Seeing double: a comparison of microstructure, biomechanical function, and adjacent disc health between double- and single-layer vertebral endplates.

STUDY DESIGN: Experimental and computational assessment of thickness, porosity, biomechanical behavior, and adjacent disc glycosaminoglycan content in double- and single-layer bony endplate samples harvested from human cadaver spines. OBJECTIVE: To determine if the second layer of bone in double-layer vertebral endplates allows the superficial layer to achieve a more optimal balance between its biomechanical and nutritional functions. SUMMARY OF BACKGROUND DATA: Proper disc health requires the endplate to balance opposing biomechanical and nutritional functions. Previous studies investigating endplate function report seeing double: some endplates have a second layer of bone. However, it remains unclear whether the second layer of bone has any functional advantage. Such information could shed light on the factors that protect against disc degeneration. METHODS: Six lumbar spines were obtained from human cadavers (32-84 yr) and scanned with magnetic resonance imaging. Cylindrical cores that included the endplate and underlying trabecular bone were harvested from the center of the superior vertebral endplates (6 double- and 12 single-layer endplates) and imaged using micro-computed tomography. The thickness and porosity of the bony endplate layers were measured for each core. High-resolution finite element analysis was performed to assess biomechanical behavior. Glycosaminoglycan content within the adjacent nucleus tissue was quantified using the dimethylmethylene blue technique. RESULTS: The superficial layer of the double-layer endplates was 50% thinner (P = 0.009) and tended also to be more porous than single-layer endplates. Strains were higher in thinner endplates; however, the second layer of bone in the double-layer endplates had a stiffening effect so that despite being thinner than single-layer endplates, the superficial layer of the double-layer endplates had a similar risk of damage. After adjusting for age, glycosaminoglycan content was significantly higher in the nucleus tissue adjacent to the double-layer endplates (P = 0.01). CONCLUSION: Compared with single-layer endplates, double-layer endplates seem to permit a more optimal balance between endplate biomechanical and nutritional functions, and may therefore offer a significant protective factor against disc degeneration.

Morphology of the human vertebral endplate.

It is presumed that poor intervertebral disc cell nutrition is a contributing factor in degeneration, and is exacerbated by vertebral endplate sclerosis. Yet, quantitative relationships between endplate morphology and degeneration are unavailable. We investigated how endplate bone microstructure relates to indices of disc degeneration, such as morphologic grade, proteoglycan content, and cell density. Intervertebral core samples [n = 96, 14 subjects, L1-L5 level, ages 35-85 (64 ± 16 years), degeneration grade 1 (n = 4), grade 2 (n = 32), grade 3 (n = 44), grade 4 (n = 10), grade 5 (n = 6)] that included subchondral bone, cartilage endplate, and adjacent nucleus were harvested from human cadaveric lumbar spines. The morphology of the vertebral endplate was analyzed using µCT and the adjacent nucleus tissue was collected for biochemical and cellular analyses. Relationships between vertebral endplate morphology and adjacent disc degeneration were analyzed. Contrary to the prevailing notion, vertebral endplate porosity increased between 50% and 130% and trabecular thickness decreased by between 20% and 50% with advancing disc degeneration (p < 0.05). We also observed that nucleus cell density increased (R(2) = 0.33, p < 0.05) and proteoglycan content decreased (R(2) = 0.47, p < 0.05) as the endplate became more porous. Our data suggest that endplate sclerosis is not a fundamental factor contributing to disc degeneration. Rather, the opposite was observed in our samples, as the endplate became progressively more porous with age and degeneration. Since ischemic disc cell behavior is commonly associated with degenerative change, this may be related to other factors such as the quality of vertebral capillaries, as opposed to decreased permeability of intervening tissues.

Human disc nucleus properties and vertebral endplate permeability.

