The geometry of animals and human IVDs varies, and thus should be taken into consideration. Geometric parameters from various animals’ IVDs have been previously measured ^{[20][21][22][23][24]}, including baboon, sheep, rabbit, rat, mouse and bovine in terms of height, lateral width, anterior–posterior width (AP width) and area. According to the results, human lumbar specimens were larger than all of the aforementioned animal specimens. The value of normalized AP width, which was scaled by the lateral width, was 0.665 for human lumbar discs, and the normalized AP widths for baboon, sheep and mouse lumbar discs were close to human lumbar discs, indicating the similar shapes of those animals’ discs with the human lumbar discs, which were all like ‘kidney bean’.

The amount and distribution of GAG in IVDs are functionally important to define swelling pressure, water content and compressive properties ^[25][26][27]. Generally speaking, GAG and water analysis of specific regions indicate a similar trend, featuring higher GAG and water content levels in the nucleus pulpous (NP) and lower amounts of GAG and water in the annulus fibrosus (AF) ^[28][29][30]. In addition, GAG and water content were significantly different across species. According to results from Jesse et al. ^[23], GAG contents in NP of IVDs from calf, porcine, sheep, rabbit, rat and cow tail were similar to those of human species (466 ± 205 μg/mg). GAG contents in AF of IVDs from calf, baboon, rabbit, cow tail indicated approximately the same or higher content with AF from human species (269 μg/mg). The water content of calf, porcine, baboon, sheep, rabbit, rat lumbar, cow tail and rat tail IVDs was similar to that of human in the NP and AF, which were 81% and 76% separately.

Jesse et al. ^[23] conducted experiments to acquire the axial compressive mechanical parameters, including stiffness, range of motion (ROM), step and creep displacement using IVDs from various animals under the same loading protocol (cyclic loading followed by creep test under 0.48 MPa for 1 h), and revealed the impact of species related factors (GAG content, water content and size) on IVDs biomechanics. Their study concluded that the compressive stiffness of the baboon (1426 ± 382 N/mm) and sheep lumbar discs (1432 ± 334 N/mm) was closed to that of humans (1734 ± 446 N/mm). The ROM of cow tails (1.24 ± 0.31 mm) was most similar to that of humans (1.21 ± 0.18 mm). The step displacement of cow tail (1.45 ±0.60 mm) was closed to that of humans (0.90 ± 0.12 mm). The creep displacement of porcine (0.55 ± 0.18 mm), baboon (0.36 ± 0.11 mm), sheep (0.24 ± 0.03 mm), rabbit (0.47 ± 0.17 mm), rat lumbar (0.19 ± 0.02 mm), rat tail (0.40 ± 0.11 mm) was similar to that of humans (0.55 ± 0.03 mm). Their study further demonstrated that axial mechanics of IVDs were similar across species, indicating that the values between species were comparable by altering the load magnitude. In experiments simulating disc herniation, sheep IVDs are good choices with a similar shape to human lumbar IVDs and also herniated at the posterolateral region ^[31][32]. Large animal models (sheep, ovine, pigs) are suitable for investigating effects of mechanical factors, implant preclinical trials and surgical techniques on biomechanics, while small animal models (rat, mouse) are suitable for studying biological processes. When IVD pressure is of focus, animal models with a smaller cross-sectional area than human IVDs can be used, and the applied load needs to be changed proportionately ^[33][34].

Specimens of in vitro creep tests should be carefully processed into bone–disc–bone structures by making parallel cuts at the upper and lower vertebral bones with a distance of ~10 mm from the IVD using a bone saw under aseptic conditions. The functional segment unit (FSU) is more likely to be used in a kinematic study (Figure 1a). While in biomechanical tests, soft tissue, pedicles and posterior elements should also be removed (Figure 1b), and vertebral bodies can be embedded in polymetheylmethacrylata (PMMA) dental cement to ensure they are paralleled during the loading period. A separate disc without endplates can be used with external restrictions to prevent extrusion.

