Characterization of an in vitro intervertebral disc organ culture system

Abstract

Intervertebral disc organ culture has the capacity to control mechanical and chemical boundary conditions while keeping the tissue largely intact, and allowing interventions that would be impossible or unethical on animal studies. Recent studies on ex vivo organ culture has mostly involved small animals, or been limited to development and validation studies. In this study, bovine caudal discs were used. The large animal model design ensures that sufficient tissue is available for measurement of multiple dependent variables on the same disc, and a similar aspect ratio, diffusion distance, composition and rate of proteoglycan synthesis to human lumbar discs. The first goal of this study was to refine a set of dependent variables capable of characterizing the response of the intervertebral disc to culturing and to develop a technique to measure cell viability in all three regions of the disc. The second goal was to use these variables to compare static and diurnal loading as a method of maintaining intervertebral disc structure, composition, and cell metabolism similar to the in vivo state. Static (0.2 MPa) and diurnal loading (0.1 and 0.3 MPa alternating at 12 h intervals) were applied and intervertebral discs were examined after 4 or 8 days with dependent variables including changes in geometry (disc height and diameter), composition (tissue water content, tissue proteoglycan content and proteoglycan content lost to the culture media), cell viability and metabolism (proteoglycan synthesis). Results indicate that there was a decrease in disc height and water content after culture regardless of culture duration or loading condition. Cell viability significantly decreased with culture duration in the inner annulus and nucleus; however, a significant reduction in cell viability for the diurnal versus static loading condition was only observed after 8 days in the nucleus region. No significant differences were seen in viability of the outer annulus region with time, or in any loading groups. We conclude that our system is capable of keeping bovine caudal discs alive for at least 8 days without significant changes in GAG content, or cell metabolism, and that static loading was slightly better able to maintain cell viability than diurnal loading. This system offers promise for the future studies on large intervertebral discs requiring measurements of multiple mechanical and biological dependent variables on the same tissue.

Fig. 1

Average ± SD percent water content by disc region for each of the testing groups (OA outer annulus, IA inner annulus, NP nucleus pulposus). All testing groups were significantly (P < 0.05) different relative to control (indicated by asterisks). Solid bars represent static loading groups and diagonal striped groups represent diurnal loading groups. Of the loading groups bars containing black are 4 days sample and gray represent 8 days sample

Fig. 2

Average ± SD GAG loss to the culture media over the course of the culture period. Values are expressed as a percentage of initial disc bulk weight. No significant effect of loading groups was detected. Bars have similar patterns as in Fig. 1, with the additional control specimen being denoted with vertical bars

Fig. 3

Average ± SD GAG content by tissue region at the end of each testing period (OA outer annulus, IA inner annulus, NP nucleus pulposus). No significant effect of loading groups was detected

Fig. 4

Average ± SD sulfate incorporation rates normalized to tissue wet weight for all testing groups (OA outer annulus, IA inner annulus, NP nucleus pulposus). No significant effect of loading groups was detected

Fig. 5

Representative viability images at ×20 magnification (black alive, white dead). Scale bar in black equals 400 μm. Columns represent test groups [C control, numbers correspond to days in culture (four or eight) and S and D represent static and diurnal loading, respectively], and rows represent tissue regions (OA outer annulus, IA inner annulus, NP nucleus pulposus)