Effects of Diurnal Loading on Intervertebral Disc using a Whole Organ in Vitro Culture Model

Abstract

Introduction Intervertebral disc (IVD) disorders associated with pain and disability are extremely prevalent and current treatment options do not result in restoration of tissue integrity or function. The exact mechanisms of IVD degeneration are not fully understood at this time although aging, injury, nutrition, metabolism, and mechanical stress are suspected to contribute. To more fully understand IVD degeneration, an in vitro whole organ model of IVD is desirable. It has been theorized that preservation of large animal IVDs in vitro is difficult due to poor diffusion of nutrients. To assess the effects of nutrient diffusion and more closely mimic in vivo loading of the human spine, we hypothesized that a diurnal loading regimen would be associated with significantly higher cell viability in IVDs compared with static loading. We further hypothesized that a supraphysiological load of 1 MPa would be associated with significant loss of cell viability and tissue morphology of IVD explants compared with a physiological load of 0.1 MPa. Material and Methods Under ACUC approval, lumbar spine segments containing L1–L5 discs were harvested from dogs ( n = 7) euthanized for reasons unrelated to this study. Soft tissue was aseptically dissected and explants were created using a diamond band saw. Explants were randomly divided into either diurnal or static load and were further divided into either 0.1 MPa load or 1 MPa load. Explants in the diurnal group were subjected to the respective load using a 12-hour cycle. After 3 days, IVDs were bisected and assessed for cell viability and viable cell density was quantified using an in-house cell counting program. Glycosaminoglycan (GAG) content and collagen content were calculated. Biomechanical analysis was determined using unconfined compression and calculated using the slope of the force is placement curve. All samples were evaluating statistically with significance set at p < 0.05 using Student t-test. Results For both static and diurnal loading, 0.1 MPa groups had significantly ( p < 0.05) higher viable cell density than 1 MPa groups after 3 days of loading. Viable cell density was significantly ( p < 0.05) higher in each diurnally loaded group compared with its statically loaded counterpart. GAG and HP contents in annulus fibrosus were significantly ( p < 0.05) higher in 0.1 MPa groups compared with 1 MPa groups. Explant stiffness was significantly ( p < 0.05) higher in the 0.1 MPa group at day 3 as compared with the day 0 controls. Conclusion To our knowledge, this is the first study to report the effects of physiological and supraphysiological static and diurnal loads on whole organ canine IVDs in culture. There were significant differences in viable cell density and extracellular matrix composition associated with level (0.1 and 1 MPa) and type (static vs. diurnal) of loading. Taken together, these data suggest that diurnal, physiological load best preserves IVD viability and integrity. Ongoing research is aimed at evaluating additional loading levels and cyclic regimens, as well as assessing the effects of insult and injury on loaded whole-organ IVD explants.