The local cytokine and growth factor response to rhBMP-2 after spinal fusion

Publication date: Available online 14 March 2018
Source:The Spine Journal
Author(s): John D. Koerner, Dessislava Z. Markova, Greg D. Schroeder, Brian P. Calio, Anuj Shah, Corbin W. Brooks, Alexander R. Vaccaro, D. Greg Anderson, Chris K. Kepler
Background contextThe systemic response regarding cytokine expression after application of recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) in a rat spinal fusion model has recently been defined¹, but the local response has not. 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 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 than the systemic response given the tissue-specific effects of rhBMP-2.PurposeThe purpose of this study was to evaluate the local cytokine and growth factor response after application of rhBMP-2 in a rat spinal fusion model.Study Design/SettingAnimal study, basic scienceMethodsThis study was partially funded by a physician sponsored grant from Medtronic. 135 Wistar rats (age 8 weeks, weighing approximately 300-400g) underwent L4-L5 posterolateral intertransverse fusion with demineralized bone graft (approximately 0.4cm³ rat demineralized bone matrix (DBM) per side). In the first group, 10µg of rhBMP-2 on an allograft collagen sponge (ACS) was added to the fusion site with approximately 0.4cm³ rat DBM per side. In the second group, 100µg of rhBMP-2 on an ACS was added to the fusion site with approximately 0.4cm³ rat DBM per side, and the third experiment was the control group, which consisted of only an ACS plus 0.4cm³ DBM per side. There were nine groups of five animals each per experiment. Each group was sacrificed at time points up to four weeks (1, 6, 24, 48 hours, and 4, 7, 14, 21, 28 days after surgery). At sacrifice, the DBM, transverse processes, and any new bone formed was harvested, immediately frozen in liquid nitrogen, and prepared for protein extraction. ELISA was performed to compare the levels of various cytokines (IL-1β, TNF-α, IL-6, IL-1ra, IL-4, IL-10) and growth factors (VEGF, IGF-1, PDGF, 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 microCT by two independent observers.ResultsThe 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 hour, 6 hours). TNF-α demonstrated significantly lower expression in the groups treated with rhBMP-2 at days 1, 2 and 4. At the early time points (1 hour, 6 hours), in the groups treated with rhBMP-2, all of the growth factors IGF-1, VEGF, PDGF-AB, TGF-B had equal or lower expression compared to 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 4/5 fused based on manual palpation and 2/5 based on microCT.ConclusionsThere 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.



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