Cells from degenerative intervertebral discs demonstrate unfavorable responses to mechanical and inflammatory stimuli: a pilot study.

OBJECTIVE: Mechanical forces and inflammatory signaling influence intervertebral disc matrix homeostasis. We hypothesized that annulus fibrosus cells from degenerative discs would have altered responses to mechanical and inflammatory stimuli compared with cells isolated from normal discs. DESIGN: Annulus fibrosus cells were isolated from New Zealand White rabbits with normal and magnetic resonance imaging-confirmed degenerative discs created by annular stab. Cells were cultured with and without inflammatory and mechanical stimuli (tensile strain). After 4 or 24 hrs, the mRNA expression of inflammatory, catabolic, and anabolic genes was measured by reverse transcription polymerase chain reaction. RESULTS: Baseline gene expression differences were noted between cells from normal and degenerative discs. Degenerative cells demonstrated a more proinflammatory response profile to inflammatory and mechanical stimuli and loss of the beneficial effects of mechanical signaling. Decreased expression of catabolic and anabolic genes was observed in degenerative cells under conditions of inflammatory and mechanical stimuli. CONCLUSIONS: These data demonstrate that degenerative cells have a decreased capacity to respond positively to beneficial levels of mechanical strain and demonstrate an exaggerated response to an inflammatory stimulus. This may, in part, help to explain differential responses to motion-based therapies in patients with intervertebral disc degeneration.

In vitro and in vivo testing of a novel regulatory system for gene therapy for intervertebral disc degeneration.

STUDY DESIGN: In vitro and in vivo testing of a gene expression control system. OBJECTIVE: The purpose of this study is to establish the ability of controlling gene expression using an adeno-associated viral vector containing a novel control system (AAV-RheoSwitch GFP [Intrexon Corp., Blacksburg, VA]) in intervertebral disc cells for potential use in gene therapy trials. SUMMARY OF BACKGROUND DATA: Gene therapy for disc degeneration shows promise; however, concern remains regarding safety. Careful control of gene expression is needed to facilitate translation into clinical trials. METHODS: Rabbit nucleus pulposus cells were treated in vitro with increasing multiplicities of infection of AAV-RheoSwitch GFP, followed by increasing concentrations of Intrexon's activator ligand, and examined for fluorescence during and after removal of ligand. New Zealand white rabbits were injected with AAV-RheoSwitch GFP and killed either before or after 5 days of daily ligand injection. Tissues were analyzed for the presence of green fluorescent protein (GFP) with fluorescence microscopy and immunohistochemical staining. RESULTS: In vitro, GFP expression was noted to be dose and time dependent, decreased 24 hours after removal of ligand, and was minimally detectable in cells after 48 hours. In vivo, increasing GFP expression was seen in animals treated with viral vector and ligand. No GFP expression was evident in tissues from rabbits that received only virus, or activator ligand alone. In addition, no GFP expression was evident in the adjacent discs, spinal cord, dura, bone, liver, or brain of any animals. CONCLUSION: These data demonstrate that in vitro ligand-induced gene expression can be stimulated and effectively turned off by removal of the ligand. In addition, we demonstrated the in vivo utility of this system through showing up-regulation of GFP without nonspecific gene expression or expression in adjacent tissues. This system, therefore, has the potential to increase the safety of gene therapy in the treatment of intervertebral disc degeneration.

Morphological analysis of the transverse carpal ligament.

Transection of the transverse carpal ligament (TCL) for carpal tunnel syndrome is commonly performed, yet actual knowledge of TCL morphology is rudimentary and the anatomical terminology is inconsistently used. The purpose of this study was to perform a morphological analysis of the TCL, to redefine the anatomical terminology concerning the TCL and surrounding structures, and to evaluate any correlation between external, measurable hand dimensions, and TCL dimensions. A silicone casting technique and digitization were employed to measure the morphology of the TCL in cadaveric specimens and to construct a three-dimensional TCL model. The TCL was the thickest distally at the midline and ulnar segments and the thickest proximally at the radial segment. External hand dimensions did not significantly correlate with TCL dimensions. The TCL thickness distribution is variable along the radioulnar axis. The thickness of the TCL was 2.1 ± 0.8 mm, ranging from 1.3 to 3.0 mm.

Morphological analysis of the carpal tunnel.

Although carpal tunnel release is one of the most commonly performed procedures in the USA, the morphology of the carpal tunnel as determined previously in the literature has been questioned. Previous methodology has been questioned for accuracy by recent studies. The purpose of this study was to perform a morphological analysis of the carpal tunnel and correlate carpal tunnel and hand dimensions. The carpal tunnels of ten cadaveric specimens were emptied of their contents and a silicone cast of the carpal tunnel was then created. This cast was then digitized, and the dimensions of the carpal tunnel were calculated. These dimensions were compared with the measured hand dimensions of the specimens. The width, depth, tilt angle, length, cross-sectional area, and volume of the carpal tunnel were 19.2 ± 1.7 mm, 8.3 ± 0.9 mm, 14.8 ± 7.8°, 12.7 ± 2.5 mm, 134.9 ± 23.6 mm(2), and 1,737 ± 542 mm(3), respectively. Width, depth, and cross-sectional area did not change significantly along the length of the carpal tunnel, but tilt angle did. The width of the palm strongly correlates with the width of the carpal tunnel. Other dimensional correlations did not reach statistical significance. The carpal tunnel is of uniform dimension along its length. The long axis of the carpal tunnel in cross-section rotates volarly from the radial side of the hand increasingly with distal progression along the carpal tunnel. Based on our analysis of ten cadaveric specimens, the width of the carpal tunnel may be estimated by the width of the palm using the equation: [Formula: see text].