Abstract
Introduction
There is a clinical need for annulus fibrosus (AF) repair to reduce re-herniation risk, restore intervertebral disc (IVD) biomechanics and promote regeneration of the IVD following herniation and discectomy surgery. Functionalized polyethylene glycol with poly(trimethylene carbonate) block copolymers (PEG-TMC3) can strongly adhere to native AF tissue. This multi-scale study evaluated PEG-TMC3 for adhesion, cytocompatiblity, biomechanics restoration and herniation risk using cell and organ culture experiments and 2 ex vivo biomechanics studies to assess (1) cytocompatibility, (2) biomechanics, (3) organ culture, and (4) failure strength. We hypothesized PEG-TMC3 allows greater AF cell proliferation rates than Dermabond (i.e., cyanoacrylate); restores IVD biomechanics to intact levels; does not herniate under rigorous IVD organ culture loading; and that IVDs repaired with PEG-TMC3 fails at levels similar to intact conditions.
Material and Methods
(1) For cytocompatibility tests, bovine AF cells from three donors were seeded in triplicate in 48 well plates; the wells were either coated with PEG-TMC3 or Dermabond, or left uncoated (No Adhesive). Cells were cultured for 1, 3 or 7 days at 37°C, 5% CO2 and ambient oxygen. Cells were then lysed and the DNA content was quantified by PicoGreen. (2) For biomechanical tests, vertebra-IVD-vertebra segments were tested under axial (0.25MPa to -0.50MPa) and torsional loading (±4°) in a repeated measures study design with Injured Control and PEG-TMC3 Repair groups (n = 6). (3) For organ culture validation tests, bovine IVDs with endplates were randomly distributed among 3 groups: Intact, Injured and PEG-TMC3 repair (n = 3). IVDs were then cultured for 4 days with 2 daily bouts of rigorous loading and a diurnal cycle (total 10,800 cycles) [4]. (4) For failure strength tests, vertebrae-IVD-vertebrae segments of Intact, Injured and PEG-TMC3 groups (n = 9) were subjected to increasing axial force under a fixed 5° bend until failure or nucleus pulposus extrusion was observed. Force and subsidence were measured through the test and extrusion was monitored by video. ANOVA with Tukey's post-hoc assessed differences between groups and time points for DNA concentration, failure strength and subsidence. Repeated measures ANOVA with Tukey's post-hoc assessed torque range and axial range of motion. p < 0.05 was significant.
Results
(1) DNA concentration significantly increased from Day 1 to 7 for PEG-TMC3 and No Adhesive groups, while no changes in DNA content was observed for Dermabond treated cells which tended to decrease. Further, DNA concentration was higher for PEG-TMC3 and No Adhesive compared with Dermabond at each time point. (2) Biomechanically, injury increased axial range of motion and torque range. PEG-TMC3 repair restored axial range of motion to intact levels and increased torque range from injured levels but not to intact levels. (3) In organ culture, 2 of 3 PEG-TMC3 repairs herniated after 4 days of culture. (4) In failure testing, PEG-TMC3 did not significantly increase herniation stresses compared injured.
Conclusion
PEG-TMC3 showed high cytocompatibility, restored axial range of motion to intact conditions and increased torque range from injury conditions, suggesting it is a good candidate for AF repair. However, the high herniation risks under cyclic organ culture and failure testing conditions indicated further optimization is necessary to prevent herniation after many cycles of loading.