*In Vitro Models for Intervertebral Disc Tissue Engineering
R.M. Ozuna, MD; M. Spector, PhD; S. Shortkroff, MS; D.M. Hastreiter, SM
Defects in the annulus fibrosis of the intervertebral disc lead to degeneration that may require discectomy or spinal fusion, surgical procedures with unfavorable long-term outcomes. Because the annulus fibrosis does not undergo spontaneous regeneration after injury, the need for an implant to facilitate regeneration is compelling. Our research focuses on a collagen-glycosaminoglycan matrix that could be implanted into an annular defect. The objectives of the proposed investigation are to determine the proliferate and biosynthetic effects of selected matrix variables on adult human and canine annulus fibrosis cells in vitro.
Cyclic Failure of Multi-Segment Pedicle Screw Systems
S.A. Yerby, PhD; J. Sun; E.J. Carragee, MD; D. Kim; D.R. Carter
Introduction. Premature failure of pedicle screw systems has been reported and often its effects result in increased morbidity and revision surgery. No definitive preoperative assessment exists which reliably predicts whether the spinal implant will fail before the patient has achieved full spinal fusion. This study seeks to define a threshold value of bone quality, above which long implant life will be predicted, and below which a construct may be susceptible to premature failure.
Pedicle screw failure generally occurs by one of two mechanisms: (1) bony failure; or (2) hardware failure. Bony failure is often reported in patients with poor bone quality, whereas hardware failure is reported in patients with good bone quality. Both of these mechanisms can occur immediately after surgery or after a longer period of time. Bony failure is often seen as a “fan-shaped” region of compressed vertebral trabecular bone with the origin or fulcrum located within the pedicle;1 whereas hardware failure is generally described as pedicle screw bending or breakage within the pedicle.2 This study was undertaken to establish a relationship between bone quality and fatigue life of pedicle screw systems loaded in a corpectomy model. Constructs were cyclically loaded in flexion and the pedicle screw angle relative to the vertebra was measured during each cycle. We hypothesized that as the bone quality increased, (1) the mode of construct failure would change from bony failure to hardware failure; and (2) the number of cycles to failure would increase.
Methods. Seven fresh human cadaver spine segments (T11-L3) were procured and stored at –20 degrees C until tested. On the day of testing, each specimen was thawed and placed on 20 cm of water. Each specimen was scanned laterally using a dual energy X-ray absorptiometry (DEXA) bone densitometer (QDR 4000, Hologic, Waltham, MA). Bone mineral density (BMD) of the T12 and L2 vertebral bodies were measured in the sagittal plane using a lumbar spine analysis protocol (Hologic, Waltham, MA).
Following scanning, each specimen was instrumented with 7mm x 45mm pedicle screws and 6/5mm rods at T12 and L2 (Synthes, Paoli, PA). A vertebral body corpectomy was created at the L1 level by removing the L1 vertebral body, while maintaining the thin posterior cortex and all of the posterior elements. The T11 and L3 vertebrae were secured with polymethylmethacrylate (PMMA) and each specimen was mounted in a materials testing machine and loaded from the neutral, unloaded position to 7.5Nm of flexion under load control at 0.5 Hz (MTS 858, MTS, Eden Prairie, MN).
Pedicle screw angles were measured by mounting an extension arm from the posterior end of each pedicle screw to a position parallel to the pedicle and 1cm lateral.
An angular displacement transducer (RVIT, Lucas Schaevitz, Hampton, VA) was then mounted to the arm and the rotary shaft of the RVIT was securely fixed to the midlateral wall of the pedicle; the body of the RVIT was rigidly fixed to the screw via the extension arm. Therefore, as the screw rotated in the sagittal plane, the RVIT measured the screw angle relative to the vertebra, assuming that the center of rotation was within the pedicle. The pedicle screw angle, construct bending moment and angular displacement data were collected at 25 Hz with a sixteen channel A/D board (MTS, Eden Prairie, MN). Each construct was loaded until one of the four screw angles reached 6/5deg or the construct survived 300,000 cycles without any of the screws toggling. The constructs were hydrated continuously and maintained at room temperature during testing. The relationship between BMD and cycles to failure was established using a least squares sigmoidal fit.
