7 Purpose : Nucleus augmentation has been proposed as an early -stage intervention for intervertebral 8 disc degeneration and involves the injection of a biomaterial into the nucleus to restore disc height 9 and functional ity. The aim of this work was to identify clinically relevant quantitative measures that 10 indicate the mechanical performance of the disc following nucleus augmentation . 11 Method : Bovine tail bone -disc-bone units (n=22) were mechanically tested under cyclic loading 12 sequentially in native, artificially degenerated and treated states. Treatment invo lved injection of a 13 peptide:glyc osaminoglycan mixture into the degenerated disc to a predetermined load using a syringe 14 driver with an integrated force sensor . The mechanical restorat ion of the treatment was determined 15 by comparing the biomechanical behaviour of the native state to the treated state of each disc. The 16 mechanical restoration was then compared against clinically quantitative parameters. 17 Results : Significant biomechanical differences were observed from the degenerate state to the native 18 and treated states. The force delivered during injection was found to ramp to a steady state, followed 19 by a final rapid increase, however a ll measures associated wi th injection force poorly correlated with 20 level of mechanical restoration. Direct measures of the disc, volume injected and change in disc height 21 from injection, had the strongest relationship to mechanical restoration. 22 Conclusion : This work showed that me asuring the injection force for injectable treatments of the disc 23 can provide lower and upper limits for delivery, but direct measures are stronger indicators of disc 24 mechanical restoration. 25 26 Keywords: Intervertebral disc degeneration, nucleus augmentation , surgical optimisation, 27 biomaterials, biomechanics. 28 Statements and declarations: 29 Funding: Financial support through the grant EPSRC grant EP/R513258/1 is gratefully acknowledged. 30 Employment, Financial interests & Non -financial interests: Nothing to declare 31 Authors’ contribution : Conceptualization (ARD, JPW, MM, RKW); Data curation (ARD, RKW); Formal 32 Analysis (ARD, JPW, MM, RKW); Funding acquisition (RKW); Investigation (ARD, JPW, MPC); 33 Methodology (ARD, JPW, MPC); Project administration (RKW); Supervision (MM, RKW); Visualization 34 (ARD, JPW); Writing – original draft (ARD, JPW); Writing – revie w & editing (ARD, JPW, MPC, AK, 35 MM, RKW) . A. Dixon and J. Warren contributed equally to this paper. 36 For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) 37 licence to any Author Accepted Manuscript version arising f rom this submission. 38 39 2 1. Introduction 40 Nucleus augmentation, in which a biomaterial is injected into the intervertebral disc without prior 41 denucleation, has been proposed as an early stage intervention for disc degeneration [1]. A number 42 of injectable biomaterials are under development for nucleus augmentation, and studies have 43 reported on both their biological and mechanical characteristics [2]. The biomechanical performance 44 of intervertebral disc specimens augmented with candidate biomaterials ha s also been evaluated 45 through a range of in vitro tests in cadaveric human and animal tissue , with some evidence of the 46 ability of the treatment to restore short -term biomechanical function [3]. In these studies, the amount 47 of biomaterial injected in the nucleus augmentation procedure has been either to a fixed volume or 48 based on the haptic judgement of the researcher [2] , and there has been little investigation to date 49 on the effects of clinical variables such as the volume of injected material or the force required to 50 deliver it. 51 In a clinical setting, if the volume of biomaterial injected into the nucleus were too low, then there 52 would be le ss restoration of disc height or biomechanical functio n, while too great a volume could 53 potentially increase the risk of herniation or end-plate infraction [4]. However, the optimum volume 54 to inject would likely be governed by the size, extracellular constituents, and degenerative state of the 55 disc. An alternative may be to measure the mechanical resistance of the disc to filling, by m onitoring 56 the force required to deliver the biomaterial [5]. 