3 84.4 79.3 84.4 79.3 Males (not subdivided) Females (not subdivided) Danzo et al. [ 28 ] 14 15 14 Rothrauff et al. [ 33 ] 758 758 758 Menisci not subdivided Present study 72.9 3.8 77.1 2.4 81.6 1.4 Fresh (Lab A) Fresh frozen (Lab B) 4.3 Limitations Several factors must be taken into account when assessing the reliability of the results of this study. As with other experiments on ex vivo preparations, the investigations presented here cannot replicate the complex load behavior of the natural knee joint and the circumferential strains that occur during loading. Therefore, the indentation behavior determined here cannot be equated with that of an intact joint with menisci. The indentation properties are influenced by the surface layer and the locally defined area around the indenter. Nevertheless, indentation is more suitable for material characterization than other methods (e.g., punching and compression tests) because it allows us to image the entire meniscus and not just parts of it (punching out, which may have been shortened to make them plane-parallel). The current settings of the Mach 1 software do not allow the influence of the substrate to be minimized, especially when only distances can be entered. This results in thin areas of the meniscus being indented disproportionately more than thicker areas. In extreme cases, this can also lead to the substrate being measured, resulting in measurements that are too large. Furthermore, indentation mapping of the entire meniscus surface allows for high spatial resolution and non-destructive testing but leads to prolonged testing times with autolysis. 5 Conclusions This round robin study showed that different in vitro test conditions (fixation methods, environmental conditions, freeze-thaw cycle) are affecting both the initial elastic and viscoelastic as well as biochemical properties of degenerated human meniscus tissue. Therefore, caution is advised when comparing one’s own results with those described in the literature. This is most relevant if the results are to be used as a basis for later measurements. These results are valuable for researchers in the field of soft tissue biomechanics. They can be used to advance the standardization of soft tissue measurements, creating a uniform basis for all researchers. Additionally, readers should be better informed about how to interpret the obtained data. Declarations Ethics statement The studies involving human participants were reviewed and approved by IRB 305/10 Albert-Ludwigs University Freiburg. The patients/participants provided written informed consent to participate in this study. Author contributions MS and AMS conceived the study. HOM participated in the design and coordination of the study. MS, MFW, LdR, BR, OP, and GT performed the preparation procedure, mechanical testing, data analysis, and statistics. MS and LdR drafted the manuscript. AMS helped in manuscript writing. All authors read and approved the final manuscript. Funding The processing fee for the article was financed by the Ministry of Science, Research, and the Arts of Baden-Württemberg and the University of Freiburg as part of the Open Access Publishing funding program. Conflict of interest The authors declare no conflict of interest. Data Availability Statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Acknowledgements The authors would like to thank Melanie Lynn Hart, a native speaker, for spell-checking and grammar correction. References Hunter DJ, Bierma-Zeinstra S, Osteoarthritis. Lancet 2019, 393 , 1745-1759.10.1016/s0140-6736(19)30417-9. Dillon CF, Rasch EK, Gu Q, Hirsch R. Prevalence of knee osteoarthritis in the united states: Arthritis data from the third national health and nutrition examination survey 1991-94. 2006, 33 , 2271–9. Nguyen U-SDT, Zhang Y, Zhu Y, Niu J, Zhang B, Felson DT. Increasing prevalence of knee pain and symptomatic knee osteoarthritis: Survey and cohort data. Annal Interal Med. 2011;155:725–32. https://doi.org/10.7326/0003-4819-155-11-201112060-00004 . Gill TM. Do the tenets of late-life disability apply to middle age? Ann. Intern. Med. 2017, 167 , 818-819.10.7326/m17-2550. Hitzl W, Stamm T, Kloppenburg M, Ritter M, Gaisberger M, van der Zee-Neuen A. Projected number of osteoarthritis patients in austria for the next decades – quantifying the necessity of treatment and prevention strategies in europe. BMC Musculoskel. Disord. 2022, 23 , 133.10.1186/s12891-022-05091-5. Walker PS, Erkman MJ. The role of the menisci in force transmission across the knee. Clin. Orthop. Relat. Res. 1975, 184-192.10.1097/00003086-197506000-00027. Kurosawa H, Fukubayashi T, Nakajima H. Load-bearing mode of the knee joint: Physical behavior of the knee joint with or without menisci. Clin Orthop Relat Res 1980, 283–90. Otani S, Kanamoto T, Oyama S, Yamakawa S, Shi W, Miyazaki R, Aihara M, Oka S, Kuroda S, Nakai T et al. , . Meniscus surface texture is associated with degenerative changes in biological and biomechanical properties. Sci. Rep. 2022, 12, 11977.10.1038/s41598-022-16206-2 Pordzik J, Bernstein A, Watrinet J, Mayr HO, Latorre SH, Schmal H, Seidenstuecker M. Correlation of biomechanical alterations under gonarthritis between overlying menisci and articular cartilage. Appl Sci. 2020;10:8673. .https://doi.org/10.3390/app10238673 . Pordzik J, Bernstein A, Mayr HO, Latorre SH, Maks A, Schmal H, Seidenstuecker M. Analysis of proteoglycan content and biomechanical properties in arthritic and arthritis-free menisci. Appl Sci. 2020;10:9012. .https://doi.org/10.3390/app10249012 . Seitz AM, Osthaus F, Schwer J, Warnecke D, Faschingbauer M, Sgroi M, Ignatius A, Dürselen L. Osteoarthritis-related degeneration alters the biomechanical properties of human menisci before the articular cartilage. 2021, 9 .10.3389/fbioe.2021.659989. Sim S, Chevrier A, Garon M, Quenneville E, Lavigne P, Yaroshinsky A, Hoemann CD, Buschmann MD. Electromechanical probe and automated indentation maps are sensitive techniques in assessing early degenerated human articular cartilage. J. Orth. Res. 2017, 35 , 858-867.10.1002/jor.23330. Lewis PB, Williams JM, Hallab N, Virdi A, Yanke A, Cole BJ. Multiple freeze-thaw cycled meniscal allograft tissue: A biomechanical, biochemical, and histologic analysis. 2007, 26 , 49–55 https://doi.org/10.1002/jor.20473 Schwer J, Ignatius A, Seitz AM. The biomechanical properties of human menisci: A systematic review. Acta Biomater. 2024;175:1–26. https://doi.org/10.1016/j.actbio.2023.12.010 . C4BioCommunity. Challenge towards consensus on characterization of biological tissue. https://c4bio.eu/ (March 31),. Hayes WC, Keer LM, Herrmann G, Mockros LF. A mathematical analysis for indentation tests of articular cartilage. J Biomech. 1972;5:541–51. Jurvelin JS, Räsänen T, Kolmonen P, Lyyra T. Comparison of optical, needle probe and ultrasonic techniques for the measurement of articular cartilage thickness. J. Biomech. 1995, 28 , 231-235.10.1016/0021-9290(94)00060-h. Veronesi F, Berni M, Marchiori G, Cassiolas G, Muttini A, Barboni B, Martini L, Fini M, Lopomo NF, Marcacci M et al. , . Evaluation of cartilage biomechanics and knee joint microenvironment after different cell-based treatments in a sheep model of early osteoarthritis. Int. Orthop. 2021, 45, 427-435.10.1007/s00264-020-04701-y Ferreira JR, Teixeira GQ, Neto E, Ribeiro-Machado C, Silva AM, Caledeira J, Leite Pereira C, Bidarra S, Maia AF, Lamghari M et al. , . Il-1β-pre-conditioned mesenchymal stem/stromal cells’ secretome modulates the inflammatory response and aggrecan deposition in the intervertebral disc. Eur Cell Mater 2021, 41. https://doi.org/10.22203/eCM.v041a28 Neidlinger-Wilke C, Ekkerlein A, Goncalves RM, Ferreira JM, Ignatius A, Wilke HJ, Teixeira GQ. Mesenchymal stem cell secretome decreases the inflammatory response in annulus fibrosus organ cultures. Eur Cell Mater. 2021;42:1–19. https://doi.org/10.22203/eCM . Ignat’eva NY, Danilov NA, Averkiev SV, Obrezkova MV, Lunin VV, Sobol’ EN. Determination of hydroxyproline in tissues and the evaluation of the collagen content of the tissues. Journal of Analytical Chemistry 2007, 62 , 51-57.10.1134/S106193480701011X. Ekiert M, Karbowniczek J, Stachewicz U, Mlyniec A. The effect of multiple freeze-thaw cycles on the viscoelastic properties and microstructure of bovine superficial digital flexor tendon. J Mech Behav Biomed Mater. 