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.

The effects of dynamic loading on the intervertebral disc

“…Static loading, static diurnal loading or a continuous dynamic loading results in different responses of the IVD [19,57,58,72,75,123]. Studies comparing the different axial loadings (static, diurnal or dynamic compression) are summarised in Table 1.…”

“…In a bovine disc organ culture model, with the endplates removed, it has been demonstrated that diurnal loading was better to maintain GAG content than static loading but little beneficial effect was found with dynamic loading in a human physiological range (0.2-1 MPa disc pressure, 1 Hz) [57,58]. It is likely that the removal of endplates from the IVD affected the cell viability and the biosynthetic activity of the disc cells [61].…”

The elastic fibre network of the human lumbar anulus fibrosus: architecture, mechanical function and potential role in the progression of intervertebral disc degeneration

“…It is likely that inclusion of elastic fibres which transversely integrate collagenous elements would enhance the ability of tissue-engineered structures to replicate the mechanical anisotropy of the anulus matrix. Organ culture systems of the kind used in tissue engineering, and those recently developed specifically for the intervertebral disc [33], also present the opportunity to investigate the types of mechanical stimuli which result in the production of elastic fibres and their distribution in the patterns described at each level of the anulus structural hierarchy.…”

Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue

“…Previous studies have measured the release of GAGs from organ cultures as an indicator of cartilaginous tissue degradation (Tchetina et al, 2006;Korecki et al, 2007). Both MMP-2 and MMP-9 have been shown to be elevated in the synovial fluid of rheumatoid arthritis patients (Cawston and Young, 2010), which might indicate that these MMPs are potential markers for the degradation of osteochondral tissues.…”