Nucleus replacement model
A standardised discectomy procedure was developed, intended to allow the evaluation of novel nucleus replacement therapies in vivo. The procedure was designed for, and performed on mature Dutch female goat intervertebral discs. Initially, the NP is evacuated with custom-made instruments (Fig. 1a). These instruments consist of tubes with increasing diameter, which are used to make an entry site laterally into the AF. Via the largest tube, with an outer diameter of 3 mm, instruments are inserted to evacuate the NP. The discectomy was always performed as complete as possible without damaging the AF or the endplates, and the result was judged by the surgeon before continuation. After evacuation, the disc space is filled with a dense collagen implant (NuRes, Arthro Kinetics AG, Esslingen, Germany) that has been described previously . Shortly, collagen gel is polymerised after which the density is increased by plastic compression. The chosen density was 25% w/w of collagen, which has a stiffness comparable to the native NP. For this study, the collagen matrix was prepared in a ‘snake’-like shape (diameter 2.5 mm, length 30 mm, volume ~0.6 cm3), allowing implantation via the tubes (Fig. 1b). After insertion of the collagen implant, the annulus defect was closed with one of the four different versions of a polyethylene closure device described below.
Annulus closure devices (ACDs)
We first performed extensive preliminary testing, using the same set up for axial compression as described below (see “Biomechanical evaluation”). These pilot experiments (data not shown) were intended to determine the optimal shape and dimensions of the annulus closure devices (Fig. 2a). All devices were intended to close a standardised 3-mm circular defect in the AF of the goat intervertebral disc, as described above. Four devices were further evaluated in the current study (Fig. 2b) since they were found to withstand axial compression forces over 1,000 N. These four ACDs were composed of polyethylene and consisted of a core (diameter 1.3 or 1.5 mm) with four or five barb rings that have a maximum diameter of 3.5 mm (Fig. 2b). The ACDs were introduced into the AF till all barb rings were inside the defect. The back end of ACDs was used to hold the implants during implantation, and this was cut after implantation (Fig. 2c).
Intradiscal pressure calibration measurements
In order to determine the relation between the applied load and the pressure inside the goat intervertebral disc, we first performed pressure measurements. This information is essential to be sure whether the resulting pressures of the applied loads in the current study are comparable to the intradiscal pressures known from studies in vivo. For these calibration experiments, the lumbar spine (L1–L6) of a goat, derived from a local abattoir, was meticulously cleaned of soft tissues. The posterior elements were left intact. The spine was separated into three separate motion segments by incision of the discs between L2–L3 and L4–L5. The ends of the vertebrae were embedded in a low melting point bismuth alloy, and the motion segments were placed in upright position in the biomechanical testing apparatus (Instron, Norwood, MA, USA). First, a pressure needle was inserted anteriorly into the core of each disc. Next, a load was applied increasing with 50 N/s to a maximum of 1,000 N or a maximum of 3 MPa as measured by the needle, whatever came first (higher pressures would result in irreversible needle damage). Both the values for load and pressure were documented. The measurements were repeated two times with the needle inserted via both lateral sides of the discs.
Biomechanical experiments were performed prior to the in vivo study on spinal segments, derived from the local abattoir. Using the same set up as described with the pressure experiments, the axial failure loads of different ACDs were tested using the standardised nucleus replacement model. After the implants were inserted, an axial compression load was applied to a maximum of 5,000 N. The experiments were ended when failure, considered as the leakage of the collagen implant or extrusion of closure devices, was observed. Each ACD was tested on three different motion segments (L1–L2, L3–L4 and L5–L6). For every experiment a freshly dissected spinal segment was used.
In addition to the failure experiments, the effects of the implants on the biomechanical behaviour of the motion segments were investigated. These experiments were mainly performed to exclude the undesired effects on the range of latero-flexion or flexion–extension due to the ACDs and to assess the possibility of the implants to restore the effects after discectomy. After fixation in bismuth, 12 motions segments (four of each different level) were multidirectionally tested using a four-point flexion–extension set up and the instron 8872 testing machine (Instron Corp., Norwood, MA, USA). The motion segments were submitted to four cycles of flexion–extension and latero-flexion under a maximum moment of 2 Nm at a speed of 1°/s. Specimens were tested before discectomy (native), after discectomy and with both implants (nucleus implant and the four rings 1.5 mm ACD) inside. During all experiments, the segments were kept moisturised by wrapping with surgical gauze drowned in 0.9% saline. Force–deformation data acquisition was performed for each direction through materials testing software (Fast Track 2, Instron Corp., Norwood, MA, USA). The range of motion-data of the third cycle of the tests was used for further calculation. The mean changes in the range of motion after discectomy and implantation of both implants were calculated as ratios compared to native values (treatment over control).
In vivo evaluation
Surgical procedure and animal care were performed in compliance with the regulations of the Dutch legislation for animal research, and the Animal Ethics Committee of the VU University Medical Center approved the protocol. Ten goats were sedated with 10 mg/kg ketamine and 1.5 mg atropine intramuscularly, followed by 0.4 mg/kg etomidate intravenously. General anaesthesia was maintained with 4 μg/kg fentanyl per hour, 0.3 mg/kg midazolam per hour and 1.5–2.5% isoflurane. Before surgery, standardised lateral thoracolumbar roentgenograms were obtained. A dorsal paravertebral incision was made. The IVDs were identified using a left retroperitoneal approach and exposed after mobilisation of the psoas muscle. Level determination was performed by identification of the lowest rib. Two discectomies, as described above, were randomly performed over the levels T13–L1, L2–L3 or L4–L5. At one of these levels, the collagen nucleus implant was inserted after evacuation of the NP, followed by closure of the annulus defect with the ACD (sterilised by γ-irradiation). At the other level the plug was inserted solely. Two variants of the ACDs were used in the 6-week follow-up group: four goats received implants with a core diameter of 1.3 mm, the remainder with a core diameter of 1.5 mm. The number (four) and diameter (3.5 mm) of the barb rings was the same for all implants.
Two of the goats were terminated by an overdose pentobarbital after 2 weeks and the remaining eight goats after 6 weeks. The latter group returned to their habitual environment from 1 week postoperative until 1 week prior to the autopsy. Evaluation was similar for all goats. After termination, the lumbar spines of the goats were harvested, and magnetic resonance imaging (MRI) of the explants was performed within 2 h. Hereafter, all soft tissue was removed, and careful macroscopic inspection was performed. Finally, a band saw was used to obtain transversal slices of the discs for macroscopic inspection. Both, macroscopic examination and MR Imaging were used to determine the position of the ACDs. The position was classified as “in situ”, partially displaced (maximum of two barb rings outside the AF), or fully displaced (at least two barb rings outside the AF).
To calculate differences between different implant groups, the Student T test was used.