57 The aim of this contribution was to investigate the relationship between clinically quantifiable 58 measures of biomaterial delivery in nucleus augmentation , and the resulting mechanical performance 59 of the augmented intervertebral disc. The study was conducted using an in vitro biomechanic al model 60 to allow the greatest control of confounding factors, and a previously developed peptide - 61 glycosaminoglycan (GAG) injectable hydrogel [6, 7] . 62 63 3 2. Materials and methods 64 2.1. Specimen preparation 65 Bovine tails (C1-C4) (n=12) were imaged and sectioned in to bone -disc-bone units (BDBU) retaining a 66 consistent thickness of vertebral bone [8]. The exposed bone was clean ed using a surgical water pik 67 (Pulsavac Plus Wound Debridement System, Zimmer Biomet, USA) and the specimens were placed in 68 an agitated anticoagulant bath at 4 °C for 24 hours (sodium citrate, 20.5 mM) . Specimens were rinsed 69 and frozen to -80 °C until the day of testing. 70 2.2. Overview 71 The prepared BDBU s were subjected to a sequential test ing protocol , where the units were 72 biomechanically tested in the native state, subjected to a rapid enzymatic degeneration procedure, 73 tested in the artificially degenerated state, injected with a hydrogel , and finally tested in the treated 74 state. A summary of the testing is shown in Figure 1A. 75 76 Figure 1 – A) Summary flow chart of test procedure, B) hold period and biomechanical test compressive loading, and C) 77 treatment injection fixture set up (not to scale) . 78 2.3. Biomechanical testing 79 BDBUs were biomechanically tested in the native, degenerate and treated states. To establish a 80 physiologically relevant osmotic equilibrium, and ensure consistency of hydration between tests, the 81 Commented [MM1]: I think we should say how many tails were used 4 BDBM units were held for a 24 hour period in a phosphate buffer saline (PBS) bath under a 40 N load 82 prior to each testing phase (steps 1, 6, and 8 in Figure 1A) . After the hold period, the BDBUs were 83 subjected to a 1,000 cycle cyclic compression test at 1 Hz between 300 N and 740 N as shown in Figure 84 1B (steps 2, 7, and 10) . Cyclic compression testing was conducted with in a PBS bath using a n 85 electromechanical testing machine (ElectroPuls E10000, 2527 Series 10 kN Bi -axial Dynacell, Instron, 86 USA) . After each cyclic compression test, specimens were imaged using micro -computed tomog raphy 87 (74 µm voxel size, µ CT100, Scanco M edical AG, Switzerland) . 88 2.4. Enzymatic degeneration 89 After the native state testing, the BDBUs were artificially degenerated by injecting 0.3 ml papain 90 (1.6 kU/ml ) with a 30G needle to non -selectively break down the collagen and proteoglycan structures 91 within the nucleus [9]. The units we re then left to digest for a 24 hour hold period under 40 N load at 92 42 °C (step 4) and then 0.3 ml of a papain inhibitor, ebselen (0.064 µmol) , was injected preventing 93 further degeneration (step 5). Following biomechanical testing (steps 6 and 7) , the unit s were frozen 94 to -80 °C until ready for treatment. 95 2.5. Nucleus augmentation 96 The nucleus augmentation procedure was carried out between the hold period and the cyclic 97 compression test (Step 9). The augmentation was performed using a peptide -GAG hybrid hydrogel 98 (P11-12 and chondr oitin sulfate , 1:20 ratio [6, 7] ). Lyophilised peptide (P 11-12, Ac -SSRFOWOFESS -NH 2) 99 was weighed out (20 mg, 0.014 mol, 95% purity, CS Bio , CHEMGO Organica AG, Switzerland ) into 100 glass vials and reconstituted in 130mM NaCl aqueous solution (500 μl). Lyophilised chondroitin 101 sulfate (CS) (MW ~ 50 kDa, ZPD , Zeria Pharmaceutical Co., Ltd, Denmark ) was weighed out (137 mg) 102 into glass vials and reconstituted in 130mM NaCl aqueous solution (500 μl). The samples were then 103 vortexed ( 30s) at 2500 rpm on a vortex mixer . After early analysis of the augmented discs, an 104 additional step was added to sonicate the peptide and GAG components to reduce the likelihood of 105 microbubble formation. The peptide and GAG components were injected separately via two 25G 106 needles in to the cen tre of the nucleus pulposus , such that mixing and gelation occurred in situ . To 107 allow radiographic analysis of the injected material in the post -test CT scans , a radio -opaque agent 108 (Ultravist ®) and UV fluorescent dye (5,6-carboxyfluorescein) were added to the gel components . The 109 injection was performed using a custom rig that connected two syringes in parallel to a syringe 110 driver such that both components were delivered at a constant rate. A 40 N axial compressive load 111 was applied to the BDBU during injection, as shown in Figure 1C. A transducer (Flexiforce B201 112 series, Tekscan Inc, USA) was inserted between the syringe driver and the rear of the custom 113 injec tion rig to measure the force applied to the syringes (‘injection force’). The volume of hydrogel 114 5 injected was measured using the syringe markings. Injection was stopped when a predetermined 115 load was reached, the syringes were empty, or if the syringe driver began to slip repeatedly . The pre - 116 and post -injection BDBU height were recorded using Vernier callipers. 