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Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 66 - 66
1 Jul 2020
Michalek A Duclos S Denning S
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Residual strain development in biological tissue is believed to result from remodeling in response to repetitive loading. This study hypothesized that differences in in-vivo loading between levels of the bovine tail result in differences in intervertebral disc (IVD) annulus fibrosus (AF) microstructural remodeling. The hypothesis was tested by quantifying tail musculature using clinical computed tomography and tissue microstructure using collagen fiber crimp period, which has previously been correlated with residual strain.

Three bovine tail segments (levels c1 through c6) were imaged using a clinical computed tomography (CT) scanner followed by removal of muscle and harvest of IVDs. The discs were frozen, and transverse cryosections were obtained. Additionally, tangential plane cryosections were obtained from the inner and outer zones of the AF.

Transverse CT slices corresponding to each joint level thresholded for both disc and muscle tissue and analyzed in MATLAB. First, the centroid of the disc image was calculated to use as an origin. Then the disc area and moments of inertia about the flexion extension axis and lateral bending axis were calculated. Total muscle area was then calculated, along with muscle moments of inertia relative to the disc centroid. All muscle parameters were normalized by those of the corresponding disc.

Cryosections were imaged using an inverted light microscope equipped with crossed polarizing filters and a digital camera. A MATLAB routine was used to perform Fourier transform analysis on user selected lines of interest in the transverse micrographs, yielding average fiber crimp period in the inner and outer AF. Micrographs from tangential sections were opened in ImageJ, and fiber orientation angles were measured manually.

Muscle moments of inertia were analyzed using a two-way ANOVA with disc level and axis as dependent variables. Normalized muscle area was analyzed with a one-way ANOVA with disc level as a dependent variable. A two-way ANOVA, with disc level and zone (inner versus outer) was used to analyze collagen fiber crimp period and collagen fiber angle.

Normalized muscle moment of inertia showed significant effects of both level and axis (p < 0 .001), decreasing at distal levels, and being lower about the flexion-extension axis than the lateral bending axis. Normalized muscle cross section showed a visible, but not significant (p=0.0721) decreasing trend with disc level. Fiber crimp period had significant effects of both level and zone (p < 0 .001), and was significantly longer in the outer zone than inner at all levels. Significant decrease in crimp period at distal levels were seen in the outer AF, but not the inner. While fiber angle was significantly (p < 0 .001) higher in the inner AF (36±6.6°) than outer AF (24±3.5°)), there was no significant effect of level.

Fiber crimp period in the AF has previously been correlated with residual circumferential strain, with larger crimp period corresponding to increased residual tension. The present study suggests that at proximal levels of the tail, where peak compressive and bending stresses in the AF (as inferred from normalized muscle area and moments of inertia respectively) are greatest, there is more accumulation of residual strain.


Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 67 - 67
1 Jul 2020
Michalek A Nguyen D Varden L
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As the intervertebral disc is largely avascular, needle injection is the most practical method for delivery of therapeutic agents used in treatments for degenerative disc disease. Intradiscal pressure increases during injection, and insufficient recovery time prior to needle retraction may result in injectate leakage. In order to determine the maximum pressure and post-injection recovery time for a given injection volume and rate, an analytical model of intradiscal injection was developed and calibrated experimentally.

A governing equation was derived defining intradiscal pressure as a function of effective permeability, initial elastic stiffness, nonlinear stiffness term, and injection rate. The equation was solved using a fourth order Runge-Kutta routine with a 0.05s time step and a ramp-dwell injection.

The model was calibrated by performing controlled intradiscal injections on five bovine caudal intervertebral discs. Three had adjacent vertebrae intact, while two were separated from vertebrae and constrained between porous stainless steel platens. A syringe driven by a linear actuator was used to inject phosphate buffered saline through a 21g hypodermic needle inserted radially into the disc to a depth of one half of the disc diameter. Injection was performed at a rate of 75μL/s to a volume of 250μL followed by a 240s dwell. Fluid pressure was recorded during both the injection phase and subsequent recovery phase. For each experimental pressure vs time trace, model parameters were varied in order to obtain an optimal fit.

The model was run with the average parameter values across a grid of possible injection protocols, with injection volume ranging from 30 to 300μL and injection time ranging from 0.1 to 5s. For each case, peak pressure and time required to reach a 1kPa threshold were recorded.

Experimentally measured peak pressure ranged from 68 to 88kPa. Pressure at the end of the 240s dwell ranged from 49 to 69kPa. There was no apparent difference between discs with and without endplates. Leakage of fluid following needle retraction was observed in all specimens. Experimental data were well fit by the analytical model, which predicted higher peak pressure and longer recovery time with increasing volume, from approximately 1500s at 30μL to nearly 3000s at 300μL. The model was nearly insensitive to injection rate.

