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With aging, the nucleus pulposus of the intervertebral disk begins to desiccate, characterized by a loss of aggrecan core proteins, glycosaminoglycan, matrix turnover, and cell numbers that eventually leads to losing the ability to imbibe water. As the nucleus pulposus loses its water content, the disk can no longer distribute forces effectively. The annulus fibrosus buckles under compressive loading, and this leads to disk collapse. Further load on the annulus fibrosus leads to fissuring and cracks. Loss of disk height also leads to overriding facet joints. Uneven loading causes osteophyte formation and joint instability. Pain associated with intervertebral disk degeneration can be caused by bulging or rupturing of the annulus with herniation of disk material irritating the pain fibers in the peripheral part of the annulus. Neural tissues are also implicated in herniated disks as a result of mechanical or chemical irritation. Finally, degenerated facet joints, together with instability, subluxation, or deformity of the functional spinal unit (FSU), can also produce pain.
If conservative modalities fail, surgical intervention is indicated, especially in those patients with significant neurologic compromise. Classically, the most common surgical treatment for lumbar disk degeneration is spinal fusion. Although effective in controlling pain, fusion leads to restriction of the spinal motion and may cause adjacent level degeneration secondary to increased stress and motion at adjacent levels. The goals of surgical treatment of lumbar spine degeneration are to relieve any neural compression and to maintain a stable FSU that is free of deformity. Thus, many different types of intervertebral disk implants have been advocated to avoid the effects of spinal fusion and to preserve motion. Artificial disks made of metals, polymers, or combinations of materials have been attempted and are gaining popularity. The early results of total disk replacement (TDR) are encouraging and are at least comparable to the results of spinal fusion. However, questions have been raised regarding the implant material, design kinematics, recipient factors, surgical precision, and long-term outcome and salvage options. An interesting long-term study of artificial disk replacement showed that the best results occurred in patients who had spontaneous fusion in the replaced disk.
Disk replacements can also be biologic, with the goals of preserving anatomy, motion, and stability. Theoretically, one could manufacture a disk scaffold using tissue engineering technology. Appropriate cells with necessary promoter growth factors can be used to populate this scaffold. Regeneration or repair of the disk by using growth factors, gene therapy, and cell therapy is being actively researched. Most experiments are focused on the rejuvenation of the nucleus pulposus by restoring the matrix production through increasing cell numbers. However, this approach has a major flaw because the annulus fibrosus is also structurally and mechanically incompetent when the disk is degenerated to the point of causing symptoms. In addition, the delivery channel in which nutrients reach the disk is also jeopardized. This limits sustained cell viability and the ability for cells to restore the matrix or to repair the damaged annulus fibrosus. Current evidence suggests that cellular therapy is unable to restore the matrix content in advanced disk degeneration but can only maintain it in mild degeneration.
The concept of disk transplantation began in 1991 when Olson and associates reconstructed a spinal column defect by using a quadruped model transplantation of a vertebral body together with the two adjacent disks to act as a spacer. Relatively normal mobility and stability of the spinal column were found because of partial revascularization of the intervening vertebral body and the intervertebral disks. The same research group followed up with a fresh autograft disk transfer in a canine model. In this experiment, the morphology and the metabolic functions of the transplanted disks were abnormal, but the structure and function were maintained. The likely cause of these findings was attributed to the rigidly fixed transplanted disks, which jeopardized disk nutrition. Further studies by Katsuura and Hukuda, as well as by Matsuzaki and colleagues, used cryopreserved allografts in quadruped models, but these investigators experienced the same limitation of rigid fixation of the grafts with plates and screws.
Around the same time as the study by Olson and colleagues, Professor Keith DK Luk and investigators at the Department of Orthopaedics and Traumatology at the University of Hong Kong had a similar idea of disk transplantation that avoided constraining the transplanted graft. Experiments were conducted in upright primates, the model closest to human biomechanics. A series of experiments was performed to verify this animal model, disk autograft, disk allograft, and fresh frozen allograft. Further studies were carried out to validate the storage processing technique and implantation technique. A small-scale clinical trial was conducted in 2000 to prove the applicability of this technique in clinical practice. The following discussion provides an account of the evolution of allograft disk transplantation from animal models to the latest clinical trial outcomes, as based on the experience of investigators at the University of Hong Kong and their collaborators.
