Effects of Level, Loading Rate, Injury and Repair on Biomechanical Response of Ovine Cervical Intervertebral Discs

Abstract

A need exists for pre-clinical large animal models of the spine to translate biomaterials capable of repairing intervertebral disc (IVD) defects. This study characterized the effects of cervical spinal level, loading rate, injury and repair with genipin-crosslinked fibrin (FibGen) on axial and torsional mechanics in an ovine cervical spine model. Cervical IVDs C2-C7 from nine animals were tested with cyclic tension-compression (- 240 to 100 N) and cyclic torsion (± 2° and ± 4°) tests at three rates (0.1, 1 and 2 Hz) in intact, injured and repaired conditions. Intact IVDs from upper cervical levels (C2-C4) had significantly higher torque range and torsional stiffness and significantly lower axial range of motion (ROM) and tensile compliance than IVDs from lower cervical levels (C5-C7). A tenfold increase in loading rate significantly increased torque range and torsional stiffness 4-8% (depending on amplitude) (p < 0.001). When normalized to intact, FibGen significantly restored torque range (FibGen: 0.96 ± 0.14, Injury: 0.88 ± 0.14, p = 0.03) and axial ROM (FibGen: 1.00 ± 0.05, Injury: 1.04 ± 0.15, p = 0.02) compared to Injury, with a values of 1 indicating full repair. Cervical spinal level must be considered for controlling biomechanical evaluations, and FibGen restored some torsional and axial biomechanical properties to intact levels.

Keywords: Annulus fibrosus; Biomechanics; Hydrogel; In vitro; Large animal; Tissue engineering.

Figure 1. Repeated measures study design for assessing biomechanical response of intact, injured and repaired ovine intervertebral discs (IVDs)

(A) IVDs from five cervical levels (C2:C7) from nine animals were distributed to two groups in a repeated measures study design. (B) The cervical IVDs were distributed to two groups independent of level. (C) IVDs distributed to Injured and FibGen groups were tested in the Intact condition and injured with 2- mm biopsy punch. IVDs in the Injured group were tested after injury. IVDs in FibGen group were repaired with injection of FibGen and tested.

Figure 2. Biomechanical testing procedures and parameter definitions for torsional and axial biomechanical testing

(A) The torque range was calculated as the peak to peak torque between ± 2° and ± 4°; the stiffness was slope of top 20% of the torque rotation curve at ± 2° and ± 4° which was averaged for clockwise and counter-clockwise stiffness values. (B) The range of motion was the total displacement between the common applied load for that frequency (for 0.1 Hz, 94 N and -227 N); the axial compliance was the slope of 20% of the displacement force curve.

Figure 3. The two cranial (C2-C3 & C3-C4) motion segments (n = 9/level) have different axial response than the two most caudal levels (C5-C6 & C6-C7)

(A) The force displacement curve of a C3-C4 motion segment has lower range of motion (ROM) and lower tensile compliance than the C5-C6 motion segment from the same animal. (B) The axial ROM of C2-C3 & C3-C4 were significantly lower than C5-C6 & C6-C7, and the ROM of C2-C3 was significantly lower than C4-C5. (C) The tensile compliance for C2-C3 was significantly lower than C5-C5 and C6-C7, and C3-C4 was significantly lower than C6-C7 but was only a trend lower than C5-C6 (p = 0.09). There were no differences in compressive compliance between levels. Lines are median, error bars are interquartile range and bars indicate significant difference (p < 0.05).

Figure 4. The two cranial (C2-C3 & C3-C4) IVDs (n = 9/level) have different torsional response than the two most caudal levels (C5-C6 & C6-C7)

(A) The torque-rotation curve for ± 2° rotation at 0.1 Hz of a C3-C4 motion segment has higher torque range and torsional stiffness than the C5-C6 motion segment from the same animal. (B) The torque range between ± 2° of the two most cranial levels (C2-C3 & C3-C4) were significantly higher than two most caudal levels (C5-C6 & C6-C7) at 0.1 Hz. (C) The torsional stiffness at ± 2° of C2-C3 and C3-C4 was significantly greater than C5-C6 & C6-C7. (D) The torque rotation curve for ± 4° rotation at 0.1 Hz of a C3-C4 motion segment has higher torque and torsional stiffness than the C5-C6 motion segment from the same animal. (E) The torque range between ± 4° of the two most cranial levels (C2-C3 & C3-C4) were significantly higher than two most caudal levels (C5-C6 & C6-C7) at 0.1 Hz. (F) The torsional stiffness at ± 4° of the C2-C3 and C3-C4 levels were significantly greater than C5-C6 and C6-C7, and C3-C4 had significantly greater torsional stiffness than C4-C5. Lines are median, error bars are interquartile range and bars indicate significant difference (p < 0.05).

Figure 5. Torsional loading rate (n = 45) increased torque range and torsional stiffness

(A) For rotation of ± 2°, the torque range of 0.4 °/s loading was significantly lower than for 4 °/s and 8 °/s. (B) The torsional stiffness at ± 2° was significantly higher for each increasing loading rate from 0.4 °/s to 4 °/s and 8 °/s. (C) Representative torque rotation curves at 0.4, 4 and 8 °/s shows the median change in torque range and torsional stiffness. (D) For rotation of ± 4°, the torque range was significantly higher for each increasing loading rate from 0.8 °/s to 8 °/s and 16 °/s. (E) The torsional stiffness at ± 4° was significantly higher for each increasing loading rate from 0.8 °/s to 8 °/s and 16 °/s. (F) Representative torque rotation curves at 0.8, 8 and 16 °/s shows the median change in torque range and torsional stiffness. Lines are median, error bars are interquartile range and bars indicate significant difference (p < 0.05).

Figure 6. Annulus injury altered some parameters of axial biomechanical response relative to intact more than the FibGen repair

(A) The range of motion (ROM) (ratio to intact) of the Injured group was higher than the FibGen group. (B) The tensile compliance ratio and (C) compressive compliance ratio did not differ between groups. (D) Representative force displacement curves of an Injured sample in intact (black) and injured (red) condition shows median effect of injury on axial ROM. (E) Representative force displacement curves of a FibGend sample in intact (black) and repaired (green) condition shows median effect of repair on axial ROM. Lines are median, error bars are interquartile range and bars indicate significant difference (p < 0.05).

Figure 7. Annulus injury altered torsional biomechanical properties relative to intact and FibGen repair restored some of these changes

(A) The ± 2° torque range (ratio to intact) of the Injured group was higher than the FibGen group. (B) The torsional stiffness of the Injured group had a trend of increase compared to the FibGen group (p < 0.1). (C) Representative ± 2° torque rotation curves of an Injured sample in intact (black) and injured (red) condition shows median effect of injury on torque range. (D) The ± 4° torque range ratio of the Injured group had a trend of increase compared to the FibGen group (p < 0.1). (E) The ± 4° torsional stiffness ratio did not differ between groups. (F) Representative ± 4° torque rotation curves of a FibGen sample in intact (black) and repaired (green) condition shows median effect of injury on torque range. Lines are median, error bars are interquartile range, bars indicate significant difference (p < 0.05) and dashed line bars indicate trends (p < 0.10).