In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies

Abstract

Study design: Whole bovine coccygeal discs were cultured under static load, with or without vertebral endplates (VEPs), and assessed for cell viability, biochemical stability, biosynthetic activity, and biosynthetic responsiveness to changes in mechanical load.

Objectives: To assess the effects of VEPs on biochemical and cellular stability of disc cells during in vitro culture of large disc explants. To determine whether cultured discs could respond to mechanical perturbation.

Summary of background data: Previous methods for culturing the intervertebral disc have focused on rabbit and rat discs, but the small size of these discs limits the relevance of these culture systems to the human condition. Bovine coccygeal discs have similar dimensions to the human lumbar disc (i.e., similar size and nominal stresses), but long-term culture of these discs has not been reported.

Methods: Bovine coccygeal discs were harvested with or without VEPs, cultured under static load (5 kg, approximately 0.25 MPa, in situ swelling pressure) for up to 1 week, and evaluated for changes in hydration, glycosaminoglycan content, cell viability, and biosynthetic activity. Additionally, the biochemical and biosynthetic response of discs cultured without VEP to increasing the load to a 20-kg (approximately 1 MPa, the estimated stress in human lumbar disc during heavy lifting) static load for 6 hours was assessed.

Results: During the first 24 hours, culturing discs with endplates was moderately better with regards to maintaining in situ anulus hydration and nucleus glycosaminoglycan levels. The endplates, however, obstructed media flow to the disc, resulting in a marked decrease in cell viability after 1 week of culture. Nucleus pulposus cell viability was maintained in discs cultured without endplates, but there was a significant drop in biosynthetic activity within 2 days of culture. Despite this drop, the disc cells in the discs without VEP remained biosynthetically responsive to changes in mechanical loading.

Conclusions: It is possible to maintain cell viability and the biosynthetic responsiveness of large discs for up to 1 week in vitro when the discs are cultured under static load and without VEP.

Figure 1.

Schematic of chambers used for culture of bovine coccygeal intervertebral disc. Design based on that of Ohshima et al.

Figure 2.

a, Hydration of anulus and nucleus of fresh and cultured bovine coccygeal discs (mean ± SEM; n = 4 for cultured discs and n = 8 for fresh discs. *P < 0.05 vs. fresh discs. b, GAG content of anulus and nucleus tissue of fresh and cultured bovine coccygeal discs. Data are mean ± SEM, normalized to the average GAG content of freshly harvested tissue; n = 3 or 4 for cultured discs and n = 8 for fresh discs. *P < 0.05 vs. fresh discs.

Figure 3.

Nucleus pulposus cell viability staining of representative areas of (a) fresh disc and discs cultured for 1 (b, c) or 7 days (d–o). The six images in d–i are from the six different discs cultured with vertebral endplate (VEP). Likewise, the six images in j–o are from the six different discs cultured without VEP. Live cells fluoresce green, and dead cells fluoresce red. Images show cells in a 50-μm slice of the tissue, starting at a minimum of 50 μm from the surface. Scale bar = 200 μm.

Figure 4.

Radiolabel incorporation measured during 6 hours of free-swelling culture. Data are mean ± SEM; n = 5. *P < 0.05 vs. fresh discs. **P < 0.05 vs. day 1. †P < 0.05, no VEP vs. VEP.

Figure 5.

Biosynthetic activity of no VEP discs radiolabeled in chambers during the last 6 hours of culture (under 5-kg static load). Data are mean ± SEM; day 0, n = 10; days 1, 2, n = 5; day 7, n = 8. *P < 0.05 vs. day 0. †P < 0.05 vs. days 0 and 1.

Figure 6.

Change in ³⁵S incorporation of no VEP discs cultured under 20-kg static load for the last 6 hours of culture compared with discs cultured under 5-kg static load. Data are mean ± SEM and are presented relative to incorporation levels measured in 5-kg cultured discs; n = 5. *P < 0.05 vs. 5 kg.