Tissue Engineering for Intervertebral Disc Repair and Regeneration

Introduction Tissue engineering aims to combine isolated cells, engineered materials and small molecule biochemical factors to generate neotissues with specific form and function to replace or repair damaged or degenerate native tissue. Intervertebral disc (IVD) degeneration directly causes back pain, and understanding how to regenerate this tissue may help alleviate this costly global problem. Several studies have combined isolated nucleus pulposus (NP) disc cells with novel scaffold materials to produce promising therapeutics toward IVD repair. We have taken three avenues toward IVD tissue engineering: culturing isolated IVD cells on continuous expansion surfaces to enhance cell phenotypes, utilizing novel injectable hydrogels for cell delivery to intact discs, and production of 3D printed scaffold geometries enhancing disc matrix production by seeded NP cells. Material and Methods Bovine NP cells were isolated from caudal discs of freshly slaughtered steer (∼ 24 months) obtained from a local slaughterhouse. Bovine NP cells are expanded on either tissue culture plastic with two passages, or on highly elastic silicone rubber dishes which slowly expands the surface from 12 to 78 cm 2 and marker gene expression is compared. Isolated cells are labeled with fluorescent membrane binding dye, and cells are mixed with an injectable, chitosan/hyaluronic acid hydrogel. Approximately 100 µL of cell/gel (10 6 cells/µL of gel) mixtures is injected into isolated whole bovine discs (obtained as described earlier, with vertebral bone drilled down to the cartilage end plate). Discs are then placed into a custom bioreactor, and dynamic diurnal loading is applied to the injected discs. After 14 days, localization and viability of injected cells is assessed by confocal microscopy. For 3D printed constructs, 10 × 10 × 0.3 mm plastic scaffolds were printed using a desktop 3D printer, with pore size of approximately 700 µm. Overall, 5 × 105 bovine NP cells were seeded on scaffolds, and cell viability, DNA content, proteoglycan content, and collagen type II content was assessed after 21 days in culture. Results When cultured on elastic silicone dishes, continuous expansion of bovine NP cells preserve cell phenotype and promotes higher aggrecan and collagen type II expression compared with tissue culture plastic controls. NP cells embedded in a hydrogel and injected into whole discs were localized throughout the discs. Cells also showed high viability after 14 days in culture. Bovine NP cells seeded on 3D printed orthogonal plastic scaffolds grow extremely well and fill in the large 700 µm pores over 21 days culture. The cultured constructs have high viability and produce ample aggrecan and collagen type II protein. Conclusion By combining three different approaches we show the following: (1) individual NP cells can be engineered to maintain superior phenotypic qualities compared with standard culture procedures, (2) combination of expanded cells with injectable hydrogels can be delivered to intact discs for potential repair, and (3) simple 3D printed scaffolds can promote NP matrix production by seeded cells. Combination of these avenues may further enhance IVD regenerative capacities.

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