1 Coupling in cyclically compressed discs

3 How osmo-viscoelastic coupling affects recovery of

4 cyclically compressed intervertebral disc
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9 Faten Fekia, Rym Taktaka, Karim Kandilb, Amil Derrouicheb, Melissa

10 Moulartc, Nader Haddara, Fahmi Zaïrib*, Fahed Zaïrid
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13
14 a
Sfax University, ENIS, Materials Engineering and Environment Laboratory
15 (LGME), 3038 Sfax, Tunisia
16 b
Lille University, Civil Engineering and geo-Environmental Laboratory (EA
17 4515 LGCgE) 59000 Lille, France
18 c
Japet Medical Device, 59000 Lille, France
19 d
Ramsay Générale de Santé, Hôpital privé Le Bois, 59000 Lille, France
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21
22 * Corresponding author.
23 fahmi.zairi@polytech-lille.fr
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25
26 The manuscript submitted does not contain information about medical
27 device(s)/drug(s)
28
29 No funds were received in support of this work.
30
31 No benefits in any form have been or will be received from a commercial
32 party related directly or indirectly to the subject of this manuscript.
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34
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39

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 5 Coupling in cyclically compressed discs
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40 Abstract
41 Study Design. Osmo-viscoelastic behavior of cyclically loaded cervical intervertebral disc.
42 Objective. Evaluate the effects of physiologic compressive cyclic loading on the viscoelastic
43 properties of cervical intervertebral disc and, examine how the osmo-viscoelastic coupling
44 affects time-dependent recovery of these properties following a long period of unloading.
45 Summary of Background Data. The human neck supports repetitive loadings during daily
46 activities and recovery of disc mechanics is essential for normal mechanical function.
47 However, the response of cervical intervertebral disc to cyclic loading is still not very well
48 defined. Moreover, how loading history conditions could affect the time-dependent recovery
49 is still unclear.
50 Methods. Ten thousand cycles of compressive loading, with different magnitudes and saline
51 concentrations of the surrounding fluid bath, are applied to eight motion segments (composed
52 by two adjacent vertebrae and the intervening disc) extracted from the cervical spines of
53 mature sheep. Subsequently, specimens are hydrated during 18-hours of unloading. The
54 viscoelastic disc responses, after cyclic loading and recovery phase, are characterized by
55 relaxation tests.
56 Results. Viscoelastic behaviors are significantly altered following large number of cyclic
57 loads. Moreover, after 18-hours recovery period in saline solution at reference concentration
58 (0.15 M), relaxation behaviors were fully restored. Nonetheless, full recovery is not obtained
59 whether the concentration of the surrounding fluid, i.e. hypo-, iso- or hyper-osmotic
60 conditions.
61 Conclusion. Cyclic loading effects and full recovery of viscoelastic behavior after hydration
62 at iso-osmotic condition (0.15 M) is governed by osmotic attraction of fluid content in the
63 disc due to imbalance between the external load and the swelling pressure of the disc. After
64 removal of the load, the disc recovers its viscoelastic properties following period of rest.
65 Nevertheless, the viscoelastic recovery, being a chemically activated process, its dependency
66 on saline concentration is governed by fluid flow due to imbalance of ions between the disc
67 tissues and the surrounding fluid.
68

69 Key words: intervertebral disc, axial cyclic loading, fluid flow, chemo-mechanical
70 coupling, recovery, osmo-viscoelastic mechanisms.
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72

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73 Mini abstract

74

75 This in-vitro study reports observations on the response of cyclically compressed cervical

76 intervertebral discs. Due to chemo-mechanical coupling, the disc viscoelastic response and

77 disc recovery capacity are found highly osmolarity dependent and modulated either by change

78 in intradiscal pressure or by imbalance of ions between disc tissues and external environment.

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98 Introduction

99 Epidemiological studies consider slow repetitive loadings as the most contributors of human

100 spine injuries1. In-vivo, repetitive loadings occur during daily activities. For example, the

101 whole body is exposed to long vibrations from industrial workplace2-3 or during athletic

102 training4. Although the attention is generally focused on the lumbar segments due to low back

103 pain issues, human neck supports also repetitive loadings. As a representative example, in

104 military occupations, personnel training and prolonged use of head-mounted devices (e.g.

