Intervertebral disc degeneration has been implicated in the etiology of low back pain; however the current surgical strategies for treating symptomatic disc disease are limited. A variety of materials have been developed to replace disc components, including the nucleus pulposus (NP), the annulus fibrosus (AF), and their combination into disc-like engineered constructs. We have previously shown that layers of electrospun poly(ε-caprolactone) scaffold, mimicking the hierarchical organization of the native AF, have functional parity with native tissue. Likewise, we have combined these structures with cell-seeded hydrogels (as an NP replacement) to form disc-like angle ply structures (DAPS). The objective of this study was to develop a model for the evaluation of DAPS in vivo. Through a series of studies, we developed a surgical approach to replace the rat caudal disc with an acellular DAPS and then stabilize the motion segment by external fixation. We then optimized cell infiltration into DAPS by including sacrificial poly(ethylene oxide) layers interspersed throughout the angle-ply structure. Our findings illustrate that DAPS are stable in the caudal spine, are infiltrated by cells from the peri-implant space, and that infiltration is expedited by providing additional routes for cell migration. These findings establish a new in vivo platform in which to evaluate and optimize the design of functional disc replacements.

Intervertebral disc degeneration is characterized by a cascade of cellular, biochemical and structural changes that may lead to functional impairment and low back pain. Interleukin-1 beta (IL-1β) is strongly implicated in the etiology of disc degeneration, however there is currently no direct evidence linking IL-1β upregulation to downstream biomechanical changes. The objective of this study was to evaluate long-term agarose culture of nucleus pulposus (NP) cells as a potential in vitro model system to investigate this. Bovine NP cells were cultured in agarose for 49 days in a defined medium containing transforming growth factor-beta 3, after which both mechanical properties and composition were evaluated and compared to native NP. The mRNA levels of NP cell markers were compared to those of freshly isolated NP cells. Glycosaminoglycan (GAG) content, aggregate modulus and hydraulic permeability of mature constructs were similar to native NP, and aggrecan and SOX9 mRNA levels were not signifi cantly different from freshly isolated cells. To investigate direct links between IL-1β and biomechanical changes, mature agarose constructs were treated with IL-1β, and effects on biomechanical properties, extracellular matrix composition and mRNA levels were quantifi ed. IL-1β treatment resulted in upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 4, matrix metalloproteinase-13 and inducible nitric oxide sythase, decreased GAG and modulus, and increased permeability. To evaluate the model as a test platform for therapeutic intervention, cotreatment with IL-1β and IL-1 receptor antagonist (IL-1ra) was evaluated. IL-1ra signifi cantly attenuated degradative changes induced by IL-1β. These results suggest that this in vitro model represents a reliable and cost-effective platform for evaluating new therapies for disc degeneration.

IntroductionInflammation plays a key role in the progression of intervertebral disc degeneration, a condition strongly implicated as a cause of lower back pain. The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with interleukin-1 receptor antagonist (IL-1ra) for sustained attenuation of interleukin-1 beta (IL-1β) mediated degradative changes in the nucleus pulposus (NP), using an in vitro model.MethodsIL-1ra was encapsulated in PLGA microspheres and release kinetics were determined over 35 days. NP agarose constructs were cultured to functional maturity and treated with combinations of IL-1β and media conditioned with IL-1ra released from microspheres at intervals for up to 20 days. Construct mechanical properties, glycosaminoglycan content, nitrite production and mRNA expression of catabolic mediators were compared to properties for untreated constructs using unpaired Student's t-tests.ResultsIL-1ra release kinetics were characterized by an initial burst release reducing to a linear release over the first 10 days. IL-1ra released from microspheres attenuated the degradative effects of IL-1β as defined by mechanical properties, glycosaminoglycans (GAG) content, nitric oxide production and mRNA expression of inflammatory mediators for 7 days, and continued to limit functional degradation for up to 20 days.ConclusionsIn this study, we successfully demonstrated that IL-1ra microspheres can attenuate the degradative effects of IL-1β on the NP for extended periods. This therapeutic strategy may be appropriate for treating early-stage, cytokine-mediated disc degeneration. Ongoing studies are focusing on testing IL-1ra microspheres in an in vivo model of disc degeneration, as a prelude to clinical translation.

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