Characterization of photocrosslinked alginate hydrogels for nucleus pulposus cell encapsulation.

Intervertebral disc (IVD) degeneration is a significant health concern in the USA. Tissue engineering strategies have the potential to provide a viable alternative to current treatments. Nevertheless, such approaches require a suitable biomaterial scaffold for IVD tissue regeneration. Calcium crosslinked alginate has traditionally been used for in vitro culture of nucleus pulposus (NP) cells of the IVD. However, such ionically crosslinked hydrogels lose structural integrity over time. Recently, various polymers have been modified with photopolymerizable functional groups to create covalently crosslinked hydrogels. This technology may be employed to maintain the structural and mechanical integrity of three-dimensional alginate hydrogels. In this study, photocrosslinkable alginate was synthesized and evaluated for material properties and the ability to maintain the viability of encapsulated NP cells. Photocrosslinked alginate at varying percent modifications and weight/volume percentages displayed equilibrium swelling ratios and Young's moduli of 30.52 +/- 1.782 to 43.50 +/- 1.345 and 0.5850 +/- 0.1701 to 8.824 +/- 0.6014 kPa, respectively. The viability of encapsulated NP cells was highest in hydrogels at lower percent modifications, and decreased with time in culture. Taken together, this study is the first to demonstrate that photocrosslinked alginate can be used for cellular encapsulation and synthesized with tunable material properties that may be tailored for specific applications.

Distinct intervertebral disc cell populations adopt similar phenotypes in three-dimensional culture.

Tissue engineering strategies have the potential to improve upon current techniques for intervertebral disc repair. However, determining a suitable biomaterial scaffold for disc regeneration is difficult due to the complex fibrocartilaginous structure of the tissue. In this study, cells isolated from three distinct regions of the intervertebral disc, the outer and inner annulus fibrosus and nucleus pulposus, were expanded and seeded on resorbable polyester fiber meshes and encapsulated in calcium crosslinked alginate hydrogels, both chosen to approximate the native tissue architecture. Three-dimensional (3D) constructs were cultured for 14 days in vitro and evaluated histologically and quantitatively for gene expression and production of types I and II collagen and proteoglycans. During monolayer expansion, the cell populations maintained their distinct phenotypic morphology and gene expression profiles. However, after 14 days in 3D culture, there were no significant differences in morphology, gene expression, or protein production between all three cell populations grown in either alginate or polyester fiber meshes. The results of this study indicate that the culture environment may have a greater impact on cellular behavior than the intrinsic origin of the cells, and suggest that only a single-cell type may be required for intervertebral disc regenerative therapies.

The effect of serial monolayer passaging on the collagen expression profile of outer and inner anulus fibrosus cells.

STUDY DESIGN: Sheep outer and inner anulus fibrosus cells were isolated and analyzed to determine the effect of serial monolayer passaging on their phenotype. OBJECTIVES: To characterize the effect of sequential serial passage on outer and inner anulus cells to determine at which point passaged cells are significantly different from freshly isolated cells. SUMMARY OF BACKGROUND DATA: Previous studies show that chondrocytic cells lose their differentiated phenotype with sequential monolayer passage. Although intervertebral disc cells are similar, to our knowledge, a complete characterization of passage effects has not been performed. METHODS: Sheep outer and inner anulus cells were isolated, serially passaged, and evaluated for changes in cellular morphology, collagen I and II gene expression and protein elaboration, and total protein and deoxyribonucleic acid content. RESULTS: Outer anulus cells displayed an elongated morphology, while inner anulus cells were initially polygonal and became more fibroblast-like with passage. At low passage, outer anulus cells showed higher collagen I expression, while inner anulus cells indicated higher collagen II expression. At high passage, collagen I expression increased for inner anulus cells and decreased for outer anulus cells, whereas collagen II expression decreased for both cell types. Immunohistochemical staining confirmed gene expression results. CONCLUSIONS: The differences in expression profiles of outer and inner anulus cells support previous findings that zonal differences exist between the cell types. Up to passage 2, both cell types were not significantly different from freshly isolated cells and maintained distinct phenotypic characteristics. However, after 6 sequential passages, outer and inner anulus cells became morphologically indistinguishable, and displayed no significant differences in collagen I gene and protein expression, thus becoming a more homogeneous population. As such, serial monolayer passaging has a marked effect on disc cell behavior, and is an important factor to consider when designing and evaluating in vitro studies and for potential cell-based therapies for disc repair.