1 Core bone diameter in an organic implant-less technique affecting the biomechanical 1 properties of the anterior cruciate ligament fixation; an in-vitro study 2 Running Title: 3 Geometry effects on BASHTI technique 4 5 Authors / Affiliations: 6 Mahdi Mohseni, M.Sc. student 7 Research Assistant, Department of Mechanical Engineering, Sharif University of Technology, 8 Tehran, IR, mahdi.mohseni@mech.sharif.edu, Phone: +989135335667 9 10 Amir Nourani*, Ph.D. 11 Assistant Professor, Department of Mechanical Engineering, Sharif University of Technology, 12 Tehran, IR, nourani@sharif.edu, Phone: +982166165687 13 14 Hossein Korani, M.Sc. student 15 Research Assistant, School of Mechanical Engineering, College of Engineering, University of 16 Tehran, Tehran, IR, Korani@ut.ac.ir, Phone: +989129494789 17 18 Hadi Moeinnia, Ph.D. student 19 Research Assistant, Mechatronics System Engineering, Simon Fraser University, Burnaby, BC 20 V5A 1S6, Canada, h.moein@student.sharif.edu, Phone: +989375151931 21 22 Amirhossein Borjali, M.Sc. 23 Research Assistant, Department of Mechanical Engineering, Sharif University of Technology, 24 Tehran, IR, a.borjali15@student.sharif.ir, Phone: +989393962780 25 26 Narges Ghias, B.Sc. student 27 Research Assistant, Department of Mechanical Engineering, Sharif University of Technology, 28 Tehran, IR, narges.ghias1395@sharif.ir, Phone: +989133178264 29 30 Mahmoud Chizari, Ph.D. 31 Senior Lecturer, School of engineering and computer sciences, University of Hertfordshire, 32 College Ln, Hatfield AL10 9AB, UK, m.chizari@herts.ac.uk, Phone: +44(0)1707284000 33 34 35 *Corresponding author 36 Amir Nourani 37 E-mail: nourani@sharif.edu 38 Phone: +982166165687, Fax: +982166000021 39 40 41 Acknowledgements 42 The authors sincerely appreciate all the help and guidance of Dr. Farzam Farahmand, director of 43 the Biomechanics Laboratory of Sharif University of Technology. 44 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 2 45 Author contributions 46 All authors contributed to the study conception and design. Material preparation, data collection 47 and analysis were performed by Mahdi Mohseni, Hossein Korani, Hadi Moeinnia, Amirhossein 48 Borjali and Narges Ghias. The first draft of the manuscript was written by Mahdi Mohseni, revised 49 by Amir Nourani and Mahmoud Chizari and all authors commented on previous versions of the 50 manuscript. All authors read and approved the final manuscript. 51 52 Declarations 53 The authors declare that they have no conflict of interest. 54 55 Compliance with Ethical Standards 56 Declarations of interest: None 57 This research did not receive any specific grant from funding agencies in the public, commercial, 58 or not-for-profit sectors. 59 60 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 3 Highlights 61  A new implant-less technique was used to reconstruct anterior cruciate ligament. 62  Artificial bone and fresh animal soft tissue used to simulate the fixation process. 63  Loading condition were carefully chosen to simulate the post-operation. 64  Components geometry had direct effect on biomechanical properties of the fixation. 65  Optimum geometry was found trough an experimental examination. 66 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 4 Abstract 67 Background: Bone and site hold tendon inside (BASHTI) is an implant-less technique that can 68 solve some of the problems associated with other anterior cruciate ligament (ACL) reconstructive 69 methods. This study aims to investigate the effect of core bone diameter variation on the 70 biomechanical properties of a reconstructed ACL using BASHTI technique. 71 Methods: A number of 15 laboratory samples of reconstructed ACL were built using bovine 72 digital tendons and Sawbones blocks. Samples were divided into three groups with different core 73 bone diameters of 8 mm, 8.5 mm, and 9 mm. The double-stranded tendon size and bone tunnel 74 diameter were 8 mm and 10 mm, respectively. A loading scenario consisting of two cyclic loadings 75 followed by a single cycle pull-out loading was applied to the samples simulating the after-surgery 76 loading conditions to observe the fixation strength. 77 Results: Results showed that the core bone diameter had a significant effect on the failure mode 78 of the samples (P = 0.006) and their fixation strength (P < 0.001). Also, it was observed that the 79 engaging length and the average cyclic stiffness (ACS) of them were influenced by the core bone 80 diameter significantly (engaging length: P = 0.001, ACS: P = 0.007), but its effect on the average 81 pull-out stiffness was not significant (P = 0.053). 82 Conclusions: It was concluded that core bone diameter variation has a significant impact on the 83 mechanical properties of ACL reconstruction when BASHTI technique is used, and it should be 84 noted for surgeons who use BASHTI technique. 85 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 5 86 Keywords: BASHTI technique, Core bone diameter, ACL reconstruction, Geometric parameters, 87 Fixation strength 88 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 6 1. Introduction 89 Ligaments are fibrous bands connecting two bones, capable of undergoing tension and a ligament 90 rupture is a common injury in the human body. This injury can be a result of extreme conditions 91 caused by high pressures or impact, usually during sports activities 1. Various techniques have 92 been proposed to reconstruct a ruptured ligament, and most of them include an external implant. 