2021;120:104582. .https://doi.org/10.1016/j.jmbbm.2021.104582 . Warnecke D, Balko J, Haas J, Bieger R, Leucht F, Wolf N, Schild N, Stein S, Seitz A, Ignatius A, et al. Degeneration alters the biomechanical properties and structural composition of lateral human menisci. Osteoarthritis Cartilage. 2020. https://doi.org/10.1016/j.joca.2020.07.004 . Herwig J, Egner E, Buddecke E. Chemical changes of human knee joint menisci in various stages of degeneration. Ann Rheum Dis. 1984;43:635–40. https://doi.org/10.1136/ard.43.4.635 . Son M, Goodman SB, Chen W, Hargreaves BA, Gold GE, Levenston ME. Regional variation in t1ρ and t2 times in osteoarthritic human menisci: Correlation with mechanical properties and matrix composition. Osteoarthritis Cartilage. 2013;21:796–805. https://doi.org/10.1016/j.joca.2013.03.002 . Morejon A, Norberg CD, De Rosa M, Best TM, Jackson AR, Travascio F. Compressive properties and hydraulic permeability of human meniscus: Relationships with tissue structure and composition. 2021, 8 .10.3389/fbioe.2020.622552. Bursac P, Arnoczky S, York A. Dynamic compressive behavior of human meniscus correlates with its extra-cellular matrix composition. Biorheology 2009, 46 , 227-237.10.3233/BIR-2009-0537. Danso EK, Mäkelä JT, Tanska P, Mononen ME, Honkanen JT, Jurvelin JS, Töyräs J, Julkunen P, Korhonen RK. Characterization of site-specific biomechanical properties of human meniscus-importance of collagen and fluid on mechanical nonlinearities. J. Biomech. 2015, 48 , 1499-1507.10.1016/j.jbiomech.2015.01.048. Seitz AM, Galbusera F, Krais C, Ignatius A, Durselen L. Stress-relaxation response of human menisci under confined compression conditions. J Mech Behav Biomed Mater 2013, 26 , 68-80.10.1016/j.jmbbm.2013.05.027. Katsuragawa Y, Saitoh K, Tanaka N, Wake M, Ikeda Y, Furukawa H, Tohma S, Sawabe M, Ishiyama M, Yagishita S, et al. Changes of human menisci in osteoarthritic knee joints. Osteoarthritis Cartilage. 2010;18:1133–43. https://doi.org/10.1016/j.joca.2010.05.017 . Nishimuta JF, Levenston ME. Adipokines induce catabolism of newly synthesized matrix in cartilage and meniscus tissues. Connect. Tissue Res. 2017, 58 , 246-258.10.1080/03008207.2017.1281258. Aggad W, Abd El-Aziz G, Halawi M, Hindi E, AlShali R, Hamdy R, Saleh H. Dimorphic comparative histological and histometric study of the lateral and medial knee menisci in male and female human cadavers. Eur J Anat. 2024;28:109–23. https://doi.org/10.52083/ZCPM8648 . Rothrauff BB, Shimomura K, Gottardi R, Alexander PG, Tuan RS. Anatomical region-dependent enhancement of 3-dimensional chondrogenic differentiation of human mesenchymal stem cells by soluble meniscus extracellular matrix. Acta Biomater. 2017;49:140–51. https://doi.org/10.1016/j.actbio.2016.11.046 . Sanchez-Adams J, Willard VP, Athanasiou KA. Regional variation in the mechanical role of knee meniscus glycosaminoglycans. 2011, 111 , 1590-1596.10.1152/japplphysiol.00848.2011. Nishimuta JF, Levenston ME. Meniscus is more susceptible than cartilage to catabolic and anti-anabolic effects of adipokines. Osteoarthritis Cartilage. 2015;23:1551–62. https://doi.org/10.1016/j.joca.2015.04.014 . Lareu RR, Zeugolis DI, Abu-Rub M, Pandit A, Raghunath M. Essential modification of the sircol collagen assay for the accurate quantification of collagen content in complex protein solutions. Acta Biomater. 2010;6:3146–51. https://doi.org/10.1016/j.actbio.2010.02.004 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 06 Aug, 2025 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted Editorial decision: Revision requested 25 Jun, 2025 Reviews received at journal 25 Jun, 2025 Reviews received at journal 19 Jun, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviews received at journal 16 Jun, 2025 Reviews received at journal 15 Jun, 2025 Reviewers agreed at journal 15 Jun, 2025 Reviewers agreed at journal 14 Jun, 2025 Reviewers agreed at journal 13 Jun, 2025 Reviewers invited by journal 13 Jun, 2025 Editor assigned by journal 12 Jun, 2025 Submission checks completed at journal 12 Jun, 2025 First submitted to journal 11 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6870107","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472070687,"identity":"8a83a374-d997-4921-ab9c-938c53b5110f","order_by":0,"name":"Michael Seidenstuecker","email":"data:image/png;base64,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","orcid":"","institution":"G.E.R.N. Research Center for Tissue Replacement, Regeneration \u0026 Neogenesis, Department of Orthopedics and Trauma Surgery, Faculty of Medicine, Medical Center University of Freiburg","correspondingAuthor":true,"prefix":"","firstName":"Michael","middleName":"","lastName":"Seidenstuecker","suffix":""},{"id":472070690,"identity":"1d44654f-f36f-4987-91e5-1a0166dc44db","order_by":1,"name":"Lisa de Roy","email":"","orcid":"","institution":"Ulm University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"de Roy","suffix":""},{"id":472070692,"identity":"8c914e73-f498-44dd-8980-ea930e887e11","order_by":2,"name":"Max F. Weiske","email":"","orcid":"","institution":"G.E.R.N. Research Center for Tissue Replacement, Regeneration \u0026 Neogenesis, Department of Orthopedics and Trauma Surgery, Faculty of Medicine, Medical Center University of Freiburg","correspondingAuthor":false,"prefix":"","firstName":"Max","middleName":"F.","lastName":"Weiske","suffix":""},{"id":472070694,"identity":"da48231b-682c-476d-b80e-39539225e787","order_by":3,"name":"Oihana Piquet","email":"","orcid":"","institution":"Ulm University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Oihana","middleName":"","lastName":"Piquet","suffix":""},{"id":472070697,"identity":"cef5bcff-ab77-420b-8d72-503ee847280d","order_by":4,"name":"Graciose Q. Teixeira","email":"","orcid":"","institution":"Ulm University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Graciose","middleName":"Q.","lastName":"Teixeira","suffix":""},{"id":472070708,"identity":"c44d0214-f91f-4f66-b501-0b8ad3febf86","order_by":5,"name":"Hermann O. Mayr","email":"","orcid":"","institution":"G.E.R.N. Research Center for Tissue Replacement, Regeneration \u0026 Neogenesis, Department of Orthopedics and Trauma Surgery, Faculty of Medicine, Medical Center University of Freiburg","correspondingAuthor":false,"prefix":"","firstName":"Hermann","middleName":"O.","lastName":"Mayr","suffix":""},{"id":472070714,"identity":"7c081ff0-179a-47cd-bf8b-16df0c1c855e","order_by":6,"name":"Bianca Riedel","email":"","orcid":"","institution":"G.E.R.N. Research Center for Tissue Replacement, Regeneration \u0026 Neogenesis, Department of Orthopedics and Trauma Surgery, Faculty of Medicine, Medical Center University of Freiburg","correspondingAuthor":false,"prefix":"","firstName":"Bianca","middleName":"","lastName":"Riedel","suffix":""},{"id":472070716,"identity":"0e490d72-19b0-4e1e-a3bb-cf9173bf1118","order_by":7,"name":"Andreas M. Seitz","email":"","orcid":"","institution":"Ulm University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"M.","lastName":"Seitz","suffix":""}],"badges":[],"createdAt":"2025-06-11 09:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6870107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6870107/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13018-025-06151-x","type":"published","date":"2025-08-06T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84756921,"identity":"114867d8-c525-4405-bf26-d039448ff4eb","added_by":"auto","created_at":"2025-06-17 04:52:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":537538,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Direct gluing of the meniscus to the specimen holder (Lab A); (B) PMMA customized confinement cast for each meniscus (Lab B); and (C) schematic representation of nine evaluated meniscus regions: anterior horn (AH); pars intermedia (PI); and posterior horn (PH)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6870107/v1/ab8d7fa458679a1ba4bb57a0.png"},{"id":84756920,"identity":"20f2a3e7-0224-45e5-8993-dfec0ca40bf6","added_by":"auto","created_at":"2025-06-17 04:52:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22316,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Maximum applied force (P\u003csub\u003emax\u003c/sub\u003e) in N, (B) instantaneous modulus (IM) in MPa, and (C) relaxation modulus (E\u003csub\u003et10\u003c/sub\u003e) in MPa obtained during the indentation measurements in both laboratories. Anatomical subdivision into the snpreviously assigned nine regions (anterior horn (AH); pars intermedia (PI), and posterior horn (PH) and further subdivision into the inner (Inner), middle (Mid), and outer (Out) region). n=9; non-parametric statistics; *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6870107/v1/80782e8ab042cbc3b5f39599.png"},{"id":84757568,"identity":"97231f44-5b4d-4296-92aa-8fed47623363","added_by":"auto","created_at":"2025-06-17 05:00:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39403,"visible":true,"origin":"","legend":"\u003cp\u003eExemplary mapping of the IM for both fixation methods. Lab A: Fixation with fully removable adhesive on the sample holder; Lab B: sample-specific PMMA substrate.