Word Count: 210 15 Main Text Word Count: 1805 16 2 Abstract 17 Background: Allograft tendons are commonly used during orthopedic surgery to 18 reconstruct tissue that is severely damaged. Soaking the tendon in a n antibiotic 19 solution , specifically vancomycin, has been shown to lower the risk of post -operative 20 infections. While some material properties of tendon and ligament after antibiotic 21 soaking have previously been characterized , extensive sub -failure allograft tendon 22 material properties after soaking in antibiotic solutions have not . 23 Methods: Forty tendons were dissected from rabbits and soaked in either a phosphate 24 buffered saline ( PBS) only solution or vancomycin and PBS solution for five or 30 25 minutes. Immediately after soaking , quasi -static tensile ex periments were performed in 26 a materials testing system. 27 Findings: Tissue nominal stress, Lagrange strain, toe-region properties and elastic 28 modulus were characterized. For all forty tendons, t he average elastic modulus was 29 found to be 455 ± 37 MPa, the average transition strain (from toe -region to linear elastic 30 region) was 0.0487 ± 0.0035, and the average transition stress was 9.71 ± 0.79 MPa. 31 No statistically significant differences in any of these material properties were found 32 acros s soaking medium or soaking time. 33 Interpretation: From these results , we conclude that soaking an allograft tendon in 34 antibiotic solution for up to 30 minutes prior to implantation does not change the tensile 35 material properties of tendons, supporting current clinical practice. 36 37 Keywords – allograft tendon, elastic modulus, digital image correlation, mechanical 38 characterization, optimization 39 3 1. Introduction 40 Allograft tendons are commonly used in reconstruction when ti ssues are severely 41 damaged . Specifically, native tissue that no longer maintains mechanical viability is 42 replaced with allograft tendons that exhibit the necessary material properties for tissue 43 function. Antibiotics, such as vancomycin, are used in an attempt to reduce the risk of 44 post-surgical infections (Banios et al., 2021; Schuster et al., 2020; Xiao et al., 2021) . 45 Recent work has shown that vancomycin soaked gauze wrapped around tendon and 46 ligament grafts a t a concentration of 5 mg/mL or greate r for at least 20 minutes were 47 able to remove bacterial contamination from the tendon grafts (Schuster et al., 20 20; 48 Schüttler et al., 2019) . Additionally, s oaking tendons in vancomycin does not cause 49 tenocyte cytotoxicity if the concentration is below 12,600 μL and soaking time is less 50 than 6 hours (Xiao et al., 2020) . However, it is unclear how the sub-failure material 51 properties of tendon – specifically characteristics of the toe and linear regions of tensile 52 stress -strain behavior – are affected by antibiotic soaking and short -term soaking time. 53 The goal of the present study is to determine if the tensile material properties of 54 hybrid New Zealand White rabbit and hybrid California White rabbit achilles tendons are 55 altered by a) soaking in vancomycin antibiot ic solution versus phosphate buffered saline 56 (PBS) only and b) soaking for five or thirty minutes . We hypothesized that vancomycin 57 soaking and soaking time would not affect the elastic modulus and toe -region properties 58 of tendon. Changes to tendon graft mechanics after soaking in an antibiotic solution 59 could alter surgical implantation procedures and in vivo tendon function, thus it is crucial 60 to understand how soaking in vancomycin solution affect tendon material properties . 61 62 4 2. Materials 63 2.1 Dissection and Sample Preparation 64 Ten (n=10) hybrid New Zealand White rabbits and hybrid California White rabbits were 65 acquired post -sacrifice from a local abattoir. No live animal handling was performed by 66 the study team and all animals were bred and sacrificed as l ivestock prior to study team 67 acquisition . Rabbit hind limbs were dissected and achilles tendons were harvested for 68 materials testing using standard dissection techniques. The tendons were randomly 69 assigned into four groups: two paired (same animal) groups were soaked in a 0.9% 70 phosphate buffered saline ( PBS) solution (pH 7.4) for five minutes or thirty minutes, and 71 the other two paired (same animal) groups were soaked in a 0.33 g/mL vancomycin 72 antibiotic and PBS solution for five or thirty minutes (Lamplot et al., 2021; Schüttler et 73 al., 2019) (Figure 1A ). 