Optimization of protein crosslinking formulations for the treatment of degenerative disc disease

Abstract

Study design: Biochemical studies aimed at optimization of protein crosslinking formulations for the treatment of degenerative disc disease and subsequent biomechanical testing of tissues treated with these formulations.

Objective: To optimize protein crosslinking formulations for treatment of degenerating spinal discs.

Summary of background data: Nonsurgical exogenous crosslinking therapy is a potential new, noninvasive technology for the treatment of degenerative disc disease. The technology is based on the injection of protein crosslinking reagents into the pathologic disc to restore its mechanical properties and also to potentially increase the permeability of the tissue and so facilitate the exchange of waste products and nutrients.

Methods: Diffusion of genipin (GP) was monitored following injection into spinal discs and the effects of surfactants on diffusion studied. Formulations for GP and methylglyoxal (MG) were biochemically optimized and used to treat bovine spinal discs. Their effects on bovine anulus tissue were evaluated using a circumferential tensile test, while the GP formulation was also tested with respect to its ability to reduce disc bulge under load.

Results: GP exhibited a distinct time-dependent diffusion and sodium-dodecyl-sulfate, but not Tween-20, enhanced diffusion by 30%. Two crosslinkers, GP and MG, were inhibited by amines but enhanced by phosphate ions. Both formulations could enhance a number of physical parameters of bovine anulus tissue, while the GP formulation could reduce disc bulge following injections into spinal discs.

Conclusion: Formulations lacking amines and containing phosphate ions appear to be promising candidates for clinical use of the crosslinkers GP and MG.

Figure 1

Example of genipin diffusion in a calf lumbar spinal disc. GP was injected into the disc of a single motion segment and incubated for 1 hour at 37°C. Arrows indicate the injection points. Bars = 10mm.

Figure 2

Diffusion kinetics of genipin. GP was injected into bovine lumbar discs and incubated for 1 (n=7), 3 (n=4) or 6 hours (n=4) at 37°C. Data are presented ± SD.

Figure 3

Effect of surfactants on diffusion of genipin. GP in PBS (n=7) or in PBS containing Tween-20 (n=3) or SDS (n=5) was injected and incubated for 1-hour. Data are presented ± SD.

Figure 4

Buffers effects on genipin and methylglyoxal crosslinking. Tissue was incubated with GP or MG at 37°C for 1 hour in either Tris or EPPS buffers at pH 9. Crosslinking was quantified and normalized to untreated tissue. Data are presented ± SD (n=5).

Figure 5

Effect of phosphate on genipin and methylglyoxal mediated crosslinking. Tissue was incubated with GP or MG at pH 8 or 9 at 37°C for 1 hour, in the presence or absence of phosphate. Crosslinking was quantified and normalized to untreated tissue. Data are presented ± SD (n=5).

Figure 6

Circumferential tensile testing of crosslinked annulus tissue. Tissue was crosslinked with GP (n=17) or MG (n=13) or in GP buffer (n=16) or MG buffer (n=12) alone and subjected to testing. Data are presented ± SD. Results marked with an asterisk denote statistical significant differences between the treated and buffer samples.