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6870107/v1/7e750e4b464688aadb7f7006.jpg"},{"id":84756924,"identity":"1574b014-3669-471e-8332-dfac6519d619","added_by":"auto","created_at":"2025-06-17 04:52:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":309825,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Comparison of mean differences (Delta Values = (IM value Lab A) – (IM value Lab B)) of IM measurements in kPa in each meniscus region; (B) Quantitative reflection of the measurement differences to the nine different zones.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6870107/v1/6b5d87f472f9e3cd4256684c.png"},{"id":84756925,"identity":"330546e0-d484-4eeb-8469-448574de7a08","added_by":"auto","created_at":"2025-06-17 04:52:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22070,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplots indicating max, min, 75% and 25% percentiles and median values and of the (A) sGAG and (B) collagen dry weight; n = 9; Tukey posthoc test, non-parametric.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6870107/v1/12d1dfb3a09dc3fce5faf457.png"},{"id":88814142,"identity":"e0b29f24-367a-46f0-8e73-f3b80b448d1b","added_by":"auto","created_at":"2025-08-11 16:07:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1960123,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6870107/v1/6ebd77ff-3c39-462e-a7d0-76418623e306.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Influence of In Vitro Test Conditions on the Biomechanical Properties of Degenerated Lateral Menisci - A Ring Study","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eKnee osteoarthritis (OA) is a prevalent condition affecting millions globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], including 37% of persons aged 60 years and older [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], with multiple knee joint tissues implicated in its development. Furthermore, it is more common in women than in men [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The prevalence of OA is expected to increase with the aging populations in the U.S. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and Europe [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The meniscus plays a crucial role in load distribution and shock absorption, and its biomechanical dysfunction may accelerate OA progression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Meniscal impairment results from traumatic injuries and degeneration driven by microdamage accumulation, inflammation, and aging. Recently, increasing attention has been given to tissue alterations and the molecular mechanisms underlying meniscal injury and degeneration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The biomechanical properties of degenerated meniscal tissue are of increasing interest in the context of OA research [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast to biomechanical testing of extracted samples under confined or unconfined conditions, spatial indentation mapping allows for non-destructive mechanical characterization of the complex, wedge-shaped meniscus [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, when testing viscoelastic, biological tissues, the in-vitro test conditions are very likely to affect the outcome measures [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Neither the U.S. Food and Drug Administration nor the American Society for Testing and Materials has published guidelines for the mechanical characterization of meniscal tissue. In their systematic review, Schwer et al. derived recommendations for mechanical testing based on a quantitative analysis of the reproducibility and reliability of the reviewed studies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These can be used as a guide for future investigations and provide a basis for standardization and reporting guidelines. Moreover, the C4Bio initiative [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] was recently launched to achieve a common consensus on test protocols for material characterization of biological tissues, which could facilitate the development of standards for meniscus testing and reporting recommendations. On the basis of these prior works [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], we derived the hypothesis that the in vitro testing conditions significantly influence the biomechanical parameters of lateral human menisci. Therefore, the aim of this study was to investigate the influence of different fixation methods and related laboratory environments on the elastic and viscoelastic properties of the same degenerated lateral menisci [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, we aimed to assess the structure-function relationship in these menisci by adding biochemical quantifications.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study design\u003c/h2\u003e \u003cp\u003eAfter IRB approval (IRB 305/10 Albert-Ludwigs University Freiburg) spatial indentation stress relaxation tests of nine degenerated lateral human menisci (70\u0026thinsp;\u0026plusmn;\u0026thinsp;9 years) were performed in two independent laboratories using a multiaxial testing machine (Mach-1 v500css; Biomomentum Inc.). For this purpose, indentation stress relaxation tests were performed with thickness measurements at the lateral menisci to calculate the instantaneous modulus (IM), the modulus after a relaxation time of 10 s (E\u003csub\u003et10\u003c/sub\u003e), and the maximum applied load (P\u003csub\u003emax\u003c/sub\u003e). The degeneration state of the menisci was assessed by determining the water, sulfated glycosaminoglycan (sGAG), and collagen contents in both laboratories and correlated with the biomechanical data. Nonparametric statistical and correlation analyses were performed to evaluate the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Biomechanical tests\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Stress relaxation tests\u003c/h2\u003e \u003cp\u003eThe lateral menisci were harvested from nine patients who were initially indicated for a total knee replacement surgery. The tissues were tested in a fresh condition at Laboratory A (Lab A, Freiburg) using a multiaxial testing machine equipped with a multiaxial load cell (70N; MA23X, ATI Industrial Automation, Apex, NC, USA)). For tissue fixation, the menisci were directly glued on the specimen holder using fully removable adhesive (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The non-residue removability was confirmed in pretests utilizing histological analyses. To ensure repeatability of the spatial mappings, both, the measurement points were marked directly on the meniscus surface using a tissue marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and the indentation maps of each meniscus were exported by Lab A and re-imported in the software of the testing machine by Lab B (Ulm). The indentation parameters for both test runs were as follows: indenter diameter\u0026thinsp;=\u0026thinsp;1 mm, indentation depth\u0026thinsp;=\u0026thinsp;0.2 mm, indentation velocity\u0026thinsp;=\u0026thinsp;0.2 mm/s, relaxation time\u0026thinsp;=\u0026thinsp;10 s [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. After the first test, the menisci were frozen and shipped at \u0026minus;\u0026thinsp;30\u0026deg;C to Lab B. After thawing the menisci were embedded in a custom-made polymethylmethacrylate (PMMA) cast to confine the meniscus along its circumference (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) as previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. During all tests, the samples were kept moist with saline solution. P\u003csub\u003emax\u003c/sub\u003e, as well as the IM representing the initial elastic response and the E\u003csub\u003et10\u003c/sub\u003e representing the initial