Contrast-enhanced microCT evaluation of degeneration following partial and full width injuries to mouse lumbar intervertebral disc

Study Design: Preclinical animal study. Objective: Evaluation of the degenerative progression resulting from either a partial- or full- width injury to the mouse lumbar intervertebral disc (IVD) using contrast-enhanced micro-computed tomography and histological analyses. We utilized a lateral-retroperitoneal surgical approach to access the lumbar IVD, and the injuries to the IVD were induced by either incising one side of the annulus fibrosus or puncturing both sides of the annulus fibrosus. The full-width injury caused dramatic reduction in nucleus pulposus hydration and significant degeneration. A partial-width injury produces localized deterioration around the annulus fibrosus site that resulted in local tissue remodeling without gross degeneration to the IVD. Methods: Female C57BL/6J mice of 3-4 months age were used in this study. They were divided into three groups to undergo a partial-width, full-width, or sham injuries. The L5/L6 and L6/S1 lumbar IVDs were surgically exposed using a lateral-retroperitoneal approach. The L6/S1 IVDs were injured using either a surgical scalpel (partial-width) or a 33G needle (full-width), with the L5/L6 serving as an internal control. These animals were allowed to recover and then sacrificed at 2-, 4-, or 8- weeks post-surgery. The IVDs were assessed for degeneration using contrast-enhanced microCT (CEμCT) and histological analysis. Results: The high-resolution 3D evaluation of the IVD confirmed that the respective injuries localized within one side of the annulus fibrosus or spanned the full width of the IVD. The full-width injury caused deteriorations in the nucleus pulposus after 2 weeks that culminated in significant degeneration at 8 weeks, while the partial width injury caused localized disruptions that remained limited to the annulus fibrosus. Conclusion: The use of CEμCT revealed distinct IVD degeneration profiles resulting from partial- and full- width injuries. The partial width injury may serve as a better model for IVD degeneration resulting from localized annulus fibrosus injuries in humans.

Ex vivo study of the intradiskal pressure in the C6-7 intervertebral disk after experimental destabilization and distraction-fusion of the C5-C6 vertebrae in canine cadaveric specimens

OBJECTIVE To evaluate intradiskal pressure (IDP) in the C6-7 intervertebral disk (IVD) after destabilization and distraction-fusion of the C5-C6 vertebrae. SAMPLE 7 cadaveric C4-T1 vertebral specimens with no evidence of IVD disease from large-breed dogs. PROCEDURES Specimens were mounted in a custom-made 6 degrees of freedom spinal loading simulator so the C5-C6 and C6-C7 segments remained mobile. One specimen remained untreated and was used to assess the repeatability of the IDP measurement protocol. Six specimens underwent 3 sequential configurations (untreated, partial diskectomy of the C5-6 IVD, and distraction-fusion of the C5-C6 vertebrae). Each construct was biomechanically tested under neutral, flexion, extension, and right-lateral bending loads. The IDP was measured with a pressure transducer inserted into the C6-7 IVD and compared between the nucleus pulposus and annulus fibrosus and across all 3 constructs and 4 loads. RESULTS Compared with untreated constructs, partial diskectomy and distraction-fusion of C5-C6 decreased the mean ± SD IDP in the C6-7 IVD by 1.3 ± 1.3% and 0.8 ± 1.3%, respectively. During motion, the IDP remained fairly constant in the annulus fibrosus and increased by 3.8 ± 3.0% in the nucleus pulposus. The increase in IDP within the nucleus pulposus was numerically greatest during flexion but did not differ significantly among loading conditions. CONCLUSIONS AND CLINICAL RELEVANCE Distraction-fusion of C5-C6 did not significantly alter the IDP of healthy C6-7 IVDs. Effects of vertebral distraction-fusion on the IDP of adjacent IVDs with degenerative changes, such as those in dogs with caudal cervical spondylomyelopathy, warrant investigation.

Mechanical Stretch Induces Annulus Fibrosus Cell Senescence through Activation of the RhoA/ROCK Pathway

Background. Intervertebral disc is responsible for absorbing and transmitting mechanical compression. Under physiological conditions, the peripheral annulus fibrosus (AF) cells are subjected to different magnitudes of transverse mechanical stretch depending on the swelling of the central nucleus pulposus tissue. However, the biological behavior of AF cells under mechanical stretch is not well studied. Objective. This study was performed to study the effects of mechanical tension on AF cell senescence and the potential signaling transduction pathway. Methods. Rat AF cells were made to experience different magnitudes of mechanical stretch (2% elongation and 20% elongation for 4 hours every day at 1 Hz) in a 10-day experiment period. The inhibitor RKI-1447 of the Rho-associated coiled-coil–containing protein kinases (ROCK) was added along with culture medium to investigate its role. Cell proliferation, cell cycle, telomerase activity, and expression of senescence markers (p16 and p53) were analyzed. Results. We found that 20% elongation significantly decreased cell proliferation, promoted G0/G1 cell cycle arrest, decreased telomerase activity, and upregulated mRNA/protein expression of p16 and p53. Moreover, the inhibitor RKI-1447 partly resisted effects of 20% elongation on these parameters of cell senescence. Conclusion. High mechanical stretch obviously induces AF cell senescence through the RhoA/ROCK pathway. This study provides us a deeper understanding on the AF cell’s behavior under mechanical stretch.

Residual strains estimation in the annulus fibrosus through digital image correlation

Up-to-date, back pain is among the most prevalent health issues and generally takes its origins from lesions of the annulus fibrosus (AF). While the AF ex vivo mechanical properties are increasingly well understood, in vivo data are still missing. In particular, very few studies have precisely measured the residual strains within the AF and thus the in vivo deformation state of the AF is still miss-interpreted and miss-evaluated. In this work, we propose an original and robust method for the AF residual strains quantification via digital image correlation technics. Ten pig annulus fibrosus were extracted from adjacent vertebrae followed by a radial incision to release the residual strains. The operations were filmed and then analyzed by a custom digital image correlation software in order to quantify the circumferential, radial and shear residual deformations. Our results show that residual strains are of the same order of magnitude than the in vivo one. The average circumferential strains are in tension on the outer periphery ([3.32; 5.94]%) and in compression on the inner periphery ([−6.4; −1.69]%). The mean radial residual strains are essentially in compression ([−10.4; 2.29]%). Locally, radial and circumferential residual strains can reach really large values up to 40% of compression. The mean shear strains remain very small (−0.04% ± 2.88%). This study also shows that circumferential and radial residual strains evolve linearly along the radius and non-linearly along the angle. We propose a simple model to predict their spatial variations. Our results and methods will allow the quantification of more realistic in vivo strains and stresses within the human intervertebral disc.