117 2.6. Data analysis 118 Specimens were excluded from the analys is if they presented less than 10% change in stiffness 119 following the degeneration procedure, or if the post -test radiographic analysis revealed that either 120 the injections were not into the nucleus, or there were observable coalesced air microbubbles . A total 121 of 22 specimens were analysed. Using an ad -hoc script (MATLAB and Statistics Toolbox Release 2021a, 122 The MathWorks, Inc., Natick, Massachusetts, United States ), the stiffness of each individual loading 123 cycle was computed from a linear fit of the load -displacement data, excluding 5 points of the extreme 124 values (corresp onding to approximately 10% of the cycle at the beginning and the end of the loading 125 phase ). From each 1,000 cycle s of testing for each BDBU state , the mean stiffness over the last ten 126 cycles was extracted for comparison between samples and states (hereaft er referred to simply as 127 “stiffness ”). Mechanical restoration following treatment was defined as the difference between the 128 native and treated stiffness. 129 Due to the paired and non -normal nature of the data, a Friedman test was applied to compar e the 130 differ ent states , with post hoc Wilcoxon sign rank tests (α=0.05) . All specimens were placed into two 131 sets of groupings related to the total volume injected (low < 0.85 ml, 0.8 5 ml < med ium < 1.25 ml, high 132 > 1.25ml) and to the maximum injection force achieved recorded during the delivery (low <30 N, 30 N 133 < med ium < 40 N, high > 40 N). Due to the small sample sizes in the subgroups, and the nature of the 134 data, only the median , maximum and minimum values for these subgroups were investigated . 135 Statistical analysis was performed using the Statistics Toolbox in MATLAB (v 2021a) . 136 The mechanical restoration was evaluated against the six measurable or calculable in clinic parameters 137 using linear regressions : volume injected, volume injected/disc cross -sectional area, injection force, 138 injection force/disc cross -sectional area, work done (integral of injection force -time plot), and change 139 in BDBU height following injection (post -injection height minus pre -injection height). 140 All data is in this study is av ailable open access at the Leeds data repository [10]. 141 142 Commented [MM2]: Add reference: Dixon, Warren, Mengoni and Wilcox, 2023, dataset: Bovine intervertebral disc longitudinal in vitro study: biomechanical effect of volume and pressure of hydrogel injection into intervertebral discs 6 3. Results 143 In all specimens and states, the s tiffness increased throughout the duration of the cyclic loading and 144 the stiffness in the degenerated state was higher than in the native state for each specimen . Typical 145 profiles for the stiffness of a specimen across the three states are show n in Figure 2A; the stiffness for 146 all specimens in each state is shown in Figure 2B. During the nucleus augmentation pr ocedure, the re 147 was an initial ramp in the injection force , followed by a period where force remained relatively steady 148 before it rose more steeply . An example showing these three phases is shown in Figure 2C. 149 150 Figure 2 – A) Typical stiffness plot for different states, and B) Comparison of stiffness in different states for all specimen C) 151 typical injection force plot 152 7 Significant differences were found in stiffness values between the degenerate and the native states 153 (post -hoc Wilcoxon test, p<0.001) or between the degenerate and the treated states (post -hoc 154 Wilcoxon test, p<0.001) , and no difference between the treated and native states (post -hoc Wilcoxon 155 test, p=0.78) . The median (max, min) of the stiffness restoration for the low, medium, and high volume 156 groups were: - 1.24 (-0.38, -1.92 ) kN/mm, 0.19 (0.70, -0.36 ) kN/mm , and 0.28 (0.75, - 0.14 ) kN/mm 157 respectively , a summary plot of the stiffness restoration is shown in Figure 3A. Similarly, the low, 158 medium and high force groups median ( max, min ) restoration value s were: -0.36 (0.50, -1.92 ) kN/mm, 159 -0.09 (0.70, -1.43 ) kN/mm, and 0.12 (0.74, -0.43 ) kN/mm respectively. Mechanical r estoration was 160 compared against parameters of interest, an example is shown in Figure 3B. The r -squared and p 161 values of linear regressions for all parameters are shown in Table 1. 