viscous response of the meniscus tissue were determined. The IM was evaluated according to Hayes et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] using the following equation for normal indentation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:IM=\\:\\frac{P}{H}\\:\\bullet\\:\\:\\frac{1-\\:\\vartheta\\:\u0026sup2;}{2ak\\bullet\\:\\left(\\frac{a}{h}\\:\\vartheta\\:\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere IM is the instantaneous modulus; P is the load; H is the depth of indentation; a is the radius of the contact region; ϑ is the Poisson\u0026acute;s ratio (assumed 0.5 for soft tissues); k is the correction factor dependent on a/h and ϑ; and h is the sample thickness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Thickness measurements\u003c/h2\u003e \u003cp\u003eThe thickness of the menisci was determined after the second test run using a needle technique, as described by Jurvelin et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For this measurement, the indenter was replaced with a cannula (Sterican 18G; B. Braun, Melsungen, Germany). To prevent bending and potential inaccuracies in thickness measurements, the needle was replaced after each measurement point. The testing parameters were set as follows:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eContact criterion: 0.1 N (indenter\u0026ndash;sample)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eStage velocity: 0.5 mm/s\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eStage repositioning: 2\u0026times; load resolution\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eTermination criterion: 5 N (to protect the load cell)\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe same scan grid as used in the indentation test was applied. In this method, the needle\u0026rsquo;s displacement between the initial contact (0.1 N) with the meniscus tissue and the break-off contact (5 N) with the metallic base was recorded and analyzed using standard software of the multiaxial testing machine. The thickness measurement was adjusted using the cosine of the indentation angle, following the approach by Sim et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and Veronesi et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The correction angle was automatically determined for all normal indentation positions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Biochemical characterization\u003c/h2\u003e \u003cp\u003eThe meniscus body was divided into three anatomical regions: anterior horn (AH), pars intermedia (PI), and posterior horn (PH), with a further subdivision in an inner, middle, and outer zone, leading in total to nine distinct regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Cylindrical samples with a diameter of 3.8 mm were collected from each region. Then, their wet weight was determined using a precision scale (AC120S, Sartorius AG, G\u0026ouml;ttingen, Germany). Consecutively the samples were lyophilized (Labconco Freezone 2.5, Labconco, Kansas City, MA, USA), dry weight and their water content calculated. Subsequently, the samples were liquefied with proteinase K and their sGAG and DNA contents were determined with the dimethyl-methylene blue (DMMB)-Assay [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The soluble collagen content was determined for the AH, PI and PH regions following an established protocol [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] for Lab B and on the determination of hydroxyproline in Lab A [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Statistical Analyses\u003c/h2\u003e \u003cp\u003eData were normally distributed; thus all results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Measurements were analyzed using one-way ANOVA (Tukey posthoc test, non-parametric) at a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Differences in the P\u003csub\u003emax\u003c/sub\u003e, IM, and E\u003csub\u003et10\u003c/sub\u003e between Lab A and Lab B were analyzed for all regions using Wilcoxon testing. All statistical analyses were performed using Origin 2023 Professional SR1 (OriginLab, Northampton, MA, USA) and Graphpad Prizm 10.2.2 (Graphpad Software Inc., Boston, MA, USA). p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Mechanical properties\u003c/h2\u003e\n \u003cp\u003eAt the inner and middle meniscus zones, statistically lower values for the P\u003csub\u003emax\u003c/sub\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were assessed in Lab B when comparing to those of Lab A, except for the AH middle zone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The P\u003csub\u003emax\u003c/sub\u003e of the outer zones at the AH, PI, and PH was by tendency higher in Lab B compared to Lab A. Analyzing the elastic IM, significantly lower values were found in the inner and middle PI zones and in the inner PH zone in Lab B (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). In the outer PI and PH zones, the IM was statistically higher in Lab B (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to Lab A. No differences for the IM were found for the AH region. Statistically lower values for the viscoelastic E\u003csub\u003et10\u003c/sub\u003e were assessed in Lab B both, at the inner and middle zones of the AH, PI, and PH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). In both test environments (Lab A, Lab B) the highest P\u003csub\u003emax\u003c/sub\u003e, IM, and E\u003csub\u003et10\u003c/sub\u003e values were assessed in the PH region of the lateral menisci. All outer zones displayed higher E\u003csub\u003et10\u003c/sub\u003e values in Lab B.\u003c/p\u003e\n \u003cp\u003eThe measured values for P\u003csub\u003emax\u003c/sub\u003e were in a similar range (0\u0026ndash;0.7 N) in both laboratories (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the samples measured by Lab A displayed higher values particularly in the inner area.\u003c/p\u003e\n \u003cp\u003eIn Lab A the inner zones were more loaded while in Lab B the outer zones experienced relatively more loading. These distinct differences were further analyzed by subtracting each according measurements to determine a qualitative map (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Starting from the yellow area, mainly all values related to the outer regions of the meniscus (in purple) were higher for Lab B. The middle area (dark blue) was in a similar range in both laboratories, being slightly higher for Lab A, while in the inner area (green to red) the values were clearly higher for Lab A.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Biochemical properties\u003c/h2\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1 Water content\u003c/h2\u003e\n \u003cp\u003eThe water content was determined in both laboratories, with a mean value of 82.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% in Lab A and 76.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9% in Lab B (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMean values of the water content in the three meniscus regions, determined in both laboratories.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eMeniscus\u003c/p\u003e\n \u003cp\u003ecompartment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eMean water content [%]\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLab A\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLab B\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e78.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e77.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2 sGAG\u003c/h2\u003e\n \u003cp\u003eThe sGAG dry weight was determined in both laboratories and indicated comparable results: In detail, at the AH sGAG, determined in Lab B was 18% higher than in Lab A. The sGAG levels for the PI did not differ between the two laboratories, whereas for the PH a 42% higher value was measured in Lab B than in Lab A (Fig.\u0026nbsp;5A). No significant difference in the sGAG dry weight between the meniscal compartments was observed (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.3 Collagen\u003c/h2\u003e\n \u003cp\u003eAt Laboratory A, the collagen dry weight varies between 75 and 83 \u0026micro;g/mg, while at Laboratory B, they ranged between 3 and 4 \u0026micro;g/mg. When percentages are calculated, both laboratories are in the same range, 3\u0026ndash;5%. For the AH, Lab B determined a value of 5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6% (p\u0026thinsp;=\u0026thinsp;0.0001) of the collagen content as that determined by Lab A. For the PI, the collagen content determined by Lab B was 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of the value as that determined by laboratory A, and for the PH, 1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of the value determined by Lab A (Fig.