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The main objective of the current investigation was to regenerate cells of the nucleus pulposus without loss of phenotype. Nucleus pulposus cells were isolated from intervertebral discs from adult rabbits, grown in monolayer culture, and then maintained as a micromass pellet in tube culture. The specimens were evaluated by transmission and light microscopy, reverse transcriptase polymerase chain reaction, and immunohistochemistry. Nucleus pulposus cells proliferated in monolayer culture. When almost confluent, the cells were transferred to a tube and sedimented to form a pellet. The cells reverted to a rounded configuration and formed cell nests surrounded by extensive extracellular matrix, similar to that seen in vivo. These cells did not proliferate. Similar to that observed in situ, cells in pellet culture also expressed aggrecan, CD44, collagen Type II, and collagen Type I, but not collagen Type X, and had low alkaline phosphatase activity. The results of the investigation indicated that nucleus pulposus cells grown in monolayer culture might revert to their original characteristics when transferred to an environment that allows three-dimensional growth, such as upon implantation, a one-step approach. The results also indicated that the two-stage culture procedure might provide an expedient technique to regenerate nucleus pulposus tissue for disc repair.

The characteristics of the nucleus pulposus cells from adult rabbits maintained in in vitro cultures were described in another study. Herein, the authors provide a parallel profile of adult rabbit nucleus pulposus in situ, therefore allowing direct comparisons between in vitro and in situ investigations. Nucleus pulposus specimens from adult rabbits were evaluated using biochemical and immunohistochemical morphologic techniques. The nucleus pulposus from adult rabbits contained cell clusters embedded in proteoglycan-collagen matrix. The cells exhibited a well-defined Golgi system, an extensive endoplasmic reticulum, and a complex vesicular system filled with beaded structures (proteoglycans). Neither necrotic nor apoptotic cells were evident. There was a lack of mitochondria. The extensive extracellular matrix contained amorphous, beaded, and fibrillar components. The fibrillar banding was indicative of Type VI collagen. The nucleus pulposus of adult rabbits expressed aggrecan, collagen Type I and Type II, and CD44, but not collagen Type X and displayed low alkaline phosphatase activity.