Results. The BMD of the specimens ranged from 0.33 to 0.73 g/cm2 with a mean and standard deviation of 0.49 g/cm2 and 0.14 g/cm2 respectively. The minimum number of loading cycles achieved was one hundred and two specimens reached the maximum number of 300,000 cycles. There was a significant (p<2x10-2) sigmoidal relationship between the vertebral BMD and the number of cycles to failure.
Discussion. There continues to be a lack of meaningful predictors for the risk of spine fixation failure. Most assessments of the holding potential of spinal implants include pullout force and insertional torque, which cannot be determined preoperatively. However, BMD, assessed through dual energy X-ray absorptiometry can be determined prior to surgery. The current study described the relationship between BMD and cycles to failure in a corpectomy at a given load. For this given load (7.5Nm), we have established a strong, nonlinear relationship between the two parameters. In addition, we have established a threshold BMD value of 0.50 g/cm2, above which a large number of cycles are needed to produce substantial screw toggle and below which the construct fails in less than 50,000 cycles. This threshold value may be useful to surgeons when preoperatively planning surgeries for patients with poor bone quality and may indicate that supplemental fixation is required.
References.
1Zindrick MR, Wiltse LL, Widell EH, et al. A biomechanical study of intrapeduncular screw fixation in the lumbosacral spine. Clin Orthop. 1986;203:99-112.
2McLain RF, Sparling E, Benson DR. Early failure of short-segment pedicle instrumentation for thoracolumbar fractures. A preliminary report. J Bone Joint Surg (AM). 1993;75(2):162-167.
The Effect of Regional Gene Therapy with Bone Morphogenetic Protein-2 Producing Bone Marrow Cells on Spinal Fusion in Rats
J.C. Wang, MD; J.R. Lieberman, MD
Background. Bone morphogenetic proteins (BMP) are now being used as bone graft substitutes for spinal fusion because of the high rates of pseudarthrosis and the morbidity associated with the harvesting of autogenous iliac crest bone grafts. However, the large doses of BMP required to induce a spinal fusion in humans suggests that the delivery of these proteins requires further optimization. We used ex vivo adenoviral gene transfer to create BMP-2 producing bone marrow cells and these autologous cells induced a posterolateral fusion of the spine in syngeneic rats.
Methods. Intertransverse spinal fusion (L4-5) was attempted in ten groups of Lewis rats with 5 x 106 BMP-2 producing rat bone marrow cells (Ad-BMP-2 cells) created via adenoviral gene transfer with guanidine hydrochloride extracted demineralized bone matrix as a carrier (eight spines, Group I); 5 x 106 Ad-BMP-2 cells on a collagen sponge carrier (seven spines, Group II); 10 micrograms of recombinant BMP-2 (rhBMP-2) on guanidine hydrochloride extracted demineralized bone matrix carrier (eight spines, Group III); 10 micrograms of rhBMP-2 on a collagen sponge carrier (seven spines, Group IV); autogenous iliac crest bone graft (fifteen spines, Group V); 5 x 106 beta-galactosidase producing rat bone marrow cells created via adenoviral gene transfer with guanidine hydrochloride extracted demineralized bone matrix as a carrier (eight spines, Group VI); decortication of the transverse processes alone (eight spines, Group VII); 5 x 106 uninfected rat bone marrow cells with a guanidine hydrochloride extracted demineralized bone matrix carrier (eight spines, Group VIII); guanidine hydrochloride extracted demineralized bone matrix only (eight spines, Group IX); or a collagen sponge alone (eight spines, Group X). Each specimen underwent plain radiographs, manual palpation, and histological analysis.