105 helmet worn, visor, night vision goggles) involve cyclic loadings on the cervical segments5-6.

106 Failure mechanisms, such as annulus tears, cracks and disc prolapses, have been observed

107 under repetitive compression4,7-15 or under repetitive torsion16-18. In-vivo studies show that a

108 physiological cyclic loading alters intervertebral disc behaviors along with a loss of disc water

109 content and disc height19-23. These changes to physiological cyclic loadings are associated to

110 recoverable mechanisms and are completely restored during recovery periods upon loading 24-

111 25
. Recovery mechanism is defined as the recuperation of original properties. It is a general

112 phenomenon observed in soft tissues with various compositions, and excludes, for in-vitro

113 studies, non-recoverable failure mechanisms. The recovery capability of the intervertebral

114 disc is of prime importance for maintaining the mechanical function of the functional spine

115 unit. Conflicting data were found in the literature, especially because the characteristic time of

116 disc recovery is strongly affected by the loading history. A full recovery of mechanical

117 behavior was not found by some investigators26-32. Other investigators found that the disc

118 water content and disc height can be reproduced in-vitro after a recovery period, if unloading

119 time is twice as long as the loading time 21,27, or if the preload magnitude is large enough to

120 reduce the initial disc hydration32. Disc recovery is a chemically activated process. Recently,

121 Bezci et al.33 indicated that full recovery requires a combination of low osmotic pressure and

122 low mechanical loading. Osmotic attraction of fluid content in nucleus pulposus and annulus

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123 fibrosus is the mechanism responsible for these loads effects and recovery 34. The role of the

124 osmotic pressure in water uptake depends on the magnitude of the applied load 31. However,

125 the effect of the magnitude of the applied load in viscoelastic recovery is still not documented.

126 Besides, salinity of the testing fluid alters the inelastic monotonic response of the disc

127 tissues35-36. Up to now, the influence of saline solution changes, from hypo-osmotic to hyper-

128 osmotic conditions, on the inelastic non-monotonic response of the disc was not established.

129 In the meantime, the influence of salinity on the time-dependent recovery behavior of the disc

130 is not clearly defined and corresponding data are rare. This is needed in order to evaluate the

131 relationship between biomechanical properties, disc hydration and recovery during loading-

132 unloading process.

133 In this in-vitro study, we aim to investigate the effect of load magnitude and osmotic loading

134 on the time-dependent behavior of cervical segments subjected to compressive cyclic loading

135 and, on the viscoelastic recovery response after long unloaded period. The time-dependent

136 mechanics are characterized by relaxation tests done before and after cycling.

137

138 Materials and methods

139 Materials and specimens

140 Eight cervical spines C3-C4 of sheep from 1- to 2- years-old were obtained between 12 and

141 36 hours postmortem from a local abattoir. The choice of sheep model is due to the good

142 similarities in biomechanics between sheep and human cervical spines37. Compared to human,

143 the range of motion of the cervical sheep spine is greatest at C3-C438. Functional spine units,

144 comprising two adjacent vertebrae and the intervening disc, were carefully isolated from each

145 spine by using dissecting scissors, and separated from muscles and ligaments in order to

146 exclude interference with the facet joints. Subsequently, each vertebra was putted in a circular

147 plastic mold using a polyurethane resin in order to obtain parallel surfaces for mechanical

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148 testing. After preparation, these spine units were enclosed and stored in a freezer at -18°C

149 until the day of testing. Although the storage effect is still a controversial subject, it was found

150 that a freeze-thaw cycle has not altered the disc biomechanical behavior 39. Nonetheless, the

151 respective effects of exact freezing temperature and duration on the behavior of the disc

152 remain undefined40.

153

154 Methods

155 Before mechanical testing, the superior and inferior surfaces of each spine unit were glued to

156 two aluminum plates in order to be maintained by the clamps of the testing machine. The

157 mechanical tests were performed using a universal axial testing machine Instron-5500

158 equipped with a 5 kN load cell. All measurements were achieved at room temperature (22°C)

159 under controlled-environment, the specimen being immerged in a bath with a saline solution

160 throughout the test.