162 163 Figure 3 - A) Summary of stiffness differences compared to native state for low, medium, and high volume specimens, 164 B) Linear regression relationship between mechanical restoration and volume/cross sectional. 165 166 Table 1 – Relationship (linear regression) between parameters and treatment stiffness mechanical restoration 167 Parameter R-squared p-value Volume injected (ml) 0.63 < 0.001 Volume injected/Cross sectional area ( µl/mm2) 0.71 < 0.001 Max Injection force (N) 0.23 0.023 Max Injection force/cross sectional area (N/mm2) 0.28 0.011 Work done ( J) 0.31 0.00 7 Change in height (pre injection – post injection) (mm) 0.79 < 0.001 4. Discussion 168 The objective of this work was to assess the use of clinically relevant quantitative measures for nucleus 169 augmentation . Three key variables were measured : the volume injected, the injection force, and the 170 chan ge in disc height following augmentation . The results showed that the nucleus augmentation 171 procedure was able to restore specimen stiffness , with no statistical ly significant difference found 172 across all the data between the native and treated states. When divided into t he subsets based on the 173 volume injected or the applied force, a minimum volume was required to achieve mechanical 174 restoration (Figure 3A). 175 Commented [MM3]: These 2 figures are not vertically aligned which I find very unpleasant to look at Commented [MM4R3]: + may be better side by side than on top of each other 8 The hydrogel was able to reduce the stiffness of the disc from the degenerate state to that of the 176 native state , with no significant differen ce. However, the degenerate model used has some limitations 177 as it is not possible to mimic all stages of degeneration in the lab . Some variation in the severity of 178 degeneration were observed due to natural tissue variation, however, the degenerate model was only 179 representative of a n early -stage degeneration level , with degeneration induced from the enzyme 180 localised to the nucleus. This may not be fully representative of degeneration where annular tears can 181 be observed clinically [17, 18]. Instead, the degenerate model was representative of degeneration 182 with minimal annular pathology [19] which would be a target degeneration level for a nucleus 183 augmentation treatment . 184 Previous in vitro studies of nucleus augmentation had stopped injection based either on reaching a 185 given volume [11–13] or haptic feedback [14–16]. Not only was the injection force dependent on the 186 state of the disc but also the fluid viscosity, the syringe, and the needle gauge and length which these 187 previous studies made reference to , but the effects of systematically varying either volume or injection 188 force were not assessed as reasons for stopping the injection. In this study, it was found that the 189 volume of biomaterial injected, the volume normalised by disc cross -sectional area, and change in disc 190 height from injection, provided good indicators of the level of mechanical restoration of disc 191 biomechanical properties. The strongest relationship identified was with the change in disc height pre - 192 and post - injection, which is unsurprising given the relationship between height and stiffness. Overall, 193 the relationship between the direct volume and height measurements and the level of stiffness 194 restoration was much better than for the measurements derived from the applied injection force. 