It should be noted that both vertebral bones and disc creep under prolonged constant loads. Although the creep of vertebral bones is tiny, several studies presented studies quantifying differences with or without bones. Schmidt performed a series of experiments ^[35][36] to compare the bone–disc–bone structure with the separate disc and demonstrated specific results with regard to displacement, stiffness and pressure under the same condition. The load regime included an 8 h preload under 0.06 MPa uniaxial compression, followed by 10 cycles of 10 min, in which the force was altered between 0.06 MPa and 0.5 MPa. Combined with the research of Oravec et al. ^[37], three types of specimen structures were used in total as follows: (1) a disc with endplates, (2) a disc without endplates and (3) an isolated vertebral body.

The disc contributed to the majority of the height loss during creep, and the bony endplates also exhibited significant impacts on the whole creep value (0.341 ± 0.269 mm under 1000 N after 2 h). Therefore, studies using bone–disc–bone specimens should exclude the height loss of vertebral bodies by appropriate methods of measurement in order to avoid larger and inaccurate results. It can be also seen that the stiffness of specimens with and without endplates was the same (700 N/mm). In the specimen without endplates, the intradiscal pressure was reduced by nearly 50% (~0.43 MPa). This may have contributed to the easy extrusion of the disc without circumferential constraints formed by the annulus and the bony structure, thus resulting in relatively unstable stress and a larger area of the cross-section. It is a reminder that a separate disc is an unsuitable structure in the study related to intradiscal pressure measurement.

Specimens for in vitro creep studies should be stored at −20 °C before testing. McMillan et al. ^[38] reported that, in case of inappropriate IVD storage (unloaded in a wet environment), swelling by 20% was noted due to the tension in the ligamentum flavum, generating pressure in NP for approximately 70 kPa ^[39]. Generally, a single freeze–thaw cycle exhibited minimal effects on the intradiscal pressure, stiffness, creep behavior of IVDs ^[40][41][42] and on the tensile property of AF ^[43]. However, after several freeze–thaw cycles, significant differences in the joint flexibility may occur ^[44], and several studies have reported mechanical differences between fresh and frozen IVDs ^[45][46]. To correct these differences, several studies verified that specimens immersed in saline solution or phosphate-buffered saline for more than 8 h prior to testing may be corrected for physiological hydration status ^[47].

During experiments, hydration should be maintained to avoid the effects of dehydration by the following guidelines: (1) testing in a humidity chamber, (2) immersing in 0.15 M phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 5.4 mM Na2HPO4, and 0.6 mM KH2PO4) or saline solution (0.9% or 0.15 mol/L NaCl), or (3) wrapping in saline-soaked gauze ^[19]. The majority of studies immersed specimens in saline solutions during tests, while other studies placed specimens in the custom build culture system, in which the environment was kept at 37 °C with the presence of 5% CO2 ^[48][49], simulating the physiological conditions. To prevent infection, 10,000 u/mL penicillin, 250 mg/L streptomycin and 1.5 mg/mL amphoterizin B should be added in experiments lasting for more than 20 h ^[50]. A previous study focused on the influence of osmotic pressure on the fluid flow of IVDs with the change in concentration of PEG or NaCl ^[51]. Those studies conducted tests in the air ^[52][53] failed to maintain the hydration status, and their results should be treated dialectically.

Temperature is another factor that needs to be controlled. It was reported that body temperature at 37 °C could cause a 10% higher creep under compression than that at room temperature ^[54], while the results have not been confirmed by other studies.

Prior to creep tests, preload is necessary for reaching physiological status and squeezing additional water. Moreover, it can prevent postmortem swelling, which often occurs on cadaveric IVDs ^[55]. The non-linear property of the IVDs suggests that stiffness can increase if a preload is applied prior to creep ^[56]. Therefore, the results of the experiments with the necessary preload period can be comparable. Although the duration of preload conditioning differed considerably, it was usually short before displacement equilibrium ^[34][57][58].

Typical forms of preload are ‘static’ and ‘cyclic’. Generally, although certain studies incorporated thousands of pre-cycles ^[59], a static preload from 10 N to 50 N or three pre-cycles was sufficient for a consistent response of the IVDs ^[50].

