Acta Polytechnica CTU Proceedings https://doi.org/10.14311/APP.2023.42.0094 Acta Polytechnica CTU Proceedings 42:94–97, 2023 © 2023 The Author(s). Licensed under a CC-BY 4.0 licence Published by the Czech Technical University in Prague EFFECT OF THE LONG-TERM STORAGE METHODS ON THE STABILITY OF CARTILAGE BIOMECHANICAL PARAMETERS Blanka Žaloudkováa,b, Šárka Sekorovác, Barbora Kopeckác, Daniel Kytýřa,∗ a Czech Academy of Sciences, Institute of Theoretical and Applied Mechanics, Prosecká 76, 190 00 Prague, Czech Republic b Czech Technical University in Prague, Faculty of Transportation Sciences, Mobile Laboratory for Transport Analysis, Konviktská 20, 110 00 Prague, Czech Republic c National Cell and Tissue Centre, Dr. Slabihoudka 6232/11, 708 00 Ostrava, Czech Republic ∗ corresponding author: kytyr@itam.cas.cz Abstract. Long-term stability of the tissue product in terms of mechanical parameters is a key factor for its expiration date. For the investigation of storage effects on the cartilage tissues the experimental mechanical loading test combined with XCT scanning for the irregular shape inspection was performed. The samples were preserved according to three different protocols using the deep-freezing and two types of saline solution preservation. The stability of the biomechanical parameters was tested within annual intervals. All samples were subjected to uni-axial compression loading using the in-house developed compact table top loading device in displacement-driven mode. Based on the measurements, the results are represented in the form of stress-strain curves and quantified as elastic modulus and ultimate compression stress. It can be concluded that no significant difference was found in neither the mechanical properties of the samples nor in the effects of each preservational method. Keywords: Stability study, cartilage, biomechanical characterisation, compression loading, XCT imaging. 1. Introduction Storage of tissues of human origin for long-term bank- ing is an essential operation of Tissue Establishments. Long-term stability of the tissue product is the key parameter for determining its expiration date. The acceptable method of tissue preservation must be capa- ble of retaining the natural properties of the graft (e.g. viability, structural integrity) for the time interval of the maximum possible storage time. One of the most common preservation methods which has been used for decades is deep-freezing. The grafts without cryoprotectants are stored in the range of temperatures from −80 ◦C to −40 ◦C. For this stor- age procedure only little impact on the biomechanical parameters was reported [1, 2]. Alternatively, the chemical preservation could be used which involves the conservation of cartilage using saline solution with the concentration typically in range 0.9–3.0 % at room temperature [3]. This method is not widely used yet and in general there is a lack of publications investi- gating the stability of biomechanical parameters of products stored in this way. This method brings po- tential risk of a significant decrease of elastic modulus and yield stress in cartilages as uniquelly reported in [4]. During the pre-storage tissue treatment the allo- grafts are commonly subjected to antibiotic-based disinfection [1] and for this study the samples stored using the saline solution were also sterilized using ionizing radiation. Gamma rays irradiation steril- ization itself can influence the biomechanical proper- ties of allografts and induce changes in the molecular structure [5, 6]. In addition, to avoid potential risk, both storage methods were examined and compared in terms of costal cartilage biomechanical stability. The aim of this study is to assess the effect of long-term storage of cartilages preserved by the two different methods, using three different protocols. These find- ings will be useful for a comprehensive assessment of the suitability of the alternative saline preservation method for costal cartilage storage. The problem of irregular shape of the tested cartilage sample [7, 8] was solved by subjecting the sample to XCT imaging to obtain full 3D geometrical model for estimation of the cross-section area together with employing of closed- loop controlled precise system for mechanical testing. For that reason the set of biomechanical experiments coupling XCT imaging and uni-axial testing [9] was performed to evaluate the time-dependent trends in changes of biomechanical parameters. 