Intradiscal Delivery of Anabolic Growth Factors and a Metalloproteinase Inhibitor in a Rabbit Acute Lumbar Disc Injury Model.

Abstract

The purpose of our study was to examine the effect of controlled delivery of TGF-β3, BMP-4, and TIMP-2 with a biocompatible biopolymer, chitosan, on an acutely injured intervertebral disc (IVD) in a rabbit model. After conducting an in vitro analysis of the chondrogenic capacity of the biomolecule cocktail use (ie, TGF-β3, BMP-4, and TIMP-2) and confirming stem cell viability in chitosan hydrogel, 15 New Zealand white rabbits underwent a lateral approach of the L1 to L4 IVDs. In each rabbit, the L2 to L3 IVD was left pristine, whereas the L1 to L2 and the L3 to L4 IVDs in each rabbit underwent nucleotomy via a 25-G needle, and the animal was subsequently randomized to no further treatment (defect only), chitosan alone, Chitosan + TGF-β3 + BMP-4, or chitosan + TGF-β3 + BMP-4 + TIMP-2. At 6 weeks after injury and intervention, the rabbits were killed and spines harvested to undergo quantitative T2 magnetic resonance imaging (MRI) and subsequent histologic analysis. In the in vitro analysis, cells treated with experimental media containing TGF-β3, BMP-4, and TIMP-2 exhibited staining indicative of GAG production and began to exhibit a chondrocytic morphology. Quantitative T2 MRI mapping demonstrates that discs treated with chitosan, chitosan containing TGF-β3 and BMP-4, or chitosan containing TGF-β3, BMP-4, and TIMP-2 had consistently higher T2 relaxation times compared with defect-only discs. When the T2 relaxation times of each treatment group and defect-only discs were normalized to the healthy control disc, it was found that the T2 relaxation time of discs treated with chitosan containing TGF-β3 and BMP-4 and discs treated with chitosan containing TGF-β3, BMP-4, and TIMP-2 were significantly greater compared with defect-only discs (P = .048 and P = .013, respectively). Histologically, animals that received chitosan only, or chitosan with TGF-β3 and BMP-4, showed a significantly higher intensity of Safranin-O staining (P = .016 and P = .02, respectively) compared with control discs, whereas the difference in staining intensity in animals that received chitosan loaded with TGF-β3, BMP-4, and TIMP-2 failed to achieve significance (P = .161). A combination of chitosan, TGF-β3, and BMP-4 was effective at promoting regeneration in an acute disc injury rabbit model, whereas TIMP-2 did not have a significant effect.