161 In this investigation, total testing time per disc was three days. For each day, six distinct test

162 phases were applied as illustrated in Figure 1. In a first phase, the specimen was hydrated in a

163 saline solution at 4°C for 16-18 hours. In a second phase, a preconditioning in compression

164 was applied in order to reduce freezing effects and prolonged immobility. It consists of 10

165 cycles with low amplitude of 0.1 mm and low rate at 0.05 mm/s to ensure fluid circulation

166 and chemical equilibrium. The reliability of this preconditioning was previously proved41. In a

167 third phase, the specimen is subjected to a compressive relaxation test at an amplitude of 0.6

168 mm (10% of the disc height). This phase was termed pre-cyclic relaxation. In a fourth phase,

169 a rest period of 30 minutes was imposed in the aim restoring both mechanical and chemical

170 equilibrium. The fifth phase constitutes the cyclic loading, in which 10, 000 compressive

171 cycles were applied at a frequency of 1 Hz. The sixth phase was to apply the same relaxation

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172 test and it was termed post-cyclic relaxation. This protocol was selected basing on previous

173 studies21-23.

174 To study the effect of cyclic loading magnitude on the disc biomechanical response, three

175 different displacement levels were compared (0.8 mm, 1.3 mm and 1.8 mm as illustrated in

176 Figure 1) while the hydration phase was kept constant at reference saline concentration of

177 0.15 M (closes to human physiological solution). To illustrate the effect of osmotic loading on

178 the disc biomechanical response and disc recovery, the cyclic loading was kept constant at 1.3

179 mm and the osmolarity of the fluid in hydration phase was increased during the three days: 0

180 M (hypo-osmotic condition), 0.15 M (iso-osmotic condition) and 0.6 M (hyper-osmotic

181 condition).

182 In the purpose to quantify the effects of amplitude and saline concentration, the relaxation

183 curves were split into two regimes and fitted using the following equations as a function of

184 the relaxation time t:

185 (1)

186 (2)

187 where is the instantaneous load, is the relaxed load at t = 400 seconds, is the average

188 relaxation time parameter and, and are parameters related to the spread of the initial and

189 final relaxation times, respectively.

190

191 Statistical analysis

192 A two-way repeated measures ANOVA was performed on the dependent variables, τ and β, to

193 test the effects of cyclic loading at different amplitudes (factor of cyclic loading and

194 amplitude) and the effects of cyclic loading at different saline concentrations (factor of cyclic

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195 loading and osmolarity). A one-way repeated measures ANOVA was performed on the pre-

196 and post-cyclic parameters τ and β with a factor of applied magnitude or osmotic loading

197 condition. A post-hoc t-test analysis was performed to determine differences between groups.

198 For all relaxation tests, a p-value less than 0.05 was considered as statistically significant.

199

200 Results

201 In Figure 2, we report the results as a function of time following the experimental protocol

202 illustrated in Figure 1. The figure presents the normalized load relaxation, the normalized load

203 softening during 10, 0000 compressive cycles and the normalized load relaxation immediately

204 measured after the cyclic loading. The results are shown for the three different amplitudes, 0.8

205 mm, 1.3 mm and 1.8 mm, after overnight hydration at reference saline concentration of 0.15

206 M. Comparing the pre-cyclic relaxation responses and the post-cyclic relaxation responses,

207 we note that the relaxation response is markedly affected by the cyclic loading (Figure 3a).

208 The relaxation time parameter τ of the pre-cyclic relaxation is significantly larger than that of

209 the post-cyclic relaxation (p < 0.001, Figure 3a, Table 1). However, there is no significant

210 effect of loading magnitude (p = 0.814) and their interactions (p = 0.589). For the relaxation

211 parameter β, also, we note a significant difference between pre- and post-cyclic loading

212 responses (p<0.001, Figure 3b, Table 1) and there is no main effect of loading magnitude, nor

213 their interactions (p = 0.492 and p = 0.277, respectively). In addition, the load during cyclic

214 loading diminishes with increasing number of cycles and it, nearly, stabilizes between 3000

215 and 4000 cycles. Comparison of the three post-cyclic relaxation responses shows a clear

216 dependency of the relaxation parameter β on the applied magnitude (p < 0.001, Figure 3b).