74 75 2.2 Tensile Materials Testing 76 After soaking and immediately prior to tensile materials testing, images of the top and 77 side of the tendon were acquired for cross -sectional area calculations using an 78 ellipsoidal area assumption (Pelled et al., 2012) . Following testing, five width and five 79 thickness measurements along the length of the tendon to acquired using Fi ji/ImageJ 80 (Schindelin et al., 2012) . The tendons were then loaded into a custom planar biaxial 81 materials testing system ( eXpe rt 8000 , ADMET, Inc. , Norwood, USA ) with a 300lb f load 82 cell and stretched at a rate of 0.05 % strain/s until failure (Lim et al., 2011) . Force data 83 were acquired at 100 Hz. Charcoal powder was applied to the surface of all samples for 84 5 digital image correlation (DIC) strain tracking, with a one second image acquisition rate 85 (Figure 1B). 86 87 88 Figure 1. A) Forty dissected tendons were split into four equally sized groups (n=10) 89 and soaked in either PBS for five or thirty minutes or soak ed in a vancomycin and PBS 90 antibiotic solution for five or thirty minutes. B) Samples were tensile tested with digital 91 image correlation strain tracking over a mid -sample region of interest. C) A custom 92 MATLAB script was written to fit a piecewise power law toe region and linearly elastic 93 region to stress strain data. 94 95 2.3 Data Analysis 96 Commercial DIC analysis software was used to measure Lagrange strain of each 97 sample over a mid -sample region of in terest (ROI) ( VIC-2D, Correlated Solutions, Inc. , 98 Columbia, USA ) (Grega et al., 2020) . Nominal stress was calculated by dividing force by 99 average cross -sectional area. Stress and strain data were smoothed with the sgolayfilt 100 smoothing function in MATLAB (Ma et al., 2009) . Visual inspect ion was used on all 101 stress -strain graphs to identify the end of the linear elastic region . A custom MATLAB 102 6 script was written to fit a piecewise power law toe region and linear elastic region to 103 stress -strain graphs (Equation 1) (Danto and Woo, 1993) . This approach numerically 104 extracted the elastic modulus (Equation 1, 𝐸 in MPa), transition strain (Equation 1 105 𝜖∗,unitless), and transition stress (Equation 1 𝜎(𝜖∗) in MPa) by minimizing the residuals 106 between the piecewise function (Equation 1) and the stress -strain data points for each 107 specimen . The script tested various in itial parameter guesses and used the lsqnonlin 108 function to generate global minima and a best fit model (Figure 1C) (Vaidya and 109 Wheatley, 2020) . 110 𝜎(𝜖)={𝑎𝜖𝑏,𝜖<𝜖∗ 𝑎𝜖∗𝑏+𝐸(𝜖−𝜖∗),𝜖∗≤𝜖 (1) 111 112 2.4 Statistics 113 Three two-way analysis of variance (ANOVA) test s were run to determine if the average 114 elastic modulus , transition strain , and transition stress were statistically different across 115 groups (p<0.05) . 116 117 3. Results 118 Nominal stress -Lagrange strain graphs were generated for forty tendons (n=10 119 per group) (Figure 2). For all forty tendons, the average elastic modulus was 455 ± 37 120 MPa, the average transition strain was 0.0487 ± 0.0035 , and the average transition 121 stress wa s 9.71 ± 0.79 MPa (Table 1). There were no statistically significant differences 122 in the average elastic modulus (p=0.12) , transition strain (p=0.76) , and transition stress 123 (p=0.31) between all four groups across both soaking time and soaking solution 124 combi ned (Figure 3). 125 7 126 Figure 2. Average nominal stress (MPa), Lagrange strain graph of the four tendon 127 groups. The transition into the linear region of the stress -strain curve occurs at 128 approximately 0.05 strain for each of the four groups . Standard error bar s shown. 129 130 Solution Soak Time (min) Elastic Modulus (MPa) Transition Strain (-) Transition Stress (MPa) PBS 5 545 ± 83 0.0539 ± 0.010 12.4 ± 1.9 30 396 ± 58 0.0436 ± 0.0039 8.55 ± 0.89 Vancomycin + PBS 5 395 ± 42 0.0516 ± 0.0049 9.27 ± 1.7 30 483 ± 100 0.0456 ± 0.0079 8.62 ± 1.5 Table 1. The average elastic modulus , transition strain, and transition stress values for 131 each of the four groups (n = 10 per group, means ± standard error). 132 133 134 8 135 Figure 3. Bar plot s for A) modulus, B) transition strain , and C) transition stresses for 136 each of the four tendon groups (n = 10 per group). There were no statistically significant 137 difference s in any measures across the four groups (p = 0.12 for modulus, p = 0.76 for 138 transition strain , and p = 0. 31 for transitio n stress ). 139 140 4. Discussion 141 Prior to implantation during tendon reconstruction surgery, tendon grafts were 142 briefly soaked in solution . Prior to this work, the extent to which short -term soaking time 143 (30 minutes or less) and soaking medium (phosphate buffered saline only or saline and 144 vancomycin antibiotic) affected the sub-failure tensile material properties of tendon was 145 9 not known . No statically significant differences were found in the elastic modulus (MPa), 146 transition strain, and transition stress (MPa) be tween four group of tendons soaked in 147 either a PBS solution only or a PBS and vancomycin solution for either five or 30 148 minutes (Figure 3, Table 1) . Our work shows that sub -failure tensile material properties, 149 including the elastic modulus (MPa), transitio n strain, and transition stress (MPa), of 150 tendons are not altered by short -term soaking in vancomycin. 