Characterization of Human Anulus Fibrosus– and Nucleus Pulposus–Derived Cells During Monolayer Expansion and in Hydrogel Cultures

Abstract

In vitro-expanded intervertebral disc (IVD) cells could be a source for disc repair. However, IVD cell characterization still remains challenging and is demanded to detect phenotypical shifts. Therefore, the aim of the present study was to determine IVD cell expression profile during two- and three-dimensional culturing in direct comparison to in situ conditions. Human IVD tissue was analyzed immunohistologically and anulus fibrosus (AF) and nucleus pulposus (NP) cells were isolated and characterized for cytoskeletal architecture and expression of typical markers (type I, II, and III collagens, aggrecan, decorin, cartilage oligomeric protein, the chondrogenic transcription factor sox9, the tendon markers scleraxis and tenascin C) during 6 monolayer passages using real-time detection polymerase chain reaction and/or immunolabellings. Cells were introduced in alginate and collagen hydrogels and cell morphology and viablility was determined after 7 days. In addition to typical extracellular matrix components, IVD tissue and isolated cells revealed scleraxis expression. In early passages of cell expansion, genes of sox9, scleraxis, and the small proteoglycan decorin were expressed higher, but type I and III collagen genes were expressed lower in NP cells compared with AF cells. However, in passage 6, actin stress fibers increased and the expression levels of sox9 were nearly similar in NP and AF cells. The immunolabeling indicated that the fibroblast marker tenascin C could only be detected in vitro in both cell types but not in situ. Decorin protein expression decreased in both cell types in vitro in passage 6. IVD cells survived in both hydrogel cultures, and some cells elongated in collagen gels.