Abstract
Introduction
A successful cell therapy of disc degeneration requires long-term survival and metabolic activity of injected cells in the degenerative disc environment that is affected by low nutrient supply, decreased osmolarity, and proinflammatory conditions. A bovine disc organ culture system with simulation of degenerative and proinflammatory conditions was evaluated with regard to its suitability for testing the fate of injected cells.
Materials and Methods
Bovine caudal disc punches (up to six discs from each specimen) were exposed to needle puncture and proinflammatory factors (LPS, interleukin [IL]1β) for 2 days after a 6-day culture without stimulus, while untreated samples were used as controls. The induction of a proinflammatory response was evaluated by quantification of prostaglandin E2 (PGE2) in culture supernatants and gene expression analysis of proinflammatory cytokines, matrix metalloproteases (MMPs), collagen type II, and aggrecan was measured in isolated disc cells. The metabolic profile of disc punches was traced by quantification of glucose consumption and lactic acid production. To simulate normal versus degenerative conditions, cultures were maintained at 5 mM glucose and 400 mOsm or exposed to low glucose (0.05 mM) and reduced osmolarity (300 mOsm). PKH-labeled cells were injected within an albumin-based hydrogel scaffold. Discs were analyzed by histochemistry and by determination of glycosaminoglycan (GAG) content after 2 weeks.
Results
PGE2 increased for LPS 10 µg/mL, IL1β 10 ng/mL or 100 ng/mL treatments of 21.7 ± 0.8-fold, 5.5 ± 0.9-fold, and 9.6 ± 0.8-fold, respectively, when compared with control (Fig. 1A). Preliminary evaluations showed that LPS- and IL1β-treated discs increased expression of IL-6 and 8, MMP1 and MMP3, and decreased collagens type II and aggrecan expression (Fig. 1B). GAG content of the disc explants decreased (62.4 ± 9%) within 2 weeks. However, treated samples and controls showed similar glucose consumption and lactic acid production, with the concentrations never reaching levels of starvation or toxic environment. Fluorescent cells could be detected at each sampling time point with only little differences between culture conditions.
Conclusion
Simulation of degenerative or inflammatory conditions increased PGE2 release and expression of MMPs and ILs, and decreased matrix protein expression by disc cells. Monitoring of injected fluorescence-labeled cells allows characterization of cell reactions in a simulated degenerative environment. This approach is suitable for in vitro testing of regenerative or anti-inflammatory treatment strategies of disc degeneration.
None declared