Characterization of nucleus pulposus-like tissue formed in vitro

In order to be able to study the metabolism of nucleus pulposus (NP) tissue, we developed a cell culture system that resulted in the formation of NP-like tissue in vitro. NP cells were isolated from sheep lumbar spines and grown on filter inserts (Millicell CM). Histological examination showed that the cells accumulated extracellular matrix and formed a continuous layer of NP-like tissue. The accumulation of sulfated proteoglycans in the NP-like tissue continued up to 10 weeks and this was paralleled by an increase in tissue thickness and dry weight. DNA content remained stable during the first 4 weeks but then decreased over time. The amount of DNA, glycosaminoglycan (GAG) and collagen per mg dry weight of the tissue generated after 10 weeks in culture were 1.25+/-0.02, 301.6+/-27.7 and 411+/-65 microg, respectively, compared with 1.04+/-0.08, 320.6+/-21.2 and 399+/-4.4 microg (mean +/- SEM) for the in vivo tissue. There was no significant difference between in vitro and in vivo tissue. The cells in culture synthesized large proteoglycans (kav = 0.26+/-0.03, mean +/- S.D.) which were similar in size to those synthesized by cells in NP tissue in ex vivo culture (kav = 0.22+/-0.02, mean +/- S.D.) as determined by Sepharose CL-2B column chromatography. The in vitro generated tissue contained type II collagen as demonstrated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and silver staining as well as Western blot analysis. NP cells grown on filters generate tissue similar in composition to the in vivo tissue, for the characteristics examined to date, and should be a suitable model to use to study NP metabolism and extracellular matrix turnover.