93 Every technique has been proved to have its own advantages and disadvantages. There are some 94 different methods to reconstruct a ruptured ACL, such as using some sutures to hold the ligament 95 next to the bone (suture anchor) 2 or fixing the ligament via a button (cortical button) 3. However, 96 the most common technique is using an interference screw 4. This is a reliable technique with 97 proper strength, in which a screw is used to fix the tissue in a bone tunnel 4–7. 98 The mentioned conventional techniques for ACL reconstruction still comes with some 99 problems. One of them is the high cost of using an external implant like the interference screw. 100 Also, using these external implants may cause some side effects such as soft tissue rotation 8, bone 101 tunnel widening 9 and interfering in magnetic resonance imaging (MRI) 10. To solve these 102 problems, recent studies have been focused on new implant-free techniques for ACL 103 reconstruction, such as the press-fit technique that uses the cylindrical bone block of patellar 104 attached to the tendon graft to fix the connective tissue 11. However, using this method may cause 105 some problems such as pain in the patellar donor area 12. 106 A new implant-free approach presented in this area is bone and site hold tendon inside 107 (BASHTI). In this technique, neither an external implant nor the patellar bone but the patient’s 108 tibia bone is used to perform the reconstruction 13. Therefore, no sign of allergic reaction is 109 observed, and the costs of using an external implant made of expensive metals or biodegradable 110 polymers are eliminated 11. Also, there would not be an implant to intervene with MRI images. To 111 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 7 carry out the process, a specially designed drill bit is utilized on the bone to provide a tunnel and 112 a cylindrical bone block called the core bone. The core bone is later used to fix the ligament inside 113 the tunnel instead of using an external implant like an interference screw. The healing process in 114 BASHTI technique can be faster 11. Hence, efforts have been made to improve this fixation 115 technique. 116 BASHTI technique was introduced in a research made on bovine bones and digital tendons 117 harvested from bovine feet. The research experimentally compared the BASHTI results with the 118 interference screw fixation. The study concluded that the strength of BASHTI technique is as high 119 as an interference screw 13. Due to previous studies, tendon compression is defined as a 120 dimensionless parameter related to the amount of volume strain of the tendon in this fixation 121 technique. Recently, it was observed that this parameter significantly affects the strength of 122 BASHTI fixation with an experimental study using bovine digital tendons and artificial bones. It 123 was showed that increasing the tendon compression up to an optimum value could improve the 124 fixation strength 14. 125 Furthermore, an investigation was performed on the effect of using a sheathed core bone on 126 the biomechanical properties of the ligament fixation created by the BASHTI technique, and it was 127 observed that using these sheathes could help to increase the length of conflict between the core 128 bone, the ligament and the tunnel, resulting in a stronger fixation and also decrease the tunnel 129 widening and core bone fracture during the insertion procedure 15. Lastly, Nourani et al. studied 130 two insertion procedures to build the BASHTI fixation for biceps tendon reconstruction using 131 different frequencies. Results showed that using frequencies below 300 beats per minute would 132 improve the strength of the BASHTI fixation significantly 16. All of the previously mentioned 133 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 8 studies used Sawbones artificial bone blocks and bovine digital tendon to simulate the real 134 conditions. 135 The purpose of this study was to evaluate the effect of core bone diameter on the mechanical 136 properties of BASHTI technique under loading conditions similar to real ACL loadings. 137 Geometrical conditions, including the diameters of the core bone and the tendon used to perform 138 the reconstruction, are to be studied. These parameters can affect the amount of compression the 139 tendon withstands. This is an experimental study about the effect of core bone diameter on the 140 strength and stiffness of BASHTI fixation for ACL reconstruction, and also on the engaging length 141 between the core bone and the fixed ligament in the tunnel that could affect the strength of fixation 142 and speed up the healing process. 