Animal Models and Graft Experimentation
Autograft Experiment
In 1992, the first autograft experiment was initiated at the Tangdu Hospital, Affiliated Hospital of the Fourth Military Medical University, Xian, China in collaboration with Dr. Dike Ruan. The animal model used was the rhesus monkey. Fourteen male monkeys were followed up for 2, 4, and 6 months, and 2 monkeys were followed up for 12 months. The L3-L4 intervertebral disk was isolated without damaging the surrounding structures, and the composite graft was repositioned into the disk space and anchored to the outer annulus. No rigid internal fixation or external immobilizer was used, and the animal was allowed to move freely. Serial radiographs were used to measure the disk height and observe for any degeneration. A gradual reduction in the disk height was noted postoperatively but was stabilized at 2 to 4 months, and some disk height was regained at the 12-month final follow-up. Autografts were retrieved from the animals and underwent biochemical, histologic, and biomechanical testing. Analysis showed no statistical significant changes of water, proteoglycan, and hydroxyproline contents with time. A continuing drop in water content was reported; an initial drop was followed by an increase in proteoglycan and persistently raised hydroxyproline in the nucleus fibrosus. Viable cells were seen at the annulus fibrosus and nucleus pulposus on histologic examination. The morphology of the annulus was found to be well preserved. The grafted disk had an initial period of hypermobility in all ranges of motion at 2 to 4 months postoperatively but returned to normal by 6 months. Cells in the composite autograft were able to withstand a transient period of ischemia and were able to recover their biochemical and biomechanical function. As a result, a bipedal animal model was found to be a successful model in studies of intervertebral disk transplantation.
Allograft Experiment
Fresh allograft transplants must be examined to see how they behave when sourced from a live or freshly dead donor. The problem of immunogenicity must also be addressed. Similar to the cornea for the eye and the meniscus for the knee, the intervertebral disk is immunologically privileged because of its avascularity. This experiment was confirmed by switching the L3-L4 disks in two monkeys. No rhesus factor or blood grouping was performed for the monkeys, and no immunosuppressants were given, based on the knowledge that allografts have already been used in joint replacement revisions or tumor reconstructions without immunosuppressant agents. The two monkeys in the experiment were of similar age and size and were operated on simultaneously by two teams of surgeons and anesthesiologists to minimize the operating time and the blood loss. In this experiment, problems of repeated subluxation and dislocation secondary to graft size mismatch were reported. From this failure, appropriate graft size matching and press-fit fixation were vital to obtaining stability of the transplant without rigid internal fixation.
Fresh Frozen Allograft
Fresh frozen allografts were used for disk transplantation to confirm the feasibility of the procedure further. Specifically, this experiment was necessary to help resolve issues of organ donation, preservation, physical size, and immunocompatibility of the grafts. Seventeen monkeys were used. Two monkeys were donors of the disks, and 3 others were used as controls. The other 12 monkeys were followed up for 2 to 8 weeks, 6 months, and 24 months. Sections from T10 to L7 were harvested and split into 1- to 2-mm segments along with adjacent end plates. The grafts were measured and were immersed in a dimethylsulfoxide (DMSO) solution and cooled stepwise to −196° C in liquid nitrogen for storage.
After the disk was removed from the recipient, an appropriately sized graft was thawed and placed to fit snugly into the defect. No immunosuppressant was used. Bony union of the end plates was obtained successfully in all cases without any complication of graft subluxation or dislocation. Up to the 24-month final follow-up, the disk height was found to have a slow and progressive reduction with secondary degenerative changes of traction osteophytes. In contrast to the autograft, the water and proteoglycan content had a steady decrease from 6 to 24 months. As compared with the controls, the grafted FSU maintained similar mechanical stability and mobility. Histologic examination was also performed to look for immunoreactivity and showed inflammatory cells infiltration with lymphocytic and fibroblastic proliferation limited only at the osteotomy site. Yet this reaction was significantly reduced at 8 weeks of follow-up. The numbers of cells in both the annulus fibrosus and the nucleus pulposus were similar at early follow-up and at 24-month follow-up, the cells of the nucleus pulposus underwent degeneration with features of irregular nuclear shape, mitochondrial swelling, and karyopyknosis.
This study confirmed that, similar to autografts, a cryopreserved allograft could retain cell viability and maintain mechanical properties. The cryopreservation process could also cause minimal or no immunoreaction during disk transplantation. The minimal immunoreaction seen in this experiment was found only at the bone interface; thus, mechanical washout of the cancellous end plates should be performed before preservation. This experiment found that degeneration of the transplanted allograft still occurred. Further research should refine the preservation protocol to increase cell viability and reduce early graft degeneration. This is important because long-term storage of allografts in a bank is a vital part of allograft transplantation procedures. The use of cryopreservation is necessary for safe preservation of the intervertebral disk allograft.