217 However, there is no significant effect of loading magnitude on the relaxation time parameter

218 τ (p = 0.307, Figure 3a). Post-hoc t-tests comparisons reveal a significantly larger β for 1.8

219 mm compared to 1.3 mm and 0.8 mm (p < 0.001), with 1.3 mm also being significantly larger

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220 than 0.8 mm (p < 0.001, Figure 3b). The pre-cyclic relaxation responses were measured after

221 a long hydration period (18 hours). Figure 3a shows that they are not affected by the

222 subsequent loading cycles. In fact, it can be observed that the pre-cyclic relaxation curves in

223 Figure 2d are nearly superimposed. No significant effect is observed on the pre-cyclic time

224 constant parameter τ (Figure 3a, p = 0.163) and on the pre-cyclic relaxation parameter β

225 (Figure 3b, p = 0.955). This result indicates that disc restored its viscoelastic behaviors after

226 recovery time. This finding is similar with previous study21,42 and is controversial with others

227 26,43-44
.

228 In Figure 4, we present the effects of saline concentration as the results of the first protocol

229 for three different osmolarity conditions: 0 M, 0.15 M and 0.6 M. As previously observed in

230 Figure 2, we note the same role concerning the cyclic loading effect on the relaxation

231 response for each concentration. It can be clearly seen a difference between relaxation

232 responses before and after cyclic loading (Figure 4d). The overall effect of cyclic loading and

233 osmolarity is significant for the relaxation time parameter τ (p < 0.001, Table 2, Figure 5a).

234 The interaction between cyclic loading and loading osmolarity is significant (p = 0.002), with

235 post-hoc comparisons revealing significantly larger relaxation time for 0 M and 0.15 M

236 concentrations between pre- and post-cyclic responses (p < 0.01). There is no significant

237 difference at the 0.6 M concentration between pre- and post-cyclic responses (p = 0.067). The

238 overall effect of cyclic loading is significant for the relaxation parameter β (p = 0.001, Table

239 2). However, no significant overall effect is found for saline osmolarity (p = 0.261) and their

240 interactions (p = 0.120). Even, the same results concerning load-time responses during 10,

241 000 cycles are reported, that is to say, the load decreases with increasing number of cycles.

242 Although in this case, the stabilized load is higher under hypo-osmotic condition, surrounding

243 fluid osmolarity has no effect on the post-cyclic relaxation responses. In fact, there is no

244 difference between different salinities on the post-cyclic relaxation parameters τ and β (p =

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245 0.054 respectively p = 0.500, Table 2, Figure 5). Conversely, the surrounding fluid osmolarity

246 significantly affects the relaxation responses after 18 hours of hydration. In Figure 5b, full

247 disc properties recovery is not observed. Indeed, significant effect of osmolarity is observed

248 on both pre-cyclic relaxation parameters τ and β (p = 0.045 and p = 0.041, respectively, Table

249 2). Relaxation time parameter decreases with the saline concentration and becomes faster

250 under hyper-osmotic solution (Figure 5a). In fact, post-hoc t-tests comparisons reveal a

251 significantly larger τ for 0 M compared to 0.15 M and 0.6 M (p < 0.01). With 0.15 M the

252 parameter is also significantly larger than for 0.6 M (p = 0.033).

253

254 Discussion

255 The aim of this in-vitro study was to evaluate the effects of physiologic axial cyclic loading

256 on the viscoelastic properties of cervical disc and, to examine how loading history affects

257 time-dependent recovery behavior after long period of unloading. We showed the effect of

258 cyclic loading, the effect of changes in cyclic loading magnitude and, the effect of changes in

259 saline salt bath concentration on the relaxation parameters.

260 Our results show that the increase of cycles upon cyclic loading leads to a decrease in the load

261 until stabilization between 3000 and 4000 cycles (Figure 2). Stabilized load under cyclic

262 loading was already found in earlier studies11,18,45. These results may be attributed to fluid

263 flow, simultaneously to loss of disc height during cyclic loading 20, since disc behavior is

264 strongly linked to fluid flow46. In fact, according to earlier research, during compressive cyclic

265 loading, the intradiscal pressure in the nucleus increases which pushes water out of the disc

266 and results loss of disc height31. Immediately, the osmotic pressure increases within the disc 47.

267 Once the equilibrium between the osmotic pressure and the applied load is reached, the fluid

268 flow ceases which could explain the stabilized load during loading.

