Quantifying Baseline Fixed Charge Density in Healthy Human Cartilage Endplate: A Two-point Electrical Conductivity Method

Abstract

Study design: Regional measurements of fixed charge densities (FCDs) of healthy human cartilage endplate (CEP) using a two-point electrical conductivity approach.

Objective: The aim of this study was to determine the FCDs at four different regions (central, lateral, anterior, and posterior) of human CEP, and correlate the FCDs with tissue biochemical composition.

Summary of background data: The CEP, a thin layer of hyaline cartilage on the cranial and caudal surfaces of the intervertebral disc, plays an irreplaceable role in maintaining the unique physiological mechano-electrochemical environment inside the disc. FCD, arising from the carboxyl and sulfate groups of the glycosaminoglycans (GAG) in the extracellular matrix of the disc, is a key regulator of the disc ionic and osmotic environment through physicochemical and electrokinetic effects. Although FCDs in the annulus fibrosus (AF) and nucleus pulposus (NP) have been reported, quantitative baseline FCD in healthy human CEP has not been reported.

Methods: CEP specimens were regionally isolated from human lumbar spines. FCD and ion diffusivity were concurrently investigated using a two-point electrical conductivity method. Biochemical assays were used to quantify regional GAG and water content.

Results: FCD in healthy human CEP was region-dependent, with FCD lowest in the lateral region (P = 0.044). Cross-region FCD was 30% to 60% smaller than FCD in NP, but similar to the AF and articular cartilage (AC). CEP FCD (average: 0.12 ± 0.03 mEq/g wet tissue) was correlated with GAG content (average: 31.24 ± 5.06 μg/mg wet tissue) (P = 0.005). In addition, the cross-region ion diffusivity in healthy CEP (2.97 ± 1.00 × 10 cm/s) was much smaller than the AF and NP.

Conclusion: Healthy human CEP acts as a biomechanical interface, distributing loads between the bony vertebral body and soft disc tissues and as a gateway impeding rapid solute diffusion through the disc.

Level of evidence: N/A.

Figure 1

Schematic view of specimen preparation. Motion segments were opened through the median plane of the IVD with a scalpel. A cylindrical plug (8 mm diameter) of NP or AF/endplate/bone was extracted from superior and inferior surfaces at four regions of the disc (center, lateral, anterior, and posterior) with a serrated-edge cutter. Each plug was then mounted onto the freezing stage of a sledge microtome (Leica SM2400, Leica Biosystem, Chicago, IL) to remove the overlying NP/AF (relatively more transparent than CEP tissue) and vertebral bone. The final disc-shaped CEP specimens were punched with a 5 mm corneal trephine for electrical conductivity measurements. Specimen preparation was performed in a humidified hood to prevent tissue dehydration.

Figure 2

Experimental flow of the two-point conductivity approach for FCD and ion diffusivity measurements in healthy human CEPs. Electrical conductivity was measured with a previously developed conductivity apparatus ^,, consisting of two stainless steel current electrodes coaxial to two Teflon-coated Ag/AgCl voltage electrodes placed on the top and bottom of a cylindrical nonconductive Plexiglass chamber (5mm diameter). Measurements were achieved by sequentially equilibrating tissue specimens in two concentrations (i.e., isotonic and hypotonic) of potassium chloride (KCl) solution and measuring the corresponding electrical conductivity of the tissue.

Figure 3

Region-dependent electrical conductivities of healthy human CEPs under isotonic and hypotonic conditions. Electrical conductivity in the central region was significantly higher than in other regions under both isotonic (p<0.0001) and hypotonic conditions (p<0.0001).

Figure 4

(A) Region-dependent FCDs from electrical conductivity measurements in healthy human CEPs. FCD in the lateral region was significantly lower (p=0.04) than in other regions. (B) The linear correlation between FCD and GAG content in healthy human CEPs was significant (r=0.472, p=0.005).

Figure 4

(A) Region-dependent FCDs from electrical conductivity measurements in healthy human CEPs. FCD in the lateral region was significantly lower (p=0.04) than in other regions. (B) The linear correlation between FCD and GAG content in healthy human CEPs was significant (r=0.472, p=0.005).

Figure 5

(A) Region-dependent relative ion diffusivities of healthy human CEPs. Ion diffusivity in the central region was significantly higher (p<0.0001) than in other regions. (B) The linear correlation between ion diffusivities and porosities in healthy human CEPs was significant (r=0.555, p=0.001).

Figure 5

(A) Region-dependent relative ion diffusivities of healthy human CEPs. Ion diffusivity in the central region was significantly higher (p<0.0001) than in other regions. (B) The linear correlation between ion diffusivities and porosities in healthy human CEPs was significant (r=0.555, p=0.001).