143 144 2. Materials and Methods 145 2.1. Materials and Specimen Preparation 146 Bovine digital tendons were harvested using surgical instruments and stored at -20˚C to ensure its 147 biomechanical properties remain unchanged 17–19 (Fig. 1 a). They were used to model human 148 ligament 14,19. After the harvesting process, the tendon was trimmed to an identical size using 149 surgical blade (Fig. 1 b). As a choice in ACL surgery, it was decided to use tendons with a double-150 stranded diameter of 8 mm. The tendons were kept moist by spraying water during preparation 151 and testing procedures maintain their mechanical properties 18. Moreover, Sawbones Polyurethane 152 blocks (1522-03, Sawbones Europe AB, Malmo, Sweden) with a density of 320 Kg/m3 (20 lb/ft3) 153 were used as the artificial bone to represent young human tibia bone 14,19 (Fig. 1 c). 154 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 9 The artificial bone was tunneled using a specially designed cannulated drill bit with an 155 outer diameter of 10 mm. The inside core bone of the tunnel was extracted after tunneling (Fig. 1 156 d and e). To perform the reconstruction, a double-stranded tendon was entered the tunnel by using 157 a suture, and the extracted core bone was hammered into it with a frequency of lower than 300 158 beats per minute 16 (Fig. 2 a and b). The first few millimeters of the core bone were chamfered to 159 more easily enter the tunnel. The core bone could not fully penetrate the tunnel, and its end part 160 was broken after hammer impacts and it could affect the strength of fixation. So the length of the 161 core bone that successfully entered the tunnel was measured after every experiment and reported 162 as the engaging length to examine its effect on the fixation strength. 163 164 165 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 10 Fig. 1 a. Bovine digital tendon extraction, b. Adjusting the tendon diameter using a surgical blade, 166 c. Sawbones artificial bone block with a density of 320 Kg/m3, d. Specially designed cannulated 167 drill bits with an outer diameter of 10 mm and inner diameters of 9 mm, 8.5 mm, and 8 mm were 168 used to extract the core bones, e. The core bone extracted from the artificial bone block with the 169 desired diameter. 170 171 15 laboratory samples of BASHTI fixation were built using the introduced protocols (Fig. 2 172 c). The samples were divided into Groups 1 to 3 with the core bone diameters of 9 mm, 8.5 mm, 173 and 8 mm, respectively, that were equal to the inner diameters of drill bits. The tunnel diameter 174 and double-stranded tendon size were considered to be 10 mm and 8 mm, respectively. These 175 values proved to have the best mechanical properties for BASHTI technique in previous studies 176 14. 177 178 179 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 11 Fig. 2 a. the double-strand tendon has entered the tunnel with a suture, b. The core bone was 180 hammered into the tunnel, c. The tendon and the core bone were entered into the tunnel, and the 181 laboratory sample of BASHTI fixation was prepared. 182 183 2.2. Biomechanical Testing 184 The looped end of the double-stranded tendon was attached to the Zwick-Roell Amsler HCT 25-185 400 tensile testing machine using a pin while the sawbones block was fixed on the table of the 186 testing machine (Fig. 3). A pull-out scenario was used so that the fixation underwent three levels 187 of loading, simulating the real loading conditions of ACL. For the preload, the fixation was 188 subjected to 10 cycles ranging from 10 to 50 N with a frequency of 0.1 Hz. Afterward, a periodic 189 loading with 150 cycles between 50 to 200 N at a frequency of 0.5 Hz was exerted on the fixation 190 20. In case that the construction sustained these two levels, the machine immediately pulled the 191 tendon with a rate of 20 mm/min, until the fixation failed 19. Each experiment was repeated five 192 times to validate the repeatability of the experiment. 193 194 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 12 195 Fig. 3 Testing a typical sample using BASHTI technique. It was assumed that when the tendon 196 displacement became more than 10 mm, the failure mode occurs on the fixation site. 197 198 2.3. Statistical Methods 199 The 95% confidence intervals of the results were calculated using Student’s t distribution. Also, 200 ANOVA one-way method was used to analyze the biomechanical properties results. The failure 201 mode results were analyzed using Chi-Square statistic method, which is commonly used for testing 202 the relationships between categorical variables. In this regard, probability value (P-value) was 203 supposed to compare different groups. In case if the P-value is equal or less than 0.05, it means 204 the differences between the two groups with 95% confidence are significant. 