Human disc nucleus properties and vertebral endplate permeability

Abstract

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.

Figure 1

Schematic of endplate specimen harvest. Specimen is cored from frozen motion segment (8.25 mm diameter). The cartilage endplate attached to the vertebral cores was separated from the nucleus pulposus tissue for microCT (μCT) and permeability measurements. The nuclear tissue was separated in three parts, they are labeled in reference to the adjacent vertebra: inferior (1), center (2), superior (3) for biochemical measurements.

Figure 2

Illustration of the permeameter. The custom made permeameter is composed of stainless steel pipes connected to a water reservoir. These pipes are also connected to a pressure transducer, a safety pressure release valve, fluid outlets, a filter and a specimen connector. The fluid is pressurized to 1 MPa and pressed through the cartilage-bone specimen. A schematic of the test specimen is shown on the top left corner where P_u is the upstream pressure, P_d1 is the pressure downstream of the cartilage endplate, and P_d2 is the pressure downstream of the subchondral bone

Figure 3

The variation of subchondral bone porosity with age (R²=0.13, p=0.004).

Figure 4

The variation of cell density (cells/mm³) with Pfirrmann degeneration grades is presented. Results are shown for mean and standard deviations (Significance using post-hoc Tukey test, *p<0.05).

Figure 5

The variation of cell density (cells/mm³) with disc height (mm) is shown. Data fitted with a non-linear fit equation of the relationship between the two parameters cell density (CD) and disc height (h); CD =16324 × h^−1.02 (R² = 0.15, p = 0.005).

Figure 6

The variation of GAG content with age is shown. Data fitted with non-linear fit equation of the two variables GAG =230118*age^−2.49 (R² = 0.83, p<0.0001).

Figure 7

The variation of cell function (GAGs/cell) was compared to Pfirrmann degeneration grading. Results are shown for mean and standard deviations. (Significance using post-hoc Tukey test, *p<0.05)