195 Clinically, the volume and height measurements may be readily implemented . Volume can be 196 moni tored on syringes and change in disc height may be approximated with fluoroscopy or other 197 available imaging modality during surgery . The volume of injected biomaterial normalised by the disc 198 cross -sectional area was better correlated to the mechanical restoration than the volume alone. The 199 curved nature of the endplates meant the consistent measures of disc height were more difficult to 200 derive from the CT data, so only the disc cross -sectional area rather than the disc volume was used to 201 normalise the injection volume , and the stiffness was not normalised (i.e. as an apparent modulus) to 202 the disc dimensions . Clinically, there could be potential to better estimate all of the disc dimensions 203 from additional magnetic resonance imaging [20]. 204 The injection force was monitored as a parameter to provide a qua ntitative measure of the haptic 205 feedback used clinically to determine intradiscal pressure. A s the hydrogel is injected into the disc, the 206 expansion of the nucleus will be resisted by the tensioning of the annulus tissues and the intradiscal 207 pressure will increase. The force required to inject the biomaterial was therefore expected to increase 208 9 during the delivery [21, 22]. This study found that the injection force initially increase d to a steady 209 value, which was maintained for a period of time before rising steeply. In the in vitro model used here, 210 the artificial degeneration of the disc caused a loss of disc height and increased stiffness during 211 mechanical testing , where our current hypothesis is the degeneration potentially caused a loss of GAG 212 and fluid content in the nucleus resulting in the altered biomechanics . There is therefore little 213 resistance to the injection initially as the disc ‘ refills’. I t is only once the annulus becomes tensioned 214 that the force increases consistently . Whilst the maximum injection force would in theory be a good 215 indicator of fill, in this study all measures associated with injection force poorly correlate d with the 216 mechanical restoration of biomechanical function. It is likely that in vivo , the progres sive degeneration 217 of the disc and remodel ling of the surrounding tissues would provide greater resistance to injection in 218 the initial stages, and the injection force would be more difficult to interpret. Therefore, injection 219 force alone does not appear to provide sufficient data to be used as a clinical measurement tool during 220 nucleus augmentation. Nevertheless, when used in conjunction with the directly measured volume 221 and height parameters, injection force could still provide a meaningful upper limit meas urement to 222 prevent damage. An alternative to measuring the injection force would be to directly monitor the 223 nuclear pressure ; however, this is associated to a range of issues and constraints [23-25]. 224 In this study, the biomaterial used for the nucleus augm entation was a previously developed peptide - 225 GAG hybrid hydrogel [6, 7] . The GAG component acts both to trigger rapid self -assembly of the 226 peptide, and also to mimic the healthy nucleus tissue’s ability to imbibe water. By having a similar 227 GAG composition to the native nucleus, and a hydrogel with a high water content, the aim was that 228 the natural balance between the load and osmotic pressure would reduce risks associated with 229 overfilling. It should, therefore, be noted that while the results of this study are applicable to other 230 nucleus augmentation materials, the ranges of the measured injection parameters likely depend on 231 the biomaterial us ed and its properties. It is likely that materials with different osmotic potential, 232 gelation mechanisms or mechanical properties could result in different biomechanic al outcomes for a 233 given injected volume. The needle gauge and length used for injection w ill also affect the applied 234 forces. Although the relationships between injection parameters and resultant biomechanical 235 performance may be unique to a given material, the parameters and techniques developed in this 236 work could readily be applied to other bi omaterials. 237 5. Conclusions 238 This research clearly demonstrates that mechanical restorative outcomes for nucleus augmentation 239 vary with injection parameters. Specifically, the maximum force required to inject the hydrogel has a 240 weak relationship with mechanical restoration but could provide upper and lower limits for injection. 241 10 Whereas the volume injected and change in height from injection have strong relationships with 242 mechanical restoration and are likely suitable outcome predictors . 243 244 11