205 206 3. Results 207 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 13 The failure mode results of all groups are shown in Fig. 4 a. It was observed that about 47% of the 208 samples failed during the cyclic loading and did not reach the pull-out loading step. According to 209 these results, all of the Group 3 samples failed in the cyclic loading step (second loading step), 210 while the loading step that caused all of Group 2 samples to fail was pull-out loading (third and 211 last step). Only among the specimens in Group 1, both of the above conditions were observed, 212 with two specimens failing in cyclic loading and three specimens in the tensile loading step. Duo 213 to these results, statistical analysis showed that the core bone diameter affects the failure of the 214 samples significantly (P = 0.006). Also, all of the failure modes occurred at the fixation site (i.e., 215 the tendon and core bone slipped out of the bone tunnel without tendon rupture) (Fig. 3), and no 216 tendon rupture was observed. One typical load-displacement graph (i.e., the fifth test of Group 2) 217 is shown in Fig. 4 b. The three loading steps are illustrated in this figure, and it can be seen how 218 much displacement has been made in the sample at each loading step. 219 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 14 220 221 Fig. 4 a. Loading step that samples reached the failure in Groups 1, 2 and 3, b. Three loading steps 222 load-displacement result of the fifth sample in Group 2. 223 224 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 15 The biomechanical results are available in Table 1. It was assumed that when the 225 deformation from the beginning of the second cyclic loading step reached 10 mm (that is about 226 30% of the average initial ACL length 21), the fixation is failed (i.e., the failure mode is fixation 227 failure). In this case, the maximum load in the 10 mm range has been reported as the fixation 228 maximum strength. Also, the average cyclic stiffness (ACS) was defined using the following 229 equation: 230 𝐴𝐶𝑆 = 𝐹𝐶 𝐷𝐶 𝑁𝐶 ⁄ (𝑁 𝑚𝑚⁄ ) (1) 231 where FC is the amplitude of the periodic loading (e.g., it is equal to 150 N for a cyclic loading 232 between 200 N and 50 N), DC is the final pure displacement of the looped end of the tendon in the 233 periodic loading step, and NC is the number of completed cycles. The ACS value quantifies the 234 behavior of the reconstructed ACL against active extension loading during the post-surgical and 235 rehabilitation period. 236 Also, the average slope of the load-displacement curve in the linear region of the pull-out 237 loading step was reported as the average pull-out stiffness (APS) that implies the reconstructed 238 ACL resistance to sudden impact loading. Note that ACS and APS values were calculated only for 239 the specimens that fulfilled the cyclic loading step. Subsequently, since none of the Group 3 240 samples reached the pull-out loading step, no values were reported as the stiffness for this group. 241 Finally, the length of the core bone that successfully entered the tunnel without any fracture and 242 slipped out of it after the failure of the structure was measured and reported as the engaging length 243 in Table 1. 244 245 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 16 Table 1. The biomechanical properties of each group calculated from the Load-Displacement curve 246 of each sample with 95% Confidence Interval 247 Group Maximum Strength (N) ACS (N/mm) Average Pull-Out Stiffness (N/mm) Engaging Length (mm) 1 193±33 2118±522 114.4±40.8 11.4±5.2 2 360±123 3270±574 79±27 9.6±0.5 3 137±62 -* -* 23.2±9.5 * No value was reported since none of the Group 3 samples reached the pull-out loading step. 248 249 The statistical analysis of the biomechanical properties in Table 1 is reported in Table 2 as 250 P-values. These analyses implied that the core bone diameter significantly affects the fixation 251 strength of BASHTI technique. It was also concluded that this parameter has a significant impact 252 on the ACS and related engaging length of fixations. But the core bone diameter variation does 253 not affect the APS of the samples. 254 255 Table 2. The results of statistical analysis for the effect on core bone diameter on the biomechanical 256 properties of BASHTI technique. A P-value less than 0.05 considered statistically significant 257 Biomechanical Property Maximum Strength (N) ACS (N/mm) APS (N/mm) Engaging Length (mm) P-value 0.000 0.007 0.053 0.002 258 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 17 4. Discussion 259 According to the results obtained, it could be concluded that the core bone diameter had a 260 significant effect on the failure mode of the samples (P = 0.006). In Group 3 samples (i.e., 8 mm 261 core bone), since the core bone failed to compress the 8 mm tendon into the tunnel wall properly, 262 all of the specimens failed in the main cyclic not even reached to step 3 pull-out loading. While in 263 Group 2 samples (i.e., 8.5 mm core bone), the core bone managed to compress the tendon fibers 264 into the tunnel wall, and all of the samples failed in the third loading step. Moreover, in Group 1 265 samples (i.e., 9 mm core bone), only 60 percent of specimens reached the step 3 loading (Fig. 4 a), 266 and 40 percent failed to do so. The reason for this occurrence is over-compression. In other words, 267 since the diameter of the tunnel was fixed, by increasing the core bone diameter, the compression 268 between core bone, tendon, and tunnel wall increased significantly. As a consequence, the over-269 compression damaged the tendon fibers and made its mechanical properties weaker. So, some 270 specimens failed in the cyclic loading step. It is in agreement with the results of previous studies 271 on this method 14. 