STUDY DESIGN: Experimental quantification of relationships between vertebral endplate morphology, permeability, disc cell density, glycosaminoglycan (GAG) content, and degeneration in samples harvested from human cadaveric spines. OBJECTIVE: To test the hypothesis that variation in endplate permeability and porosity contributes to changes in intervertebral disc cell density and overall degeneration. SUMMARY OF BACKGROUND DATA: Cells within the intervertebral disc are dependent on diffusive exchange with capillaries in the adjacent vertebral bone. Previous findings suggest that blocked routes of transport negatively affect disc quality, yet there are no quantitative relationships between human vertebral endplate permeability, porosity, cell density, and disc degeneration. Such relationships would be valuable for clarifying degeneration risk factors and patient features that may impede efforts at disc tissue engineering. METHODS: Fifty-one motion segments were harvested from 13 frozen cadaveric human lumbar spines (32-85 years) and classified for degeneration using the magnetic resonance imaging-based Pfirrmann scale. A cylindrical core was harvested from the center of each motion segment that included vertebral bony and cartilage endplates along with adjacent nucleus tissue. The endplate mobility, a type of permeability, was measured directly using a custom-made permeameter before and after the cartilage endplate was removed. Cell density within the nucleus tissue was estimated using the picogreen method, while the nuclear GAG content was quantified using the dimethylmethylene blue technique. Specimens were imaged at 8 µm resolution using microCT; bony porosity was calculated. Analysis of variance, linear regression, and multiple comparison tests were used to analyze the data. RESULTS.: Nucleus cell density increased as the disc height decreased (R² = 0.13; P = 0.01) but was not related to subchondral bone porosity (P > 0.5), total mobility (P > 0.4), or age (P > 0.2). When controlling for disc height, however, a significant, negative effect of age on cell density was observed (P = 0.03). In addition to this, GAG content decreased with age nonlinearly (R² = 0.83, P < 0.0001) and a cell function measurement, GAGs/cell, decreased with degeneration (R² = 0.24; P < 0.0001). Total mobility (R² = 0.14; P < 0.01) and porosity (R² = 0.1, P < 0.01) had a positive correlation with age. CONCLUSION: Although cell density increased with degeneration, cell function indicated that GAGs/cell decreased. Because permeability and porosity increase with age and degeneration, this implies that cell dysfunction, rather than physical barriers to transport, accelerates disc disease.

Mechanical profiling of intervertebral discs.

Despite recent advances in imaging diagnostic technology and additional treatment options our ability to prevent or inhibit discogenic back pain has not drastically improved. The challenge of linking early degenerative patterns to dysfunction and pain remains. Using a novel material testing device designated the tissue diagnostic instrument (TDI) we measured the local stiffness and strain energy absorption in the radial direction of 13 intact intervertebral discs; effectively generating a mechanical profile of each disc. Prior to measuring mechanical properties, an MR image was taken of each spine segment and the discs were radiologically scored according to the Pfirrmann scale. After testing, a sagittal portion of each L1-L2 disc was excised from each of four spines for histology. No significant correlations were found between Pfirrmann grade and mechanical data. However, polarized light microscopy images of disc sections indicated correlations between local tissue modulus measured with the TDI and the clarity and density of lamellar striations.

Stab incision for inducing intervertebral disc degeneration in the rat.

STUDY DESIGN: The degenerative response of rat tail and lumbar intervertebral discs to a stab incision was evaluated. OBJECTIVE: To examine and compare the postinjury degenerative response of lumbar and tail discs. SUMMARY OF BACKGROUND DATA: Although successful in larger animals, a stab incision for inducing disc degeneration in rats has not been evaluated. Rodents are desirable models for disc repair studies due to their low cost, ease of care, and fast healing times. METHODS: Lumbar and tail discs were exposed surgically and stabbed with a number 11 blade. Disc architecture, levels of IL-1beta, IL-6, and TNF-alpha, and biomechanical properties were analyzed. A functional disability secondary to multilevel lumbar disc injury was quantified and compared with that of rats undergoing sham surgery. RESULTS: Histologic evaluation of stabbed tail discs demonstrated a nucleus pulposus size decrease, anular collagen layer disorganization, and cellular metaplasia of anular fibroblasts to chondrocyte-appearing cells. Besides the continued presence of the stab injury tract, few changes were observed in the lumbar disc histology. Cytokine measurements indicated a transient peak in IL-1beta in tail discs 4 days following injury. No significant changes in IL-1beta, IL-6, or TNF-alpha were measured. No significant differences in biomechanical properties were observed between stab injury and sham surgery discs. Yet, despite insignificant differences in histologic, cytochemical, or biomechanical properties in the lumbar discs, the rats with lumbar stab injury had a significant decrease in walking ability 28 days after surgery. CONCLUSIONS: Tail disc stab injury was successful in creating morphologic signs of degeneration and transient high concentrations of IL-1beta. However, the degenerative response in the lumbar discs was much slower, suggesting that site-specific factors, such as increased stability due to posterior elements and torso musculature, helped facilitate healing. Yet, functional assessment indicated that the rats were partially disabled by multiple lumbar injuries.