Firstly, the load magnitude is of importance since disc height and intradiscal pressure are directly related ^[60][61]. Moreover, the strain in the annulus or bulge of the annulus fibrosus could be affected ^[62]. The magnitude of compressive force applied to the IVD varies in magnitude with changes in body posture, body weight, muscle activity and external loads ^[63][64][65]. In a study performed in eight healthy subjects, Nachemson and Morris et al. ^[66][67][68] demonstrated that the in vivo pressures in NP ranged from 0.091 MPa to 0.539 MPa when lying in prone or supine positions, from 0.46 MPa to 1.33 MPa in a seated position and from 0.5 MPa to 0.87 MPa in a standing position. Wilke et al. ^[61] reported that the highest pressure in the NP, 2.3 MPa, was recorded in a standing subject who was flexing forwards while simultaneously holding a 20 kg mass. These pressure values can be converted into corresponding load values by multiplying the area of the cross-section, which are ~100 N for L45 human IVDs during bedtime rest ^{[20][36][60][61][69][70]} and 750–1200 N during daytime activities. The loads for cervical spines are relatively lower than those for lumbar spines. For bovine tails (e.g., C23, C23), 27.7–209.1 N (0.06–0.28 MPa) and 211.6–373.4 N (~0.5 MPa) were simulated for loads of rest and daily activity ^[20][36][71]. For sheep lumbar, 40–60 N (~0.45 MPa) and 80–180 N (~1.05 MPa) were equal to nighttime unloading and daytime loading, respectively ^[34][60][72].

The representative loading regimes are shown in Figure 2, Figure 3 and Figure 4 and these can be divided into static, quasi-static and dynamic forms. Static load is the most simplified form of daily activities (e.g., sleep and sit) and is widely conducted in studies focusing on the mechanical properties of IVDs during creep. The quasi-static and dynamic loads are used to simulate the human body in physical exercise, driving and other daily activities. In addition, according to the frequencies of the vehicle under normal transportation conditions, the frequencies of vibration can be selected from 0 to 8 Hz in the dynamic creep experiments. Previous studies ^[73][74] suggested that, due to the resonant frequency of the human body, certain loading frequencies (4–6 Hz) may be harmful. Table 3 summarizes the magnitudes and durations of preloads, loads and the major results from studies.

Secondly, the physiological condition, which is characterized by 8 h-preload and 16 h-load, can be shortened in the in vitro creep study. It has been shown that the measured displacement was more than 80% of the equilibrium displacement following creep for 4 h ^[47]. However, it should be noted that the creep behavior reached equilibrium within ~12 h ^[78] and that the time constant of human discs under creep was estimated at 14 h as reported by O’Connell et al. ^[47].

The static loads from 100 N to 1200 N are usually applied to simulate the diurnal load from the aggregated literature ^{[51][71][75][76][78]}. The large range of the selected loads is mainly due to significant differences in the area of cross-section of the specimens used in studies. Moreover, it should also correspond to the physiological conditions of the species according to published in vivo studies. The selection of the duration varied from 10 min to 24 h. Some studies conducted tests for 12–24 h to simulate responses of IVDs diurnally ^[51][75]; other studies even lasted for several days and were thought to be time-consuming ^[78]; the reduction to 10 min may be too short to explore the creep behaviors ^[77]. A previous study indicated that the majority of the equilibrium displacement (>80%) occurred within 4 h ^[47], and the duration of their creep study was also 4 h, leading to the advice that the duration of 4 h was suitable to reduce the time cost.

The forces in the quasi-static creep experiments are alternated with high and low loads with a certain interval. The loading regimes in Figure 3 are typical quasi-static patterns, which contain more than 10 cycles of 15 min, including a high load (~0.5 MPa) and a low load (~0.06 or 0.28 MPa) in a cycle. This enables the simulation of the behaviors of the IVDs under loads with frequencies in daily activities. The quasi-static load always lasts for 2–4 h to replicate the in vivo status of the daytime loading period.

Dynamic loading regimes can be used to replicate the creep response of IVDs under various physiological frequencies. According to the range of frequency of daily activities (e.g., doing sports or driving), it could be selected from 0 to 8 Hz to simulate the in vivo conditions ^[80]. In the study from Barrett et al. ^[79], the mean force of sinusoidal load was 1500 N (Figure 4), which was relatively higher than that of the physiological loads. 130 N and 200 N were used in the studies of Vergroessen et al. ^[72] and Yang et al. ^[80] which were considered as rationally based on physiology.