151 The present study was not without limitations. While rabbit tendons have been 152 commonly used as a viable animal (Burgio et al., 2022) , greater clinical impact would be 153 achieved with human cadaver studies. Additionally, a common procedure for allograft 154 tendons is to thaw the tissue in an antibiotic solution, whi ch was not employed here. 155 However, there has been extensive work showing that the material properties of 156 tendons are not altered by a freeze -thaw cycle (Clavert et al., 2001; Hu ang et al., 2011; 157 Jung et al., 2011; Lee and Elliott, 2017) . Additionally, failure properties were not 158 characterized due to the challenges of producing mid -sample failure during materials 159 testing of whole tendons in vitro . Finally, with a sample size of n=10 for all groups, 160 greater statistical power could be achieved with more samples, however it is unlikely 161 that a greater sample size would change the conclusions of the present study . 162 While this work presents the first study of the effect of vancomycin s oaking on 163 tendon material properties, v arious studies have shown that briefly (<30 minutes) 164 soaking tendon and ligament grafts in vancomycin solutions reduces the incidence of 165 post-operation infection (Banios et al., 2021; Schuster et al., 2020; Schüttler et al., 166 2019; Xiao et al., 2021) . Prior work has shown that extended soaking times and certain 167 solutions, such as NaCl and sucrose, can affect tendon mechanics by altering tissue 168 10 hydration (Safa et al., 2017) . However, our work suggests that short -term soaking 169 typically employed in a pre-surgical setting is unlikely to alter sub-failure properties such 170 as, the toe -region properties and elastic modulus of tendon. Schüttler et al. 171 characterized the failure properties of porcine tendons soaked in antibiotic wrapped 172 gauze for up to 20 minu tes and found no changes in those material properties (Schüttler 173 et al., 2019) , similar to our findings here. In compariso n to Schüttler et al, we evaluated 174 sub-failure properties as tendon grafts more commonly experience non -failure loads in 175 vivo and properties such as tendon laxity affect muscle -tendon unit mechanics 176 (Delabastita et al., 2018) . In comparison to tendon, o ther studies of ligament material 177 properties after soaking in a similar vancomycin antibiotic solution found no changes in 178 ligament graft material properties (elastic modulus or elongation strain) (Lamplot et al., 179 2021) . In comparison to the present study , these findings are unsurprising as both 180 tendon and ligament ar e primarily composed of type I collagen fibers (Amiel et al., 181 1983) . 182 While similar in composition to ligament, tendon has a highly organized structure 183 that consists of densely packed connective tissue mainly comprised of a parallel fibers 184 of collagen (Amiel et al., 1983; Hudson et al., 2021) . Contrastingly, ligament structure 185 consists o f less organized bundles of collagen fibers (Dourte et al., 2008) . These 186 microstructural differences and the differences in connective roles in the bod y – tendon 187 as a force transmitter from muscle to bone and ligament as a bone to bone stabilizer 188 (Nordin et al., 2022) require independent mechanical evaluations (Woo et al., 2000) . 189 Future work to increase the duration of soaking time in conjunction with cell viability 190 11 studies would provide further detail into the mechanobiological effects o f vancomycin on 191 biological soft tissues. 192 193 5. Conclusions 194 Soaking tendons in an antibiotic solution is a critical step in reducing the risk of 195 post-operative infection . The present study measured the material properties of tendons 196 soaked in PBS and vancomycin solutions for five or thirty minutes. Statistical analysis 197 suggested no differences in tendon material properties after soaking across all four 198 groups. These results suggest that during tendo n reconstruction surgery, clinicians may 199 soak tendons in a vancomycin and PBS solution for up to thirty minutes without altering 200 tendon material properties. 201 202 Acknowledgements 203 Funding for equipment used in this work was provided by the National Science 204 Foundation under Major Research Instrumentation Grant No. 1828082. Authors would 205 like to thank the Bucknell -Geisinger Research Initiative for funding to support this work. 206 12