The experimental data confirm pressurization of the disc during injection and injectate leakage resulting from insufficient recovery time. The model predicts that the time required to recover to below threshold leakage pressure is impractically long for both laboratory and clinical injection protocols. Similar behavior with and without endplates confirms that fluid flow is limited by permeability of the tissue itself, not the boundary conditions. Slow recovery is likely attributable to the fact that peak injection pressures were lower than the hydraulic swelling pressure of the nucleus pulposus, which has been reported to be approximately 140kPa.

Due to the high swelling pressure of the nucleus pulposus, it is unlikely that intradiscal injection procedures can be performed without substantial injectate leakage following needle retraction.


Orthopaedic Proceedings
Vol. 92-B, Issue SUPP_I | Pages 23 - 23
1 Mar 2010
Mwale F Demers CN Michalek A Latridis JC Goswami T Beaudoin G Beckman L Alini M Platz D Antoniou J
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Purpose: Since the human intervertebral disc (IVD) is loaded in compression for approximately 16h per day, we investigated the effect of 16h compression loading on the magnetic resonance imaging (MRI) parameters, biochemical contents, and mechanical properties, of IVDs.

Method: Bovine caudal discs (2–3 years-old; non-degenerated) from 3-disc motion segments were injected in the NP with either 5 mg of trypsin in 40 μl Tris buffer or with Tris buffer only. The motion segments were placed in bags containing saline solution and antibiotics and were kept at 37°C throughout the experiment. The motion segments were subjected to either 16h of cyclic compression loading or were left unloaded for 16h. The motion segments were then paraffin embedded for MRI examinations, which were carried out in a 1.5T machine. The IVDs were dissected and the NP and AF were separated for biochemical and mechanical analyses. The NP and AF tissues were analyzed for contents of water, glycosaminoglycan (GAG), total collagen, and denatured collagen. Swelling pressure, compressive modulus HA, and hydraulic permeability were also measured.

Results: Loading had a significant effect on the MRI parameters (T1, T2, T1rho, MTR, ADC) of both the NP and AF tissues. Loading had a greater effect on the MR parameters and biochemical composition of the NP than trypsin. In contrast, trypsin had a larger effect on the mechanical properties. Localized trypsin injection predominantly affected the NP. T1rho was sensitive to loading and correlated with the water content of the NP and AF but not with their proteoglycan content.

Conclusion: Few studies have been directed towards developing an objective and accurate diagnostic tool in the detection and quantification of matrix and mechanical changes in early IVD degeneration. In this report, we demonstrated that MR parameters were influenced by compression loading. We also show showed specific correlations between T1rho and the structural and compositional changes in the disc. Further studies are required to determine the potential of the T1rho technique to be used as a non-invasive diagnostic tool of the biochemical and mechanical changes occurring in disc degeneration.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 128 - 128
1 Mar 2008
Antoniou J Demers C Michalek A Iatridis J Goswami T Beaudoin G Beckman L Alini M Mwale F
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Purpose: Quantitative MRI is currently being tested as an early and non-invasive diagnostic tool of disc problems prior to the appearance of symptoms. The aim of the present study was to determine the effects of cyclic loading and enzymatic digestion on quantitative MRI, biochemical composition, and mechanical properties of intervertebral disc tissue.

Methods: Bovine tail segments consisting of three discs were subjected to 16h of cyclic compression loading (50N–300N–50N at 1Hz) or left unloaded for 16h while in saline solution at 37°C. Prior to loading, the nucleus pulposus were injected with either a trypsin or buffer solution. MR examinations were carried out in a 1.5T Siemens` Avanto system to measure T1 and T2 relaxation times, magnetization transfer ratio (MTR), and trace of the apparent diffusion coefficient (TrD). The nucleus pulposus and annulus fibrosus were dissected and analyzed for contents of water, glycosaminoglycan, total collagen, and denatured collagen. Cylindrical nucleus pulposus and annulus fibrosus tissue plugs were harvested, prepared, and tested under confined compression to measure compressive modulus (HA) and hydraulic permeability (k). ANOVA and linear regression analyses were performed (p< 0.05).

Results: Loading decreased the T1, T2, and TrD of NP while it increased MTR. Only water content in the nucleus pulposus was significantly influenced by loading. T1, water content, and k of the annulus fibrosus tissue were significantly reduced with loading.|Enzymatic treatment of the nucleus pulposus had no effect on its MR properties, but increased the percent of denatured collagen and thus decreased HA. None of the biochemical, mechanical, and MR parameters of the annulus fibrosus changed with trypsin treatment.

Conclusions: Dynamic loading of the disc segments for 16h decreased the permeability of both disc tissues. This was consistent with the measured drop in tissue hydration and was observed as a decrease in T1. Targeted trypsin digestion of the nucleus pulposus was confirmed with no detectable changes in the biochemical, biomechanical, or MR properties of the annulus fibrosus. Future studies will address additional quantitative MR parameters such as T1-rho, a higher strength magnet, and different enzymatic treatments. Funding: Other Education Grant Funding Parties: Canadian Institutes of Health Research, McGill William Dawson Scholar Award, and Whitaker Foundation