NITRIC OXIDE MODULATES RHBMP-2 INDUCED CORTICOCANCELLOUS AUTOGRAFT INCORPORATION IN RAT INTERTRANSVERSE FUSION

Abstract

Nitric oxide (NO) is a free radical labile gas which has important physiological functions and is synthesised by the action of a group of enzymes called nitric oxide synthases (NOS) on L- arginine. We have shown that nitric oxide modulates fracture healing¹. Bone morphogenic proteins (BMP) are potent differentiating factors that augment the process of new bone formation. Recombinant human BMP-2 (rhBMP-2) enhances spinal fusion². With progression of fusion there is a remodelling of the fusion mass bone accompanied with a decrease in the fusion mass size. It is not known whether nitric oxide has a role in spinal fusion or rhBMP-2 enhanced spinal fusion.

We studied this in a novel rat intertransverse fusion model using a defined volume of bone graft (7 caudal vertebrae) along with 157 mm3 of absorbable Type-1 collagen sponge (Helistat®) carrier, which was compacted and delivered using a custom jig for achieving a similar graft density from sample to sample. The control groups consisted of a sham operated group (S, n=20), an autograft + carrier group (AC, n=28) and a group consisting of 43 μg of rhBMP-2 (Genetics Institute, Andover, MA) mixed with autograft + carrier (ACB, n=28). Two experimental groups received a nitric oxide synthase (NOS) inhibitor, N^G-nitro L-arginine methyl ester (L-NAME, Sigma Chemicals, St Louis, MO) in a dose of 1 mg/ml ad lib in the drinking water (ACL, n=28) and one of these experimental groups had rhBMP-2 added to the graft mixture at the time of surgery (ACLB, n=28). Rats were sacrificed at 22 days and 44 days, spinal columns dissected and subjected to high density radiology (faxitron) and decalcified histology. The faxitrons were subjected to image analysis (MetaMorph).

On a radiographic score (0–4) indicating progressive maturation of bone fusion mass, no difference was found between the AC and ACL groups, however, there was a significant enhancement of fusion when rhBMP-2 was added (ACB group, 3.3±0.2) when compared to the AC group (1±0) (p< .001). However, on day 44, the ACLB group (3.3±0.2) showed significantly less fusion progression when compared to the ACB group (4±0) (p< 0.01). There was a 25% (p< 0.05) more fusion-mass-area in day 44 of ACLB group (297±26 mm³) when compared to day 44 of the ACB group (225±16 mm³) indicating that NOS inhibition delayed the remodelling of the fusion mass. Undecalcified histology demonstrated that there was a delay in graft incorporation whenever NOS was inhibited (ACL and ACLB groups).

Our results show that the biology of autograft spinal fusion and rhBMP-2 enhanced spinal fusion can be potentially manipulated by nitric oxide pathways.