Introduction

The nucleus pulposus (NP) of a healthy intervertebral disc (IVD) is rich in collagen type II fibers that are arranged in a random fashion to entrap the highly anionic proteoglycan aggrecan which confers the swelling properties important for resistance to compression. A hallmark of IVD degeneration is the decrease in proteoglycan content. Matrix degradation and proteoglycan loss from the NP result in a decrease in weight bearing capacity and loss of disc height. In the final stages of IVD degradation fissures appear in the annular ring allowing extrusion of the NP. It is crucial to understand the interplay between mechanobiology, disc composition, and metabolism to understand the underlying cause of disc degeneration and to be able to study ways to regenerate the degenerate disc. To address such questions, a bioreactor has been developed that facilitates organ culture of intact discs in a controlled dynamically loaded environment.¹ The bioreactor can be used in combination with an isolation and degeneration method which maintains the integrity of the intervertebral discs by preserving the noncalcified part of the cartilage endplate.^2,3 To allow repair strategies to be studied, a degeneration method utilizing trypsin injection to deplete proteoglycans (PGs) from the matrix has been established.²

Materials and Methods

Intact bovine and human discs were prepared as previously described.^2,3 Degeneration was induced by trypsin in the bovine discs.² Human and bovine discs were loaded in the bioreactor system over 14 days. Cell viability or metabolic activity was assessed using a Live/Dead and/or Alamar Blue metabolic assay. The proteoglycan content was assessed by DMMB assay and Safranin O staining of histological sections. CHAD and collagen type II were examined by western blotting. Stress profilometry was performed at 0.6 MPa static load.⁴

Results

After PG depletion, the discs were loaded in the bioreactor under physiological cyclic dynamic load. Cell viability and metabolic activity at the end of the culture period was assessed. Bovine discs treated with trypsin maintained high cell viability when cultured for 14 days both loaded and unloaded. PG content was measured using the DMMB assay and by Safranin O staining of tissue sections. Unloaded discs lost approximately 60% of their proteoglycan content, whereas discs loaded under physiological dynamic load completely replenished the proteoglycan content. Collagen type II and CHAD protein levels were also increased under physiological dynamic loading. In a separate set of discs, stress profilometry was performed at 0.6 MPa static load and stress profiles were generated. No significant change in the load profiles were found for a low trypsin dose, however, the discs treated with the experimental dose of trypsin showed an 11% reduction of the internal pressure.

The system has also been used to culture and load intact human discs under three different load magnitudes (high 0-3-1.2 MPa, medium 0.1-0.6, and low 0.1-0.3 MPa). The load curves and cell viability were followed over a 2-week period. Discs cultured under low load maintained a viability of > 90%, discs loaded under medium load showed a viability of > 86%, whereas the cell viability decreased to below 55% in discs loaded under high loads.

Conclusion

This study shows that physiological load has the ability to stimulate PG synthesis and to fully restore PG content after 14 days of axial dynamic loading at a physiological level. It also allows the response to load to be evaluated. The bioreactor can also be used to evaluate changes in mechanical properties of the disc following biologically induced changes, or to induce biomechanical stimulus of the disc to generate a biological change. As such, it is equally useful for studying the role of load in inducing disc degeneration or the role of biological stimuli in restoring disc function. It provides an experimental platform useful to evaluate if biologic repair is feasible over a range of loading conditions, or is impaired outside this range. Such knowledge is important for patient advice on lifestyle following a biological repair procedure.

Disclosure of Interest

None declared

References

Haglund L, Moir J, Beckman L, et al. Development of a bioreactor for axially loaded intervertebral disc organ culture. Tissue Eng Part C Methods 2011;17(10):1011–1019

Jim B, Steffen T, Moir J, Roughley P, Haglund L. Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential. Eur Spine J 2011;20(8):1244–1254

Gawri R, Mwale F, Ouellet J, et al. Development of an organ culture system for long-term survival of the intact human intervertebral disc. Spine 2011;36(22):1835–1842

McNally DS, Adams MA. Internal intervertebral disc mechanics as revealed by stress profilometry. Spine 1992;17(1):66–73

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The Ability of Physiological Load to Restore Disc Function