\u0026nbsp;5B). No significant difference was found for the collagen values determined by Lab A. The collagen content determined by Lab B indicated a significant difference between the AH and PH (p\u0026thinsp;=\u0026thinsp;0.029).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe aim of this study was to investigate the influence of different fixation methods and related laboratory environments on the elastic and viscoelastic properties of the same degenerated lateral menisci. Moreover, the degeneration state of the meniscus samples was identified by means of biochemical quantifications. The fixation method in Lab A was based on directly gluing the menisci to the specimen holder, while in Lab B a confining cast, mad of PMMA was utilized. The results displayed higher IM values in the inner zones of the glued meniscus fixation, while the PMMA embedded menisci indicated higher IM values in the outer zones, thus corroborating our hypothesis. In addition, differences in biochemical parameters were observed which could be attributed to the additional freezing/thawing cycle as well as to different measurement techniques for the collagen content.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Mechanical properties\u003c/h2\u003e \u003cp\u003eThis study indicated that both, the initial elastic and viscoelastic properties are influenced by the meniscus fixation method during indentation stress relaxation testing. At the inner zone, where the meniscus is thin, the defined indentation depth of 0.2 mm could have led to very high, unphysiologic strains in the meniscus tissue which led to higher P\u003csub\u003emax\u003c/sub\u003e and IM values (particularly for the fixation method applied in Lab A) compared to the outer zones. A similar result is seen by Pordzik et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] \u0026ndash; there, the thinner outer areas of the meniscus were rated above average in terms of IM as well. Seitz et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] came to similar conclusions regarding mildly degenerated menisci. They used the same fixation method as Lab B. Fixation B confined the circumference without the need to glue the sample to the older, explaining the lower P\u003csub\u003emax\u003c/sub\u003e, IM, and E\u003csub\u003et10\u003c/sub\u003e values in the inner zones and slightly higher values in the outer zones. A cast would be preferable to gluing to the test holder, but it should be much less rigid than the PMMA cast used in the present study. In our opinion, a silicone cast, mimicking the confinement ability of the joint capsule might be the best choice. Furthermore, strain-controlled indentation testing would be superior for spatial indentation testing of viscoelastic tissues compared to distance-controlled testing. In this way, unphysiologically high strains could also be avoided, particularly in the case of anatomically thin structures such as the inner zone of the lateral meniscus. Furthermore, each meniscus underwent a freeze-thaw cycle and was tested twice, which is known to decrease the intrinsic compressive resistance of meniscal tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], potentially impacting the biomechanical outcome parameters. Ekiert et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] demonstrated a correlation between the number of freeze-thaw (F/T) cycles and the tensile properties of subcutaneous deep frontalis tendon fascicle bundles, and that as the number of F/T cycles increase, the tensile properties decrease. Following one F/T cycle, the measured values were already 8.5% less, after two cycles 14% smaller, and increasing to 19% after three cycles. This influence can be expected to be transferred to other soft tissues in a similar manner as described above, but with different characteristics, which would explain the differences in the E\u003csub\u003et10\u003c/sub\u003e readings between Labs A and B.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Biochemical properties\u003c/h2\u003e \u003cp\u003eThe water content tended to decrease slightly after freezing in the measurements of Lab B. Other authors similarly showed that the water content may depend on the degree of degeneration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Son et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] determined the water content of OA menisci to be similar to ours at 79.6%, as did Morejon et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] with 76.8%. Finally, while there was a slight difference in the water content between the two laboratories, no significant difference was found.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean water content as a percentage of the menisci of human knee joints; values from the literature in comparison with the results of the present study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAuthor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eWater content (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eComments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e \u003cp\u003elat/med\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLateral\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMedial\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHerwig et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncreased water content as degeneration progressed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBursac et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33/25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHealthy allograft group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSon et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOA menisci\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDanso et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeitz el al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25/25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLewis et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWarnecke et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncreased water content as degeneration progressed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorejon et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKatsuragawa et al.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNishimuta [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeitz et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36/36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSeverely degenerated\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresent study\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.0\u003c/p\u003e \u003cp\u003e76.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFresh (Lab A)\u003c/p\u003e \u003cp\u003eFresh frozen (Lab B)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLike a previous study on lateral degenerated menisci [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], no zone-specific dependencies were found for the collagen or sGAG content. For both laboratories, the measured values for sGAG were in the same measurement range with no significant difference, since similar assays based on the DMMB assay were used (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The sGAG content was determined by Aggad et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] also using a DMMB assay. Unfortunately, the meniscus was not divided into compartments, but is given as a total value of 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 \u0026micro;g/mg. These values differ by a factor of 16\u0026ndash;20 from our measurements (Labs A and B). However, this may be due to the fact that Aggad et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] used a slightly different method. Instead of freeze-drying the samples, they digested them with papain at 65\u0026deg;C for 6 hours and subsequently incubated them with chondroitin lyase for 30 minutes before adding them to the DMMB complex. Rothrauff et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] obtained slightly lower dry matter values for sGAG than ours, but they were still in the same range. They also performed a DMMB assay. Sanchez-Adams et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] obtained a similar range as those in the present study with slightly lower values, which can be explained by using a chondroitinase assay to determine the sGAG content.