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Intervertebral disc (IVD) degeneration is a common pathology recognized nowadays as the major cause for low back pain. Current treatments still cannot address the underlying problem of disc degeneration and can hardly restore the normal spine biomechanics. Cell-based therapies have been attempted in human trials, but the effect of cells posttransplantation is still poorly understood. 1,2  On the contrary, the use of cell carriers such as hydrogels have been able to improve cell viability, and also promoted cell differentiation into NP-like cells (i.e., expression of aggrecan and collagen type II). 3,4  Nevertheless, the long-term survival of the implanted cells remains unknown and doubtful. Therefore, the development of new regenerative treatments focused on endogenous cell targeting, activation, and recruitment seems to be a feasible strategy for IVD degeneration. The aim of the present study was to investigate human mesenchymal stem cells (hMSCs) recruitment toward a chemokine-delivery system in a degenerating IVD. For that purpose, we have incorporated stromal cell-derived facto-1 (SDF-1), a well-known chemokine which is able to recruit hMSCs, 5  into hyaluronan-poly(N-isopropylacrylamide) HA-pNIPAM hydrogel 6  and assessed: (1) SDF-1 release from HA-pNIPAM hydrogel; (2) hMSCs migration in an ex vivo model of degenerating IVD; and (3) the impact of hMSCs donor age in cell migration.Materials and Methods   HA-pNIPAM was used as a platform for the delivery of SDF-1. Briefly, HA-pNIPAM was reconstituted in PBS (pH 7.4) at a concentration of 10% wt/vol, containing different amounts of SDF-1 (Fig. a). Release studies were performed in vitro and in the ex vivo model system. The amount of released chemokine was quantified by enzyme-linked immunosorbent assay (ELISA). To assess hMSCs migration, an ex vivo model was used. IVDs with vertebral endplate (VEP) were isolated from young (5-8 months old) bovine tails ( n = 10) and hMSCs were obtained from human bone marrow ( n = 5, 19/21/24/40/64 years old). A model of IVD injury through the VEP (“cavity”) was adopted to mimic the process of disc degeneration. This cavity was filled with different formulations of the gel or remained empty and then sealed with cement. hMSCs previously labeled with PKH26 (red) were seeded (1 × 106 cells/disc) on the top of VEP (opposite to the injury) and then incubated in DMEM 4.5 g/L glucose, 2% FBS, 1% Pen/Strep, 1% ITS and 1, 0.1% Primocin during 48 hours (Fig. b). The IVDs prepared as described were randomized for the defined six groups in the study: (1) intact disc (intact); (2) discs with an empty cavity (cavity); (3) discs with a cavity filled with HA-pNIPAM gel only (HAP); (4) discs with a cavity filled with HA-pNIPAM with 5 ng/µL of SDF-1 (HAP SDF-1 (100)); (5) discs with a cavity filled with HA-pNIPAM with 10 ng/µL of SDF-1 (HAP SDF-1 (200)); (6) discs with a cavity filled with 5 ng/µL of SDF-1 solution in PBS (SDF-1 Sol (100)). Cell migration was evaluated by fluorescence microscopy and cells were counted manually using AxioVision Software 4.8 (Fig. b).Results   HA-pNIPAM was able to release SDF-1 in both in vitro and ex vivo systems, thus providing a suitable carrier for the chemokine and moreover to create a SDF-1 gradient to the cells in the IVD. Migration of MSCs was observed in all the six conditions tested; nonetheless significant enhancement of migration was observed in comparison to the controls, in the presence of SDF-1-hydrogels (Fig. c). The percentage of hMSCs found in the annulus fibrosus (AF) and nucleus pulposus (NP) was very similar, showing that cells are capable to degrade the matrix of both tissues (Fig. d). Cells from younger donors ( n = 3, 19/20/21 years old) showed a 3.7 ± 0.5 fold increase in migration in comparison to those from older donors ( n = 2, 40/64 years old), suggesting that hMSCs isolated from younger donors are much more prone to migrate, especially in response to the injury created (the cavity), when compared with older ones (Fig. e).Conclusion   Overall, this study demonstrates that the thermoreversible HA-PNIPAM hydrogel is a suitable carrier for SDF-1 delivery in the IVD being able to recruit hMSCs toward the degenerating disc. The results presented here constitute a first proof of concept for an innovative therapy for IVD degeneration. With this strategy we expect to overcome the disadvantages associated with cell transplantation therapies, namely cell survival, taking advantage of hMSCs potential. In the future, we expect to investigate if cell recruitment could be a valuable approach for IVD regeneration in vivo, in particular in the enhancement of ECM synthesis. Acknowledgments AO SPINE Research Network (AO SRN) for funding. FCT-Fundação para a Ciência e Tecnologia for the grants SFRH/BD/85779/2012 and SFRH/BPD/85651/2012. Disclosure of Interest None declared References Orozco L, Soler R, Morera C, Alberca M, Sánchez A, García-Sancho J. Intervertebral disc repair by autologous mesenchymal bone marrow cells: a pilot study. Transplantation 2011;92(7):822–828 Yoshikawa T, Ueda Y, Miyazaki K, Koizumi M, Takakura Y. Disc regeneration therapy using marrow mesenchymal cell transplantation: a report of two case studies. Spine 2010;35(11):E475-E480 Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials 2006;27(3):335–345 Crevensten G, Walsh AJ, Ananthakrishnan D, et al. Intervertebral disc cell therapy for regeneration: mesenchymal stem cell implantation in rat intervertebral discs. Ann Biomed Eng 2004;32(3):430–434 Gonçalves RM, Antunes JC, Barbosa MA. Mesenchymal stem cell recruitment by stromal derived factor-1-delivery systems based on chitosan/poly(γ-glutamic acid) polyelectrolyte complexes. Eur Cell Mater 2012;23:249-260, discussion 260–261 Mortisen D, Peroglio M, Alini M, Eglin D. Tailoring thermoreversible hyaluronan hydrogels by “click” chemistry and RAFT polymerization for cell and drug therapy. Biomacromolecules 2010;11(5):1261–1272

Stromal Cell-Derived Factor-1 Delivery System: A New Approach for the Recruitment of Mesenchymal Stem Cells in Degenerating Intervertebral Disc