Objective: This study aimed to characterize the mesenchymal stem cell (MSC) subpopulation migrating towards a degenerated intervertebral disc (IVD) and to assess its regenerative potential. Design: Based on initial screening for migration towards CC motif chemokine ligand 5 (CCL5), the migration potential of CD146þ and CD146-mesenchymal stem cells (MSCs) was evaluated in vitro and in a degenerated organ culture model (degeneration by high-frequency loading in a bioreactor). Discogenic differentiation potential of CD146þ and CD146-MSCs was investigated by in vitro pellet culture assay with supplementation of growth and differentiation factor-6 (GDF6). Furthermore, trypsin degenerated IVDs were treated by either homing or injection of CD146þ or CD146-MSCs and glycosaminoglycan synthesis was evaluated by Sulphur 35 incorporation after 35 days of culture. Results: Surface expression of CD146 led to a higher number of migrated MSCs both in vitro and in organ culture. CD146þ and CD146-pellets responded with a similar up-regulation of anabolic markers. A higher production of sulfated glycosaminoglycans (sGAG)/DNA was observed for CD146þ pellets, while in organ cultures, sGAG synthesis rate was higher for IVDs treated with CD146-MSCs by either homing or injection. Conclusions: The CD146þ MSC subpopulation held greater migration potential towards degenerative IVDs, while the CD146-cells induced a stronger regenerative response in the resident IVD cells. These findings were independent of the application route (injection vs migration). From a translational point of view, our data suggests that CD146þ MSCs may be suitable for re-population, while CD146-MSCs may represent the primary choice for stimulation of endogenous IVD cells.

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Introduction Nucleus pulposus (NP) replacement offers a minimally invasive disc regeneration treatment alternative to traditional fusion or total disc replacement. An ideal NP replacement material should maintain motion and disc height, thus slowing down further disc degeneration.1 Various biomaterials have been investigated for their use as NP replacement, including hydrogel and nonhydrogel scaffolds, while no direct comparison study has been performed. Recently, a novel fibrin-hyaluronan (FBG-HA) conjugate hydrogel was developed to mimic native NP extracellular matrix. In addition, a novel polyurethane (PU) scaffold with swelling capability in situ was designed to restore native disc thickness and mechanical properties. The present study aimed to assess the FBG-HA hydrogel, the PU scaffold, and the combination of both in an organ culture system with dynamic load for the purpose of NP replacement. Materials and Methods FBG-HA conjugate solution was synthesized with 235 KDa HA at FBG/HA w/w ratio of 17:1 via a two-step procedure as described elsewhere.2 FBG-HA hydrogels were prepared by mixing two-thirds volume of FBG-HA conjugate solution with one-third volume of thrombin solution (5.2 U/mL) and allowed to polymerize at 37°C for 20 minutes. The PU scaffold was designed to have a flat discoid/ravioli shape that is composed of a based core with swelling capacity. Electrospinning nanofibers technology was used to manufacture an envelope sheet using a blend of polyurethane carbonates (ChronoFlex and HydroMed) that were dissolved in a mixture of dimethylformamide and dioxane. The envelope was cut to discs at diameter of 6 mm. The PU hydroMed was dissolved in 95% ethanol and dried to form a film. The film was cut to discs at diameter of 3 mm. One film disc was placed between two envelope discs and the circumference was heat welded to form a scaffold with ravioli shape. Caudal bovine intervertebral discs (IVDs) with endplates were obtained from calves (4-8 months) directly after slaughter. The discs were nucleotomized by incisions through the endplate. A 4-mm endplate core and the NP tissue below the endplate stopper were removed. The nucleotomized region was refilled with either (1) 50 to 80 µL FBG-HA hydrogel, (2) dry PU scaffold, or (3) PU scaffold surrounded by FBG-HA hydrogel. To close the defect, the removed endplate stopper was re-inserted, and the crack between the stopper and the remaining endplate was sealed by polymethyl methacrylate. Empty discs served as negative controls. To assess the mechanical compatibility of the different biomaterial implants, dynamic compressive stiffness modulus and disc height were measured for each disc at different time points: Intact, after nucleotomy, after refilling with biomaterial and free swelling culture overnight, after 3 hours dynamic load at 0 to 0.1 MPa, 0.1 Hz within a bioreactor system,3 and after free swelling recovery overnight. Results Dry PU scaffold was able to trap water and swell in situ already after 24 hours culture in the disc, as indicated by weight ...