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverview of mean sGAG content in the present study compared to other studies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAuthor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003esGAG content [\u0026micro;g/mg]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eComments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAggad et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMenisci not subdivided\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOtani et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBlyscan Glycosaminoglycan Assay Kit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRothrauff et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMenisci not subdivided into AH/PI/PH\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSanchez-Adams et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNishimuta et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMenisci not subdivided\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorejon et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMenisci not subdivided\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNishimuta et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMenisci not subdivided\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresent study\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.8\u003c/p\u003e \u003cp\u003e69.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.0\u003c/p\u003e \u003cp\u003e50.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.6\u003c/p\u003e \u003cp\u003e69.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFresh (Lab A)\u003c/p\u003e \u003cp\u003eFresh frozen (Lab B)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhen measuring collagen content, a difference was observed between the values obtained by the two laboratories in the present study (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This can be attributed since Lab A used the hydroxyproline content to determine the total collagen content, whereas Lab B used a Sircol collagen assay (SCA). Lareu et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] demonstrated that the SCA can overestimate collagen content by a factor of 3\u0026ndash;24 compared to the hydroxyproline method of determining collagen content. Aggad et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] also determined the amount of hydroxyproline in their study and obtained a similar order of magnitude as Lab A. However, they distinguished between males and females as well as between lateral and medial menisci, finding 84.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2 \u0026micro;g/mg in lateral menisci of males and 79.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7 \u0026micro;g/mg in females.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverview of mean collagen content in the present study compared to other studies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAuthor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eCollagen content [\u0026micro;g/mg]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eComments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAggad et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.4\u003c/p\u003e \u003cp\u003e79.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84.4\u003c/p\u003e \u003cp\u003e79.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84.4\u003c/p\u003e \u003cp\u003e79.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMales (not subdivided)\u003c/p\u003e \u003cp\u003eFemales (not subdivided)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDanzo et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRothrauff et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMenisci not subdivided\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresent study\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.9\u003c/p\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.1\u003c/p\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81.6\u003c/p\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFresh (Lab A)\u003c/p\u003e \u003cp\u003eFresh frozen (Lab B)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Limitations\u003c/h2\u003e \u003cp\u003eSeveral factors must be taken into account when assessing the reliability of the results of this study. As with other experiments on ex vivo preparations, the investigations presented here cannot replicate the complex load behavior of the natural knee joint and the circumferential strains that occur during loading. Therefore, the indentation behavior determined here cannot be equated with that of an intact joint with menisci. The indentation properties are influenced by the surface layer and the locally defined area around the indenter. Nevertheless, indentation is more suitable for material characterization than other methods (e.g., punching and compression tests) because it allows us to image the entire meniscus and not just parts of it (punching out, which may have been shortened to make them plane-parallel). The current settings of the Mach 1 software do not allow the influence of the substrate to be minimized, especially when only distances can be entered. This results in thin areas of the meniscus being indented disproportionately more than thicker areas. In extreme cases, this can also lead to the substrate being measured, resulting in measurements that are too large. Furthermore, indentation mapping of the entire meniscus surface allows for high spatial resolution and non-destructive testing but leads to prolonged testing times with autolysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThis round robin study showed that different in vitro test conditions (fixation methods, environmental conditions, freeze-thaw cycle) are affecting both the initial elastic and viscoelastic as well as biochemical properties of degenerated human meniscus tissue. Therefore, caution is advised when comparing one\u0026rsquo;s own results with those described in the literature. This is most relevant if the results are to be used as a basis for later measurements. These results are valuable for researchers in the field of soft tissue biomechanics. They can be used to advance the standardization of soft tissue measurements, creating a uniform basis for all researchers. Additionally, readers should be better informed about how to interpret the obtained data.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eEthics statement\u003c/h2\u003e\n\u003cp\u003eThe studies involving human participants were reviewed and approved by IRB 305/10 Albert-Ludwigs University Freiburg. The patients/participants provided written informed consent to participate in this study.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eMS and AMS conceived the study. HOM participated in the design and coordination of the study. MS, MFW, LdR, BR, OP, and GT performed the preparation procedure, mechanical testing, data analysis, and statistics. MS and LdR drafted the manuscript. AMS helped in manuscript writing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe processing fee for the article was financed by the Ministry of Science, Research, and the Arts of Baden-Württemberg and the University of Freiburg as part of the Open Access Publishing funding program.\u003c/p\u003e\n\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank Melanie Lynn Hart, a native speaker, for spell-checking and grammar correction.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHunter DJ, Bierma-Zeinstra S, Osteoarthritis. \u003cem\u003eLancet\u003c/em\u003e 2019, \u003cem\u003e393\u003c/em\u003e, 1745-1759.10.1016/s0140-6736(19)30417-9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDillon CF, Rasch EK, Gu Q, Hirsch R. Prevalence of knee osteoarthritis in the united states: Arthritis data from the third national health and nutrition examination survey 1991-94. 