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269 Increasing the applied magnitude decreases the stabilized load during cyclic loading (Figure

270 2). This result points out hardening of the disc23, which explains reversing slopes of the post-

271 relaxation curves (Figure 5b). This is conceivable. In fact, an increase of applied loading

272 magnitude leads to an increase of the quantity of water squeezed out of the disc and an

273 increase of loss disc height 31. Moreover, we found that cyclic loading affects significantly the

274 relaxation behavior of the disc. Nevertheless, these behaviors were restored after a long period

275 of unloading at reference saline concentration (Figure 4a). This is in line with Johannessen et

276 al.21 study. Presumably, during unloading, the fluid gets back into the disc, and the loss of disc

277 height is regained. Therefore, the disc restores its viscoelastic properties. Yet, our findings

278 show no influence of cyclic loading magnitude on recovery of viscoelastic behavior.

279 Interestingly, our results show that the recovery of relaxation behavior is highly sensitive to

280 the surrounding fluid concentration. Recovery is a chemically activated process. In fact, after

281 18 hours of hydration at iso-osmotic condition (i.e 0.15 M), the relaxation behavior of the disc

282 is found close to the pre-cyclic relaxation behavior. Nevertheless, recovery of relaxation

283 behavior is not observed after long hydration phase at 0 M or 0.6 M. We found that the

284 relaxation time for hypo-osmotic condition is twice than that of hyper-osmotic condition

285 (Figure 5a). In the previous case, the disc recovers its viscoelastic behavior by dint of fluid

286 back into the disc during unloading at reference osmolarity. Nonetheless, the fluid transfer

287 process is highly osmolarity-dependent33 since diffusion process depends on the difference

288 between negatively charged proteoglycans and mobile ions outside the disc. Therefore, the

289 full recovery is deleted. The osmotic dependency of the relaxation highlights the osmo-

290 viscoelastic coupling effect on the time-dependent recovery of a previously cyclically loaded

291 disc. During long hydration in hypo-osmotic condition, osmotic gradient between the disc and

292 surrounding fluid increases, and the disc swells due to fluid in-flow 48 resulting in an increase

293 in disc height. Thus, during relaxation test, the disc needs a larger characteristic time.

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294 However, during long hydration in hyper-osmotic condition, fluid is pushed out of the disc

295 resulting in loss in disc height49-50, and then the relaxation time constant increases41. By

296 contrast, Berzi et al.33 found that the increase in saline osmolarity increases the disc height.

297 Furthermore, the stabilized load during cyclic loading is different for the three osmolarity

298 conditions and it decreases from 0 M to 0.6 M. Also in this case, we note the hardening of the

299 disc and the reversing slope of the post-relaxation curves. This result points out the chemo-

300 mechanical coupling of cyclic loading response. Figure 6 presents a schematic representation

301 in which we illustrate the induced disc height changes during cyclic loading and the inherent

302 osmo-viscoelastic coupling effect on disc height recovery.

303 Several limitations ought to be mentioned. The use of sheep spine model may be a limitation

304 of this study. However, sheep model was often used for in-vivo and in-vitro experiments 51-52.

305 Another limitation was the cutting of all musculature and soft tissue, which excludes their

306 contribution in resisting to cyclic loading effects. Furthermore, room temperature and bath

307 with NaCl saline solution do not reflect the physiological environment; using phosphate-

308 buffered saline with an osmolarity of 0.15 M at 37°C would be more relevant. Finally, while

309 our interpretation of results based on the fluid flow within the disc is consistent with previous

310 studies, water content at different disc subparts (nucleus/annulus) and disc height could be

311 measured to be more pertinent.

312 In summary, axial cyclic loading responses of the cervical intervertebral disc and recovery are

313 highly dependent on the osmotic pressure, generated either by change in intradiscal pressure

314 or by alteration in the ions equilibrium between disc and external environment. For both, the

315 fluid flow and the induced disc height changes are the responsible mechanisms. The

316 loading/unloading history-dependent effects are related to the complex organization of the

317 disc components. However, more work is needed to evaluate relation between osmo-

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318 viscoelastic coupling effect on the disc behavior recovery and microstructures of the cervical

319 intervertebral disc at different scales.

320

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