Nutrient supply and nucleus pulposus cell function: effects of cartilage endplate permeability and potential implications for intradiscal biologic therapy

Abstract

BACKGROUND CONTEXT Intradiscal biologic therapy (stem cell, gene, or growth factor) is a promising strategy for disc regeneration. However, these therapies require a rich nutrient supply, which may be limited by the permeability of the cartilage endplate (CEP). PURPOSE The goals of this research were to: 1) investigate how physiologic fluctuations in CEP permeability impact nutrient diffusion and nucleus pulposus (NP) cell survival and function; 2) identify compositional traits of the CEP matrix that coincide with reduced permeability; and 3) test if matrix modification of the CEP via enzymatic treatment with human MMP-8 can improve nutrient transport and NP cell survival. We hypothesized that deficits in CEP composition that hinder nutrient diffusion would associate with impaired NP cell survival and function, and that ameliorating those CEP compositional deficits via MMP-8 treatment would enhance nutrient diffusion and cell survival. STUDY DESIGN/SETTING This basic science study involved ex vivo analyses of cadaveric human CEP tissues, bovine NP cell culture, and biomechanical testing. METHODS Human CEP tissues harvested from six cadaveric lumbar spines (38-66 years old; 56 ± 10 years; Pfirrmann grades II-IV) were treated with 2.0 U/mL recombinant MMP-8 or control buffer (n = 6-12 CEP samples per group) for 18 h at 37°C and placed at the open sides of diffusion chambers. Bovine NP cells cultured inside the chambers were nourished by nutrients diffusing through the CEP tissues. After 72 hours in culture, depth-dependent NP cell viability and gene expression were measured using a live/dead assay and in situ hybridization. These cell viability and function outcomes were related to CEP transport properties and CEP biochemical composition, determined using fluorescence recovery after photobleaching (solute: 376 Da fluorescein) and Fourier transform infrared spectroscopy imaging. To test the effects of CEP matrix modification in whole discs, one CEP from each of six bovine discs was superficially treated with 0.75 U/mL collagenase or control buffer (n = 3 discs/group). Solute concentration inside the discs was measured after submerging the discs in fluorescein baths and applying dynamic compression (0.2 MPa for 6 h, then 0.4-0.8 MPa 0.5 Hz for 1.5 h). RESULTS Solute diffusivity in the CEPs varied nearly 4-fold (14.8 ± 5.1 μm2/s; range: 5.9–21.5 μm2/s), and chambers with the least permeable CEPs had up to 51% shorter viable distance from the CEP/nutrient interface. CEPs that permitted the least solute diffusion also attenuated the expression of aggrecan, type 2 collagen, and MMP-2 by the NP cells in the chambers. Doubling the chamber cell density shortened the viable distance; however, this effect depended on CEP transport properties. For CEPs with low diffusivity ( CONCLUSIONS Cartilage endplate permeability has a significant effect on NP cell survival and function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply. Treatment of the CEP with matrix-modifying enzymes can ameliorate these matrix changes and thereby improve disc nutrient supply for biologic therapy. FDA DEVICE/DRUG STATUS Human recombinant MMP-8, Clostridium collagenase (Not approved for this indication).