2006, \u003cem\u003e33\u003c/em\u003e, 2271\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen U-SDT, Zhang Y, Zhu Y, Niu J, Zhang B, Felson DT. Increasing prevalence of knee pain and symptomatic knee osteoarthritis: Survey and cohort data. Annal Interal Med. 2011;155:725\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7326/0003-4819-155-11-201112060-00004\u003c/span\u003e\u003cspan address=\"10.7326/0003-4819-155-11-201112060-00004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGill TM. Do the tenets of late-life disability apply to middle age? \u003cem\u003eAnn. Intern. Med.\u003c/em\u003e 2017, \u003cem\u003e167\u003c/em\u003e, 818-819.10.7326/m17-2550.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHitzl W, Stamm T, Kloppenburg M, Ritter M, Gaisberger M, van der Zee-Neuen A. Projected number of osteoarthritis patients in austria for the next decades \u0026ndash; quantifying the necessity of treatment and prevention strategies in europe. \u003cem\u003eBMC Musculoskel. Disord.\u003c/em\u003e 2022, \u003cem\u003e23\u003c/em\u003e, 133.10.1186/s12891-022-05091-5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalker PS, Erkman MJ. The role of the menisci in force transmission across the knee. \u003cem\u003eClin. Orthop. Relat. Res.\u003c/em\u003e 1975, 184-192.10.1097/00003086-197506000-00027.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurosawa H, Fukubayashi T, Nakajima H. Load-bearing mode of the knee joint: Physical behavior of the knee joint with or without menisci. Clin Orthop Relat Res 1980, 283\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOtani S, Kanamoto T, Oyama S, Yamakawa S, Shi W, Miyazaki R, Aihara M, Oka S, Kuroda S, Nakai T et al. ,\u003cem\u003e. Meniscus surface texture is associated with degenerative changes in biological and biomechanical properties. Sci. Rep. 2022, 12, 11977.10.1038/s41598-022-16206-2\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePordzik J, Bernstein A, Watrinet J, Mayr HO, Latorre SH, Schmal H, Seidenstuecker M. Correlation of biomechanical alterations under gonarthritis between overlying menisci and articular cartilage. Appl Sci. 2020;10:8673. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.https://doi.org/10.3390/app10238673\u003c/span\u003e\u003cspan address=\".10.3390/app10238673\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePordzik J, Bernstein A, Mayr HO, Latorre SH, Maks A, Schmal H, Seidenstuecker M. Analysis of proteoglycan content and biomechanical properties in arthritic and arthritis-free menisci. Appl Sci. 2020;10:9012. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.https://doi.org/10.3390/app10249012\u003c/span\u003e\u003cspan address=\".10.3390/app10249012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeitz AM, Osthaus F, Schwer J, Warnecke D, Faschingbauer M, Sgroi M, Ignatius A, D\u0026uuml;rselen L. Osteoarthritis-related degeneration alters the biomechanical properties of human menisci before the articular cartilage. 2021, \u003cem\u003e9\u003c/em\u003e.10.3389/fbioe.2021.659989.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSim S, Chevrier A, Garon M, Quenneville E, Lavigne P, Yaroshinsky A, Hoemann CD, Buschmann MD. Electromechanical probe and automated indentation maps are sensitive techniques in assessing early degenerated human articular cartilage. \u003cem\u003eJ. Orth. Res.\u003c/em\u003e 2017, \u003cem\u003e35\u003c/em\u003e, 858-867.10.1002/jor.23330.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis PB, Williams JM, Hallab N, Virdi A, Yanke A, Cole BJ. Multiple freeze-thaw cycled meniscal allograft tissue: A biomechanical, biochemical, and histologic analysis. 2007, \u003cem\u003e26\u003c/em\u003e, 49\u0026ndash;55\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jor.20473\u003c/span\u003e\u003cspan address=\"10.1002/jor.20473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwer J, Ignatius A, Seitz AM. The biomechanical properties of human menisci: A systematic review. Acta Biomater. 2024;175:1\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actbio.2023.12.010\u003c/span\u003e\u003cspan address=\"10.1016/j.actbio.2023.12.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC4BioCommunity. Challenge towards consensus on characterization of biological tissue. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://c4bio.eu/\u003c/span\u003e\u003cspan address=\"https://c4bio.eu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (March 31),.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayes WC, Keer LM, Herrmann G, Mockros LF. A mathematical analysis for indentation tests of articular cartilage. J Biomech. 1972;5:541\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJurvelin JS, R\u0026auml;s\u0026auml;nen T, Kolmonen P, Lyyra T. Comparison of optical, needle probe and ultrasonic techniques for the measurement of articular cartilage thickness. \u003cem\u003eJ. Biomech.\u003c/em\u003e 1995, \u003cem\u003e28\u003c/em\u003e, 231-235.10.1016/0021-9290(94)00060-h.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeronesi F, Berni M, Marchiori G, Cassiolas G, Muttini A, Barboni B, Martini L, Fini M, Lopomo NF, Marcacci M et al. ,\u003cem\u003e. Evaluation of cartilage biomechanics and knee joint microenvironment after different cell-based treatments in a sheep model of early osteoarthritis. Int. Orthop. 2021, 45, 427-435.10.1007/s00264-020-04701-y\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerreira JR, Teixeira GQ, Neto E, Ribeiro-Machado C, Silva AM, Caledeira J, Leite Pereira C, Bidarra S, Maia AF, Lamghari M et al. ,\u003cem\u003e. Il-1β-pre-conditioned mesenchymal stem/stromal cells\u0026rsquo; secretome modulates the inflammatory response and aggrecan deposition in the intervertebral disc. Eur Cell Mater 2021, 41.\u003c/em\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22203/eCM.v041a28\u003c/span\u003e\u003cspan address=\"10.22203/eCM.v041a28\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeidlinger-Wilke C, Ekkerlein A, Goncalves RM, Ferreira JM, Ignatius A, Wilke HJ, Teixeira GQ. Mesenchymal stem cell secretome decreases the inflammatory response in annulus fibrosus organ cultures. Eur Cell Mater. 2021;42:1\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22203/eCM\u003c/span\u003e\u003cspan address=\"10.22203/eCM\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIgnat\u0026rsquo;eva NY, Danilov NA, Averkiev SV, Obrezkova MV, Lunin VV, Sobol\u0026rsquo; EN. Determination of hydroxyproline in tissues and the evaluation of the collagen content of the tissues. \u003cem\u003eJournal of Analytical Chemistry\u003c/em\u003e 2007, \u003cem\u003e62\u003c/em\u003e, 51-57.10.1134/S106193480701011X.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEkiert M, Karbowniczek J, Stachewicz U, Mlyniec A. The effect of multiple freeze-thaw cycles on the viscoelastic properties and microstructure of bovine superficial digital flexor tendon. J Mech Behav Biomed Mater. 2021;120:104582. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.https://doi.org/10.1016/j.jmbbm.2021.104582\u003c/span\u003e\u003cspan address=\".10.1016/j.jmbbm.2021.104582\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarnecke D, Balko J, Haas J, Bieger R, Leucht F, Wolf N, Schild N, Stein S, Seitz A, Ignatius A, et al. Degeneration alters the biomechanical properties and structural composition of lateral human menisci. Osteoarthritis Cartilage. 2020. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2020.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2020.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerwig J, Egner E, Buddecke E. Chemical changes of human knee joint menisci in various stages of degeneration. Ann Rheum Dis. 1984;43:635\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/ard.43.4.635\u003c/span\u003e\u003cspan address=\"10.1136/ard.43.4.635\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSon M, Goodman SB, Chen W, Hargreaves BA, Gold GE, Levenston ME. Regional variation in t1ρ and t2 times in osteoarthritic human menisci: Correlation with mechanical properties and matrix composition. Osteoarthritis Cartilage. 2013;21:796\u0026ndash;805. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2013.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2013.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorejon A, Norberg CD, De Rosa M, Best TM, Jackson AR, Travascio F. Compressive properties and hydraulic permeability of human meniscus: Relationships with tissue structure and composition. 2021, \u003cem\u003e8\u003c/em\u003e.10.3389/fbioe.2020.622552.