Bupivacaine decreases cell viability and matrix protein synthesis in an intervertebral disc organ model system

Abstract

Background context: Bupivacaine is a local anesthetic commonly used for back pain management in interventional procedures. Cytotoxic effects of bupivacaine have been reported in articular cartilage and, recently, in intervertebral disc cell culture. However, the relevance of these effects to discs in vivo remains unclear. This study examines the effect of bupivacaine on disc cell metabolism using an organotypic culture model system that mimics the in vivo environment.

Purpose: To assess the effect of bupivacaine on disc cell viability and matrix protein synthesis using an organotypic model system and to determine whether this anesthetic has toxic effects.

Study design: Mouse intervertebral discs were isolated and maintained ex vivo in an organotypic culture then exposed to clinically relevant concentrations of bupivacaine, and the impact on disc cell viability and matrix proteoglycan (PG) and collagen syntheses were measured in the presence and absence of the drug.

Subjects: Mouse functional spine units (FSUs) were isolated from the lumbar spines of 10-week-old mice.

Outcome measures: Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Total PG and collagen syntheses were determined by measuring the incorporation of radioactive (35)S-sulfate and (3)H-l-proline into PG and collagen, respectively.

Methods: Organotypic cultures of mouse FSUs were exposed to different concentrations (0%-0.5%) of bupivacaine for variable amounts of time (0-2 hours). Cell viability within disc tissue was quantified by MTT staining and histologic assay. Matrix protein synthesis was measured by incorporation of radioactive (35)S-sulfate (for PG synthesis) and (3)H-l-proline (for collagen synthesis).

Results: Untreated mouse disc organs were maintained in culture for up to 1 month with minimal changes in tissue histology, cell viability, and matrix protein synthesis. Exposure to bupivacaine decreased cell viability in a dose- and time-dependent manner. Exposure to bupivacaine at concentrations less than or equal to 0.25% did not significantly affect matrix protein synthesis. However, at 0.5% bupivacaine, collagen synthesis was reduced by fourfold and PG synthesis by threefold.

Conclusions: Mouse discs can be successfully maintained ex vivo for upward of 4 weeks with little cell death, change in histologic structure, or matrix protein synthesis. This organotypic model system closely mimics the in vivo environment of the disc. Exposure of these cultures to bupivacaine dramatically decreased cell viability and matrix protein synthesis in a dose- and time-dependent manner. These findings corroborate those previously reported by Lee et al. using disc cell culture and demonstrate that this anesthetic at clinically relevant doses is toxic to intervertebral discs in both cell culture and disc organ models representative of the native architectural context.

Figure 1. Histological features and cell viability in disc organotypic culture

Disc structure remains intact after 28 days in organtotypic culture (A) as assessed by H&E staining and (B) Safrinin ) staining indicates minimal loss of PG in the nucleus pulposus (NP), but substantial loss in the growth plates (GP) even by 7 days (arrow). (C–D) MTT assay on FSU sections revealed no decline in cell viability at any of the time-points. The inset shows a positive control using dehydrated disc tissue to induce complete cell death. AF, annulus fibrosus.

Figure 2. Matrix synthesis of disc organotypic culture

Synthesis of proteoglycan synthesis (³⁵S-sulfate incorporation) and collagen (³⁵H-proline incorporation) by FSUs after being cultured for 0, 7, 14 or 28 days were determined as described in Material and Method. PG synthesis (fmoles ³⁵S-sulfate/ng DNA) showed no change after 14 days and a slight increase after 28 days. Collagen synthesis (fmoles ³⁵H-proline/ng DNA) remained constant during 28-day culture period. Values shown are averages of three independent trials + SE.

Figure 3. Time- and dose-dependent effect of bupivacaine on disc cell viability

Top, MTT staining of disc sections from FSUs exposed to bupivacaine or saline vehicle control. Black spots reveal viable cells and the insets stained with DAPI reveal the total cell number. Bottom, the percent cell death as a function of bupivacaine concentration (left) and time of exposure (right). Values are shown averages of six independent experiments + SE. Statistical significance (asterisk) was noted at 30, 60, and 120 minutes (Bottom Right) as well as in comparing each concentration (Bottom Left). NP, nucleus pulposus.

Figure 4. Dose-dependent effects of bupivacaine on matrix synthesis

Matrix synthesis by disc organotypic culture done by dual labeling with ³⁵S-sulfate and ³H-L-proline. Synthesis was quantified as the amount of incorporated radiolabel per unit DNA (fmoles ³⁵S-sulfate/ng DNA for proteoglycan, and fmoles ³⁵H-proline/ng DNA for collagen). Y axis, matrix synthesis as the percent of untreated control. Bupivacaine exposure did not affect PG synthesis except at a concentration of 0.5% (Black bars). Collagen synthesis was affected at a concentration of 0.25% or greater (Gray bars). Values shown are averages of three independent trials + SE. Statistical significance (asterisk) was noted at 0 and 0.5% bupivacaine for both collagen (Col) and proteoglycan (PG) synthesis.