Cryopreservation Experiments
Two further studies were published to improve knowledge of the cryopreservation process further. Cryopreservation must retain both mechanical properties and cellular activity, and so this method was refined with different cooling rates, solutions for immersion, and incubation times. The first study, published in 2010, was able to optimize survival of disk cells by modulating cooling rates, cryoprotective agents (CPAs) concentration, and incubation time in porcine lumbar disks. In this study, 52 porcine lumbar disks (L2-L3 to L4-L5) were obtained from 22 pigs. Three different rates of cooling were tested by immersing disk samples in cryopreservation solution in either a precooled glass container filled with 80° C isopropanol, a precooled glass container filled with 4° C isopropanol, or a 16 × 11.5 × 21 cm polystyrene box that was 1.6 cm thick. Three different cryopreservation formulas were also tested, including the traditional formula of 10% DMSO, 10% DMSO with 10% propylene glycol, and 10% DMSO with 0.1% Supercool X-1000. Different precooling intubation time periods between 2 and 4 hours in the cryopreservation solution were also tested. Metabolic activity, mechanical property, and histologic features of the allografts were evaluated by comparing them with fresh specimens. The authors found that a slow cooling rate (−0.3° C/minute), a combination of cryoprotective agents (10% DMSO and 10% propylene glycol), and a limited cryoprotective agent incubation time of 2 hours favored the overall metabolic activity of disk cells up to 60% of the fresh control. The mechanical property and matrix organization were maintained with this method.
The second study, published in 2011, further analyzed the variable cryoprotective agents and their effects on the biomechanical properties of the allografts. Forty disks (from L1 to L6) were harvested from 9 pigs. Corneal Potassium TES 2 solution (CPTES2) was used as the cryoprotective agent carrier solution. Different cryoprotective agent concentrations were used in combination with CPTES2 for cryopreservation. These included CPTES2 only, 10% DMSO in CPTES2 solution, and 10% DMSO with 10% propylene glycol in CPTES2 solution. Disks were incubated at 4° C for 2 hours and were stored after freezing to −80° C and in liquid nitrogen for 4 weeks. All disks were thawed to 37° C in a saline bath before analysis. Uniaxial compression testing and viscoelastic properties were investigated. The results showed that allografts that were cryopreserved with cryoprotective agents were able to preserve the normal elastic modulus and viscous modulus of an intervertebral disk, whereas allografts without cryopreservatives were stiffer. Although this study further confirms that cryopreservation can preserve the mechanical properties of an intervertebral disk allograft, only human studies can truly validate this finding.
Biomechanical Studies on Graft Positioning
Besides the issues surrounding storage, the technical aspects of allograft disk placement and the determination whether malpositioning of the allograft would affect the kinematics of the FSU and lead to early failure were equally important. A biomechanical study addressed the effect of remodeling on the kinematics of the malpositioned disk allograft transplantation. Eighteen male goats were used in this study. Three goats were selected as donors of their intervertebral disks, whereas the other goats were assigned randomly to control, allograft, and malpositioned allograft groups. All goats were followed up for 6 months. The 3 donor goats were sacrificed, and the entire spinal column of T13 to S1 was harvested en bloc. Preparation included osteotomy at the end plates 1 to 2 mm above and below the disks and washing of the grafts with saline and immersion in 10% DMSO and 10% calf serum for 2 hours at 4° C to preserve cellular viability. The disks were then placed at −15° C for 1 hour, −40° C for 1 hour, and −80° C for 1 hour, after which the disk grafts were preserved in liquid nitrogen at −196° C until implantation.
L4-L5 diskectomy and complete removal of the posterior annulus with preservation of the posterior longitudinal ligament were performed in the recipient goats. The preserved frozen disk allograft of the most compatible size was selected and was positioned into the disk space. For the well-aligned groups, the disk allograft was positioned and aligned to the anterior vertebral margin of the excised disk. For the malaligned group, the allograft implant was placed proud anteriorly by 25% of the allograft’s anteroposterior length. Sutures were used to fix the allografts in place by attaching them to the outer annulus.
In vitro three-dimensional kinematics was performed by placing a pure moment of 5 Nm to the top vertebra. This continuous moment was applied at 0.5 degrees per second in the axis of flexion and extension, bilateral and lateral bending, and axial rotation. Five complete loading cycles were applied, with the first four used for preconditioning and the fifth for analysis. Analysis found no significant differences in flexion, axial rotation, and lateral bending. A significant increase in extension motion was observed in both the aligned allograft group and the malpositioned allograft group as compared with the control group. This difference was likely caused by early degeneration in the transplanted allograft in response to its more fibrotic nucleus pulposus resulting from decreased water content. No significant differences in range of motion were noted between the aligned and malpositioned groups. In summary, intervertebral disk allograft transplantation did not compromise the stability of the lumbar spine or motion parameters. In this study, precise positioning of the allograft did not affect the overall survival of the FSU. Despite these promising findings, human studies are ultimately required to validate findings.
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