2. Materials and methods 2.1. Samples For costal cartilages of human origin three types of tissue preservation for the long-term storage were used: (i) deep-freezing and storage in range temperatures 94 https://doi.org/10.14311/APP.2023.42.0094 https://creativecommons.org/licenses/by/4.0/ https://www.cvut.cz/en vol. 42/2023 Cartilage long-term stability from −80 ◦C to −40 ◦C and cooled solid CO2 during the transportation, (ii) preserved by 3.0 % NaCl solution and stored at room temperature, (iii) preserved by 0.9 % saline and stored at room temperature. Before the storage process, all the samples were treated by antibiotic-based disinfection bath. More- over, the samples stored in the saline solution were sterilised by gamma rays. Two testing samples were prepared by manual sectioning from each cartilage with height of 5–7 mm and plan-parallel (± 0.1 mm) contact faces fitting in circumscribed circle of 12 mm. Because of the irregular shape of the samples, 3D models were used for identification of cross-section area Ac used in stress calculation. Individual sam- ples were subjected to X-ray microtomography (XCT) scanning just before the loading experiment. The scanning procedure consisted of 800 projections with acquisition time of 400 ms, in total 8 mins including rotation and read-out time. The sample was scanned in polymer container partly filled with water but the sample was surrounded only by air. This setup ensures that the imaging process does not affect the structure and biomechanical properties of the sample, utilizing an X-ray source with an acceleration voltage of 75 kV and tube current of 120 µA. The sample placed in XCT scanned is depicted in Figure 1. Figure 1. The environmental container with the sam- ple placed in XCT scanner. X-ray source (left), sample with container and holder on rotary table (middle), flat panel detector (right). To obtain proper Ac size, volumetric data were reconstructed using cone-beam FDK algorithm [10] implemented in VGStudio Max (Volume Graphics, Germany) software and exported as image stack with pixel size of 9.5 µm. On the volume of interest the tis- sue was segmented by thresholding and application of gaussian smoothing and erosion filtering (see Figure 2). Minimal Ac was taken for further stress calculations. In all cases minimal Ac reached at least 95 % of me- dian cross-sectional area. This fact minimizes the risk of corrupted data processing. Figure 2. Visualization of the cartilage sample based on XCT imaging. Figure 3. Compression test arrangement 2.2. Biomechanical analysis Biomechanical parameters, namely compressive mod- ulus E, ultimate stress σu and corresponding strain level εu were taken for evaluation of the long-term storage effect on the tissues. For that purpose uni- axial quasi-static compression tests were performed using in-house developed compact loading device (see Figure 3) in detail described in [9]. To ensure the op- timal accuracy of the measurement load-cell LCM300 (Futek, USA) with the nominal capacity of 1.1 kN was employed. For the displacement driven experiment the loading rate was set to 10 µm s−1 and maximal displacement 3500 µm corresponding to 50 % strain for the highest sample. Read-out frequency was set to 200 Hz for raw data logging employing in-house developed control system [11]. Automated Matlab (MathWorks, USA) script was used for the data pro- cessing to obtain the stress-strain curves representing the deformation behaviour and assess to the biome- chanical parameters. 95 B. Žaloudková, S. Sekorová, B. Kopecká, D. Kytýř Acta Polytechnica CTU Proceedings For deformation behavior the stress was considered as engineering stress obtained using σeng = F Ac , (1) where F is load-cell output. Engineering strain εeng was calculated by the following formula εeng = u h0 , (2) where u represents displacement measured by encoder and h0 is the initial height of the sample. 3. Results and discussion To evaluate effect of the long-term storage of carti- lages under deep frozen condition, biomechanical char- acteristics assessed from uni-axial compression test performed in February 2022 and February 2023 were used. The deformation behaviour of all tested samples is represented by stress-strain curves in Figure 4. 0 2 4 6 8 10 0 0.05 0.1 0.15 0.2 0.25 0.3 S tr e ss [ M P a ] Strain [-] frozen Feb 2022 Feb 2023 Figure 4. Deformation characteristic of all deep frozen samples The mean values of material parameters with cor- responding standard deviation are summarized in Ta- ble 1. Based on the obtained results, no significant Date Feb 2022 Feb 2023 E [MPa] 57.24 ± 0.87 65.45 ± 13.69 σu [MPa] 6.84 ± 0.62 7.97 ± 1.88 εu [-] 0.1821 ± 0.0286 0.1582 ± 0.0311 Table 1. Time dependence of biomechanical parame- ters of deep frozen samples long-term storage effect was proven. The difference in biomechanical parameters of each individual sample is higher than the influence of the storage effect. To evaluate the effect of the long-term storage of cartilages preserved in 0.9 % NaCl, biomechanical char- acteristics were assessed using the same tasting proce- dure. Resulting stress-strain curves are presented in Figure 5. 