The local cytokine and growth factor response to recombinant human bone morphogenetic protein-2 (rhBMP-2) after spinal fusion.

BACKGROUND CONTEXT

The systemic response regarding cytokine expression after the application of recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rat spinal fusion model has recently been defined, but the local response has not been defined. Defining the local cytokine and growth factor response at the fusion site will help explain the roles of these molecules in the fusion process, as well as that of rhBMP-2. Our hypothesis is that the application of rhBMP-2 to the fusion site will alter the local levels of cytokines and growth factors throughout the fusion process, in a manner that is different from the systemic response, given the tissue-specific effects of rhBMP-2.

PURPOSE

The purpose of this study was to evaluate the local cytokine and growth factor response after the application of rhBMP-2 in a rat spinal fusion model.

STUDY DESIGN/SETTING: This was a basic science animal model study.

METHODS

This study was partially funded by a physician-sponsored grant from Medtronic. A total of 135 Wistar rats (age 8 weeks, weighing approximately 300-400 g) underwent L4-L5 posterolateral intertransverse fusion with demineralized bone graft (approximately 0.4-cm rat demineralized bone matrix [DBM] per side). In the first group, 10 µg of rhBMP-2 on an allograft collagen sponge (ACS) was added to the fusion site with approximately 0.4-cm rat DBM per side. In the second group, 100 µg of rhBMP-2 on an ACS was added to the fusion site with approximately 0.4-cm rat DBM per side, and the third experiment was the control group, which consisted of only an ACS plus 0.4-cm DBM per side. There were nine groups of five animals each per experiment. Each group was sacrificed at time points up to 4 weeks (1, 6, 24, and 48 hours, and 4, 7, 14, 21, and 28 days after surgery). At sacrifice, the DBM, transverse processes, and any new bone formed were harvested, immediately frozen in liquid nitrogen, and prepared for protein extraction. ELISA was performed to compare the levels of various cytokines (interleukin [IL]-1β, tumor necrosis factor alpha, IL-6, IL-1RA [IL-1 receptor antagonist], IL-4, and IL-10) and growth factors (vascular endothelial growth factor [VEGF], endothelia growth factor [EGF], insulin-like growth factor-1 [IGF-1], platelet derived growth factor [PDGF], transforming growth factor beta [TGF-β]) that are known to be involved in the fusion-fracture healing process. Fusion was evaluated on the rats sacrificed at 28 days by manual palpation and microcomputed tomography (microCT) by two independent observers.

RESULTS

The expression of cytokines and growth factors varied throughout the fusion process at each time point. In the groups treated with rh-BMP-2, IL-6 and IL-1RA had higher expression in the early time points (1 and 6 hours). Tumor necrosis factor alpha demonstrated significantly lower expression in the groups treated with rhBMP-2 at Days 1, 2, and 4. At the early time points (1 and 6 hours), in the groups treated with rhBMP-2, all of the growth factors IGF-1, VEGF, platelet derived growth factor AB (PDGF-AB), TGF-β had equal or lower expression compared with controls. At 24 hours, there was a peak in IGF-1, VEGF, and PDGF-AB. These growth factors then declined, with IGF-1 and PDGF-AB having a second peak at Day 7. At 4 weeks, all of the rhBMP-2-treated animals fused based on manual palpation and microCT. The control group had four of five rats fused based on manual palpation and two of five rats based on microCT.

CONCLUSIONS

There is significant variability in the expression of cytokines throughout the fusion process after treatment with rhBMP-2. The inflammatory response appears to peak early (1 and 6 hours), followed by a significant decrease with rhBMP-2 treatment. However, the growth factor expression appears to be suppressed early (1 and 6 hours), followed by a peak at 24 hours, and a second peak at Day 7.