The effect of perfusion with and without mechanical loading on an in vitro model of the outer annulus...

Intervertebral disc (IVD) degeneration is a prevalent condition contributing to lower back pain, with limited treatment options that fail to restore full disc function. Tissue engineering shows promise, but most work relies on static culturing methods, which do not mimic the dynamic IVD environment or enable large-scale tissue growth. In this study, we developed an in vitro outer annulus fibrosus (OAF)-cartilage endplate (CEP) interface model and evaluated the effects of perfusion bioreactor culture, with or without cyclic hydrostatic pressure (HP), on tissue integration. Bovine OAF cells were seeded onto electrospun polycarbonate urethane (PCNU) scaffolds and combined with deep zone chondrocytes on 3D membranes. After one week of static culture, constructs were transferred to a perfusion bioreactor (5 mL/min flow) for two weeks, with a subset exposed to dynamic HP (0.1 MPa, 0.5 Hz, 1 hr, 3 times per week). Histological and immunohistochemical analyses confirmed compositional similarity to the native interface, with collagen type I, type II, and aggrecan distributions resembling physiological patterns. Perfusion significantly increased interfacial strength (35.2 ± 14.8 kPa vs 14.8 ± 6.1 kPa static) and DNA content. Although HP reduced DNA content, it did not alter Ki-67 or TUNEL-positive cell percentages. Overall, culturing in a perfusion bioreactor improved the OAF-cartilage integration by increasing tissue growth due to more cellularity, resulting in a stronger interface. These findings demonstrate that perfusion supports development of physiologically relevant IVD constructs and addresses a key translational gap in the field, providing a foundation for scaling engineered tissues to clinically relevant sizes. STATEMENT OF SIGNIFICANCE: Perfusion bioreactors can ameliorate the shortcomings of static culturing methods, and their use could address a translation gap in the field of IVD tissue engineering as they may support the scale-up of tissues to physiological sizes. In this study we demonstrate that OAF and cartilage remain integrated in perfusion culture and it both improves tissue formation and significantly increases the mechanical strength of the interface. Additionally, the application of cyclic HP influences the proliferative state of these tissues without impacting cell viability. This study demonstrates that perfusion culture can be used to bioengineer IVD tissues which may be crucial in facilitating the development of an implant that approximates the features of the native disc.