0 2 4 6 8 10 0 0.05 0.1 0.15 0.2 0.25 0.3 S tr e s s [ M P a ] Strain [-] 0.9% NaCl Feb 2022 Feb 2023 Figure 5. Deformation characteristic of 0.9 % NaCl preserved samples The mean values of material parameters with cor- responding standard deviation are summarized in Ta- ble 2. Date Feb 2022 Feb 2023 E [MPa] 46.80 ± 11.24 53.38 ± 4.38 σu [MPa] 5.00 ± 0.81 4, 88 ± 0.25 εu [–] 0.1625 ± 0.0459 0.1305 ± 0.0537 Table 2. Time dependence of biomechanical parame- ters of 0.9 % NaCl preserved samples From the obtained results a slight increase of the magnitude of the elastic modulus over a longer time can be seen. This finding is in contrary to the hy- pothesis of time-dependent degradation of the biome- chanical parameters. There is negligible difference in ultimate stress and non-significant drop of the strain at the ultimate strain level. Because of the nearly 40 % strain variation (0.1322 ± 0.0515) result it can’t be taken as a proof of the time effect. Finally the effect of saline saturation was evaluated. Resulting stress-strain curves are presented in Figure 6. The mean values of material parameters with cor- responding standard deviation are summarized in Ta- ble 3. Based on the obtained results no significant Salinity 0.9 % NaCl 3.0 % NaCl E [MPa] 37.55 ± 5.66 56.04 ± 2.25 σu [MPa] 4.67 ± 0.81 5.33 ± 0.93 εu [-] 0.1928 ± 0.0056 0.1322 ± 0.0515 Table 3. Biomechanical parameters depending on saline solution long-term storage effect was proven. The difference in biomechanical parameters of each individual sample is higher than the influence of the storage effect. The ulti- mate stress difference is minimal (under 3.0 %). Slight 96 vol. 42/2023 Cartilage long-term stability 0 2 4 6 8 10 0 0.05 0.1 0.15 0.2 0.25 0.3 S tr e s s [ M P a ] Strain [-] saline solution 0.9% NaCl 3.0% NaCl Figure 6. Deformation characteristic of 0.9 % NaCl and 3.0 % NaCl preserved samples increase of elastic modulus together with decrease of the strain for ultimate stress could be recognized but on negligible level. But it has to be kept in mind that this study is not statistically relevant because of limited number of delivered samples. 4. Conclusions It can be concluded that no significant difference in mechanical properties was observed within one year measurement interval despite the findings for saline preservation presented by Zhang et al. [4]. For that reason all proposed preservation methods are suitable for long-term storage of the allografts. The gamma dose caused by XCT scanning cannot influence long- term biomechanical parameters because the imaging was performed just before the mechanical testing. The same testing procedure will be applied on the cartilage samples in the next two years to complete the stability study. Acknowledgements This research was supported by Strategy AV21 - Break- through technologies for the future. References [1] A. Paolin, L. Spagnol, G. Battistella, D. Trojan. Evaluation of allograft decontamination with two different antibiotic cocktails at the treviso tissue bank foundation. PLOS ONE 13(8):e0201792, 2018. https://doi.org/10.1371/journal.pone.0201792 [2] I. Szabó, B. Patzai, D. Lőrinczy. Effects of long-term deep freezing on human femoral cartilage: differential scanning calorimetric (DSC) analysis and histopathological evaluations. 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Acta Polytechnica CTU Proceedings 18:15–19, 2018. https://doi.org/10.14311/app.2018.18.0015 97 https://doi.org/10.1371/journal.pone.0201792 https://doi.org/10.1007/s10973-021-11070-0 https://doi.org/10.1016/j.jor.2019.02.018 https://doi.org/10.1016/j.clinbiomech.2018.06.003 https://doi.org/10.1016/j.clinbiomech.2018.06.003 https://doi.org/10.7150/ijms.22644 https://doi.org/10.1016/j.idc.2016.04.002 https://doi.org/10.1016/j.jbiomech.2022.111242 https://doi.org/10.1302/2046-3758.78.bjr-2018-0025.r1 https://doi.org/10.1302/2046-3758.78.bjr-2018-0025.r1 https://doi.org/10.1088/1748-0221/13/11/c11021 https://doi.org/10.1364/josaa.1.000612 https://doi.org/10.14311/app.2018.18.0015 Acta Polytechnica CTU Proceedings 42:93–96, 2023 1 Introduction 2 Materials and methods 2.1 Samples 2.2 Biomechanical analysis 3 Results and discussion 4 Conclusions Acknowledgements References