272 Also, it was observed that the core bone diameter had a significant effect on the maximum 273 strength of the reconstructed ACL (P-value = 0.000). Based on previous studies, the strength of a 274 reconstructed ACL should be more than 200 N, which is the physiologic requirement for the leg’s 275 passive motion during rehabilitation 22. It was observed that just the results of Group 2 samples 276 had the strength values higher than the 200 N limit. The critical point was that as the core bone 277 diameter increased from 8 mm to 8.5 mm, the strength of the structure increased. When the 278 diameter increased to 9 mm, due to over-compression, the result showed a lower strength than 8.5 279 mm samples. As discussed, it is believed that the over-compression of the tendon with increasing 280 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 18 the tendon size from 8.5 mm to 9 mm would decrease the fixation strength, and the core bone with 281 8.5 mm is the best choice for 8 mm tendons. 282 In addition, it was seen that the core bone diameter had a significant effect on the ACS of 283 reconstructed ACL (P-value = 0.007). The ACS means the resistance of the tendon graft to the 284 displacement in cyclic loading, and it shows the functionality of the reconstruction in daily 285 activities. It was shown that the BASHTI structure for 8 mm tendon had the best ACS when the 286 core bone diameter was 8.5 mm. As a result, this core bone diameter provided better properties 287 against the active extension loadings immediately after the ACL reconstruction surgery and during 288 the rehabilitation period. On the other hand, the core bone diameter had an insignificant effect on 289 the APS (P-value = 0.053). It is noteworthy that the APS is related to the resistance of the 290 reconstructed connective tissue to sudden impact loadings (e.g., heavy activities in soccer, 291 basketball). 292 Moreover, the core bone diameter had a substantial effect on the engaging length of the 293 samples (P = 0.002). Still, there was no significant difference between the engaging length of 294 Groups 1 and 2 (P = 0.431). It is believed that when the core bone diameter increases and the 295 tunnel size kept constant, the tendon compression and the friction between tendon and tunnel wall 296 would increase 14. Thus, a higher amount of hammer impacts is needed to insert the core bone 297 inside the tunnel. This may increase the risk of core bone fracture, and therefore a shorter length 298 of core bone would be entered into the tunnel. According to Table 1, since the Group 3 had the 299 smallest core bone diameter, no excessive hammer impacts were needed to insert the core bone 300 into the tunnel; hence, the engaging length in this group was significantly higher from Groups 1 301 and 2. Besides, in this group, although the core bone did not encounter massive strikes during the 302 insertion process, and almost remained undamaged after the fixation, since there were not 303 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 19 sufficient forces to fix the tendon into the tunnel, all of the samples in this group failed in the main 304 cyclic loading step. 305 It is concluded that the geometrical parameters had a significant effect on the 306 biomechanical properties of reconstructed ACL with BASHTI technique. While increasing the 307 core bone diameter could improve the strength and stiffness of the ACL, this diameter exceeding 308 its critical value would reduce the biomechanical properties of the reconstructed ACL due to over-309 compression. This study had some limitations that might affect the results; such as lack of human 310 tibia bone and soft tissue for more accurate modeling, absence of a comparison of the results with 311 interference screw as a gold standard of ACL reconstruction in the same conditions, and absence 312 of investigation of the interactions between core bone and tendon diameters. 313 314 5. Conclusions 315 This study aimed to find out the effect of core bone diameter on the biomechanical properties of 316 BASHTI fixation, which is an implant-less technique for ACL reconstruction. It also investigated 317 the optimum size of the core bone for a specific ligament size which was proven to have the best 318 outcome in previous studies. A series of experimental examinations were performed using 319 BASHTI technique to model the reconstructed ACL fixation and loading condition. It was 320 observed that the core bone diameter had a significant effect on almost all of the biomechanical 321 properties of the reconstructed samples. The study introduced a threshold and a critical value for 322 the optimum diameter of the core bone. Although this is not a clinical study, the outcome of this 323 study is useful to amend the ACL reconstruction treatment method. Due to the results of the 324 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452098doi: bioRxiv preprint 20 optimum diameter of core bone, BASHTI technique can be an alternative ACL reconstruction 325 method for patients. 326 327 References 328 1. Fleming BC, Hulstyn MJ, Oksendahl HL, Fadale PD. 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