Segmental variation in the in vitro cell metabolism of nucleus pulposus cells isolated from a series of bovine caudal intervertebral discs.

STUDY DESIGN: This study focuses on the association between cell metabolism and molecular matrix composition of nucleus pulposus (NP) tissue with spine level in sequential bovine caudal intervertebral discs. OBJECTIVE: To explore the hypothesis that the molecular composition of NP tissue and corresponding cell metabolism varies with caudal spine. A secondary hypothesis is tested that potential cellular differences are maintained after monolayer culture. SUMMARY OF BACKGROUND DATA: In articular cartilage, cell metabolism and molecular matrix composition are influenced by loading history. This may also be a feature of intervertebral discs in series. METHODS: NP cells (nonpooled or level pooled) were isolated from four sequential bovine caudal intervertebral discs (levels 3-4, 4-5, 5-6, and 6-7) and cultured in alginate beads immediately or following monolayer culture. Levels of 3H-TdR (proliferation) and 35SO4 (GAG synthesis) incorporation were determined from 14 animals. In a separate set of 6 animals, total content of water, DNA, collagen (and type), GAG (and type) were also determined. RESULTS: The rate of 3H-TdR and 35SO4 incorporation in freshly isolated NP cells increased nonlinearly from level 3-4 to 6-7 (P < 0.05). Monolayer cultured cells retained level-specific differences for 35SO4 and 3H-TdR incorporation similar to that of freshly isolated cells. GAG content and chondroitin sulfate proportion decreased distally (P < 0.05); however, total collagen and Type I proportion increased distally (P < 0.05). No significant differences in water or DNA content could be determined. CONCLUSIONS: The results support the hypothesis that level-specific differences in NP cell metabolism and molecular composition are dependent on spine level potentially reflecting subtle mechanical differences between levels. Retention of level-specific differences in monolayer may suggest a certain level of cell "programming." This may be important for cellular strategies to repairspecific sites of degeneration.

Passage in monolayer influences the response of chondrocytes to dynamic compression.

This study tests the hypothesis that expansion by passage in monolayer influences the response of isolated articular chondrocytes to dynamic compression. Chondrocytes, isolated from bovine articular cartilage, were seeded in monolayer and passaged 4 times (P1-4). For assessment of chondrocytic and fibroblastic phenotype, freshly isolated and passaged cells were seeded on glass coverslips or in 2% alginate beads and cultured for 7 days in DMEM + 10% FCS. Samples were assayed for DNA and GAG content and stained for collagen types I and II. In separate experiments, freshly isolated or passaged chondrocytes were seeded at 10 x 10(6) cells.ml(-1) in 4% cylindrical agarose constructs and subjected to 15% dynamic compressive strain at 1 Hz for 24 hours. [(3)H]-thymidine incorporation, SO(4) incorporation and nitrite release were analysed. Immediately following isolation (P0), chondrocytes seeded in alginate expressed high levels of type II collagen, but did not stain for type I collagen. Following repeat passage the cells expressed enhanced levels of type I collagen, with an associated reduction in type II collagen staining. These data indicate a modulation to a fibroblastic phenotype during monolayer expansion which was not rapidly reversed by culture in a 3D hydrogel. Dynamic compression down-regulated SO(4) incorporation at P0, but did not affect [(3)H]-thymidine incorporation. By contrast the incorporation of both SO(4) and [(3)H]-thymidine was enhanced by dynamic compression at both P1 and to a lesser extent P2. SO(4) and [(3)H]-thymidine incorporation were inhibited at P3 and P4. Nitrite release was down-regulated by dynamic compression at all passages. These data demonstrate a clear modulation in the response of bovine articular chondrocytes to dynamic compression following passage in monolayer.

Effect of nicotine on spinal disc cells: a cellular mechanism for disc degeneration.

STUDY DESIGN: Experimental investigation to determine the effect of nicotine on intervertebral spinal disc nucleus pulposus (NP) cells cultured in vitro. OBJECTIVES.: To evaluate the effects of nicotine on cell proliferation, extracellular matrix production, and viability of NP cells in three-dimensional alginate constructs cultured in vitro. SUMMARY OF BACKGROUND DATA: Numerous studies confirm that smoking is a strong risk factor for back pain. The most widely accepted explanations for the association between smoking and disc degeneration is malnutrition of spinal disc cells by carboxy-hemoglobin-induced anoxia or vascular disease. Nicotine, a constituent of tobacco smoke, present in most body fluids of smokers is known to have detrimental effects on a variety of tissues. It may also be directly responsible for intervertebral disc (IVD) degeneration by causing cell damage in both the nucleus pulposus and anulus fibrosus. The effect of nicotine on IVD cells has not previously been investigated. METHODS: Bovine chondrocytic intervertebral disc cells were isolated by sequential digestion of nucleus pulposus and seeded in 2% alginate. The constructs were cultured for 21 days either in growth medium containing freebase nicotine (Sigma) at concentrations found in the serum of smokers (25 nmol/L-300 nmol/L) or in standard nicotine free-medium as controls. Samples were collected at time points 3, 7, 14, and 21 days and a quantitative assay was performed for DNA, glycosaminoglycans (GAG), and hydroxyproline. Samples were also processed for qualitative histologic analysis including immunolocalization of collagen types I and II. RESULTS: There was both a dose- and time-dependent response to nicotine, with constructs cultured in low-nicotine concentration media demonstrating an early increase in DNA, GAG, and collagen content, while constructs cultured in high nicotine concentration media demonstrated a late decrease in these parameters. At 25 nmol/L dose of nicotine, there was a significant increase (P < 0.05) in the above parameters at day 7 compared with the controls. At higher doses, there was a significant dose-dependent decrease (P < 0.05) in these parameters compared to controls; however, this was only significant at day 14 for the 300 nmol/L group and at day 21 for the 100 nmol/L, 200 nmol/L, and 300 nmol/L groups. Adverse morphologic changes were observed on histology, which included reduced cell proliferation, disrupted cell architecture, disintegration of cells, and extracellular matrix. Immunohistochemistry revealed the presence of type I collagen in the extracellular matrix rather than the normal type II collagen seen in the controls. CONCLUSIONS: Nicotine has an overall detrimental effect on NP disc cells cultured in vitro. There was significant inhibition of cell proliferation and extracellular matrix synthesis. Nicotine in tobacco smoke may have a role in pathogenesis of disc degeneration.