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBursac P, Arnoczky S, York A. Dynamic compressive behavior of human meniscus correlates with its extra-cellular matrix composition. \u003cem\u003eBiorheology\u003c/em\u003e 2009, \u003cem\u003e46\u003c/em\u003e, 227-237.10.3233/BIR-2009-0537.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanso EK, M\u0026auml;kel\u0026auml; JT, Tanska P, Mononen ME, Honkanen JT, Jurvelin JS, T\u0026ouml;yr\u0026auml;s J, Julkunen P, Korhonen RK. Characterization of site-specific biomechanical properties of human meniscus-importance of collagen and fluid on mechanical nonlinearities. \u003cem\u003eJ. Biomech.\u003c/em\u003e 2015, \u003cem\u003e48\u003c/em\u003e, 1499-1507.10.1016/j.jbiomech.2015.01.048.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeitz AM, Galbusera F, Krais C, Ignatius A, Durselen L. Stress-relaxation response of human menisci under confined compression conditions. \u003cem\u003eJ Mech Behav Biomed Mater\u003c/em\u003e 2013, \u003cem\u003e26\u003c/em\u003e, 68-80.10.1016/j.jmbbm.2013.05.027.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatsuragawa Y, Saitoh K, Tanaka N, Wake M, Ikeda Y, Furukawa H, Tohma S, Sawabe M, Ishiyama M, Yagishita S, et al. Changes of human menisci in osteoarthritic knee joints. Osteoarthritis Cartilage. 2010;18:1133\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2010.05.017\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2010.05.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishimuta JF, Levenston ME. Adipokines induce catabolism of newly synthesized matrix in cartilage and meniscus tissues. \u003cem\u003eConnect. Tissue Res.\u003c/em\u003e 2017, \u003cem\u003e58\u003c/em\u003e, 246-258.10.1080/03008207.2017.1281258.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAggad W, Abd El-Aziz G, Halawi M, Hindi E, AlShali R, Hamdy R, Saleh H. Dimorphic comparative histological and histometric study of the lateral and medial knee menisci in male and female human cadavers. Eur J Anat. 2024;28:109\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.52083/ZCPM8648\u003c/span\u003e\u003cspan address=\"10.52083/ZCPM8648\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRothrauff BB, Shimomura K, Gottardi R, Alexander PG, Tuan RS. Anatomical region-dependent enhancement of 3-dimensional chondrogenic differentiation of human mesenchymal stem cells by soluble meniscus extracellular matrix. Acta Biomater. 2017;49:140\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actbio.2016.11.046\u003c/span\u003e\u003cspan address=\"10.1016/j.actbio.2016.11.046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez-Adams J, Willard VP, Athanasiou KA. Regional variation in the mechanical role of knee meniscus glycosaminoglycans. 2011, \u003cem\u003e111\u003c/em\u003e, 1590-1596.10.1152/japplphysiol.00848.2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishimuta JF, Levenston ME. Meniscus is more susceptible than cartilage to catabolic and anti-anabolic effects of adipokines. Osteoarthritis Cartilage. 2015;23:1551\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2015.04.014\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2015.04.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLareu RR, Zeugolis DI, Abu-Rub M, Pandit A, Raghunath M. Essential modification of the sircol collagen assay for the accurate quantification of collagen content in complex protein solutions. Acta Biomater. 2010;6:3146\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actbio.2010.02.004\u003c/span\u003e\u003cspan address=\"10.1016/j.actbio.2010.02.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-orthopaedic-surgery-and-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"josr","sideBox":"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)","snPcode":"13018","submissionUrl":"https://submission.nature.com/new-submission/13018/3","title":"Journal of Orthopaedic Surgery and Research","twitterHandle":"@MSKmedBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"meniscus, osteoarthritis, mechanical examination, instantaneous modulus, elastic modulus, different fixation methods, sGAG, collagen","lastPublishedDoi":"10.21203/rs.3.rs-6870107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6870107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe biomechanical properties of degenerated meniscal tissue are increasingly being studied in the context of osteoarthritis research. Spatial indentation testing using a multiaxial testing machine allows non-destructive characterization of viscoelastic properties. However, in vitro testing conditions can significantly influence the results. The purpose of this round robin study was to evaluate the effects of different fixation methods and laboratory environments on the viscoelastic properties of degenerated lateral menisci.\u003c/p\u003e \u003cp\u003eSpatial normal indentation tests were performed on nine degenerated human lateral menisci in two laboratories using a multiaxial testing machine. Key parameters, including the maximum applied force (P\u003csub\u003emax\u003c/sub\u003e), instantaneous modulus (IM), and elastic modulus (E\u003csub\u003et10\u003c/sub\u003e), were analyzed across different meniscus regions. Significant differences in the IM, E\u003csub\u003et10\u003c/sub\u003e, and P\u003csub\u003emax\u003c/sub\u003e were observed between the laboratories, highlighting the influence of testing conditions on biomechanical results.\u003c/p\u003e \u003cp\u003eThe results indicated that variations in fixation methods, environmental conditions, and freeze-thaw cycles significantly affect the elastic and viscoelastic properties of meniscal tissue. Unphysiological strains in the inner region of the menisci suggested that strain-controlled indentation may be preferable to distance-controlled testing. These results underscore the importance of standardizing in vitro conditions for meaningful comparisons with the existing literature.\u003c/p\u003e","manuscriptTitle":"The Influence of In Vitro Test Conditions on the Biomechanical Properties of Degenerated Lateral Menisci - A Ring Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 04:52:41","doi":"10.21203/rs.3.rs-6870107/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-25T15:15:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-25T15:02:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-19T16:46:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16938997526503809627788911643421786130","date":"2025-06-17T12:45:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-16T15:03:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-15T08:43:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7086120278655540834076067224993106508","date":"2025-06-15T08:40:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186784287538101595204687246758678418258","date":"2025-06-14T10:13:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229066201792543846035116111514411098938","date":"2025-06-13T08:48:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-13T08:35:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-12T06:53:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-12T04:43:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Orthopaedic Surgery and Research","date":"2025-06-11T09:14:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-orthopaedic-surgery-and-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"josr","sideBox":"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)","snPcode":"13018","submissionUrl":"https://submission.nature.com/new-submission/13018/3","title":"Journal of Orthopaedic Surgery and Research","twitterHandle":"@MSKmedBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ee167da-b465-42a9-9b21-f0685a2beeeb","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T16:01:20+00:00","versionOfRecord":{"articleIdentity":"rs-6870107","link":"https://doi.org/10.1186/s13018-025-06151-x","journal":{"identity":"journal-of-orthopaedic-surgery-and-research","isVorOnly":false,"title":"Journal of Orthopaedic Surgery and Research"},"publishedOn":"2025-08-06 15:57:27","publishedOnDateReadable":"August 6th, 2025"},"versionCreatedAt":"2025-06-17 04:52:41","video":"","vorDoi":"10.1186/s13018-025-06151-x","vorDoiUrl":"https://doi.org/10.1186/s13018-025-06151-x","workflowStages":[]},"version":"v1","identity":"rs-6870107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6870107","identity":"rs-6870107","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}