Results. All 15 spines in Groups I and II (BMP-2 producing bone marrow cells) and all fifteen spines in Groups III and IV were fused four weeks postoperatively. In contrast, none of the 55 spines in the other groups fused at a minimum of eight weeks after implantation. Histological analysis of the specimens revealed that spines that had received BMP-2 producing bone marrow cells (Groups I and II) were filled with coarse trabecular postoperatively, whereas in those that had received rhBMP-2 (Groups III and IV) the fusion mass was thin and lace-like. Spines that had been treated with autogenous iliac crest bone (Group V), bone marrow cells producing beta-galactosidase (Group VI), decorticated transverse processes (Group VII), uninfected bone marrow cells (Group VIII), guanidine hydrochloride extracted demineralized bone matrix only (Group IX) or collagen sponge only (Group X) produced minimal or no bone formation.
Conclusion. This study demonstrated that BMP-2 producing bone marrow cells, created by adenoviral gene transfer, produce sufficient protein to induce an intertransverse fusion of the rat spine.
Clinical Relevance. Regional gene therapy can be used to induce spinal fusion. This strategy using transduced bone marrow cells created via ex vivo gene transfer with a BMP-2 containing adenovirus could be adapted to enhance spinal fusions in humans.
The Natural History of Spondylolysis and Spondylolisthesis: Age 5 to Late 40’s
B.E. Fredrickson, MD; D. Baker; A.M. Murtland, MD; C. Sweeney; W.J. Beutler, MD, FACS
Status of Research Project. The initial research project identified 30 children in adolescence with spondylolysis and/or spondylolisthesis. The current project is an attempt to contact all of these individuals to obtain current medical records, X-rays and magnetic resonance images (MRIs). To date, four are deceased and one possibly. Thirteen have completed all of their requirements; six have been contacted, but not yet completed various aspects secondary to distance and other commitments. Three have been noncompliant, however, we continue to contact them in an attempt to entice them to complete their required studies; four have been lost and are currently in a national search database. A new database has been created and all data and new data have been entered to date.
Significant Findings or Conclusions to Date. No specific findings can be concluded yet as the data has not been analyzed with the limited material we have. One interesting finding to date is the death rate of at least 10% which seems excessively high.
Stimulated Autologous Macrophages in Spinal Cord Injury
V.K.H. Sonntag, MD; F.F. Marciano, MD, PhD; N. Theodore, MD, MS; S.P. Javedan, MD
Introduction. Autologous activated macrophage therapy (AMT) has been proposed as a treatment for spinal cord injury. AMT has demonstrated functional, electrophysiological and histopathological evidence of regeneration in a rodent spinal cord transaction model.
Methods. Six adult beagles were subjected to left T13 spinal cord hemisection. Microinjections (20uL) of activated macrophages (four dogs), or empty cell media (two dogs) were made into the cut ends. Animals were allowed to recover for nine months. Hind limb electromyographic (EMG) recordings were made from Vastus lateralus using trans-cranial magnetic stimulation (TCMS) of motor cortex before hemisection, after hemisection and during recovery. Prior to termination, all animals were injected with 16nL wheatgerm horseradishperoxidase (WG-Hrp) at the L2 level and perfused at 48 hours. Histological examination of tetra-methyl-benzidine (TMB)-reacted sections of the spinal cord and brainstem was performed to assess for axonal regeneration.
Results. TCMS revealed some recovery of left hind limb potential in ¾ AMT-treated animals and ½ control-treated animals. A greater mean Z-score recovery was seen in the AMT-treated animals (p=0.008). Retrograde tracer histology showed minimal uptake to the ipsilateral red nucleus (0-1.4% of control side) in both cell-implanted and control groups. In addition, using WG-Hrp fiber tracing no local crossing fibers were seen.
Conclusions. Canines subjected to lower thoracic spinal cord hemisection are capable of variable electrophysiological recovery over nine months. Injection of activated macrophages may enhance electrophysiological recovery. AMT was not associated with any significant rubrospinal tract regeneration to two segments below the injury. In addition, local fiber regeneration across the hemisection site was not seen.
*Abstracts/permission forms not received at the time of publication
**Current and ongoing research