Characterizing the host response to rhBMP-2 in a rat spinal arthrodesis model.

STUDY DESIGN: Prospective, randomized, controlled preclinical trial. OBJECTIVE: This study seeks to characterize the localized and systemic host response to recombinant human bone morphogenetic protein-2 (rhBMP-2) in a well established rodent spine arthrodesis model utilizing cytokine analysis and magnetic resonance imaging (MRI). SUMMARY OF BACKGROUND DATA: Although high fusion rates are achieved with rhBMP-2 in the spine, several complications have also been reported, including a localized response leading to radiculitis and seroma formation. The mechanism in which this occurs clinically is yet unknown. METHODS: One hundred female Fischer rats underwent a posterolateral intertransverse lumbar spinal fusion, with paraspinal muscle tissue resection, using iliac crest autograft, type I absorbable collagen sponge (ACS), 10- or 100-µg rhBMP-2/ACS. The animals underwent magnetic resonance imaging evaluation, serum cytokine analysis, manual palpation, and gross tissue inspection at 2, 4, 7, 10, and 21 days, postoperatively. RESULTS: Qualitative evaluation of MR images demonstrated a transient fluid collection at the surgery site in the rhBMP-2 animals as early as 4 and 7 days that was greater than the autograft or ACS groups. Quantitative analysis on T2-weighted axial images demonstrated greater signal intensity in the rhBMP-2 animals compared with the ACS and autograft groups in a time-dependent fashion. Higher concentrations of several cytokines were also detected at 2, 4, and 7 days, including interleukin 1ß, interleukin 18, tumor necrosis factor α, macrophage inflammatory protein 1α, and monocyte chemotactic protein 1 in animals treated with rhBMP-2/ACS relative to ACS alone. CONCLUSION: Our data suggest that the in vivo host response to rhBMP-2 in an animal model may be associated with circulating proinflammatory and osteoclastic cytokines.

Quantitative assessment of angiogenic responses by the directed in vivo angiogenesis assay.

One of the major problems in angiogenesis research remains the lack of suitable methods for quantifying the angiogenic response in vivo. We describe the development and application of the directed in vivo angiogenesis assay (DIVAA) and demonstrated that it is reproducible and quantitative. This assay consists of subcutaneous implantation of semiclosed silicone cylinders (angioreactors) into nude mice. Angioreactors are filled with only 18 micro l of extracellular matrix premixed with or without angiogenic factors. Vascularization within angioreactors is quantified by the intravenous injection of fluorescein isothiocyanate (FITC)-dextran before their recovery, followed by spectrofluorimetry. Angioreactors examined by immunofluorescence show cells and invading angiogenic vessels at different developmental stages. The minimally detectable angiogenic response requires 9 days after implantation and >/=50 ng/ml (P < 0.01) of either fibroblast growth factor-2 or vascular endothelial growth factor. Characterization of this assay system demonstrates that the FITC-labeled dextran quantitation is highly reproducible and that levels of FITC-dextran are not significantly influenced by vascular permeability. DIVAA allows accurate dose-response analysis and identification of effective doses of angiogenesis-modulating factors in vivo. TNP-470 potently inhibits angiogenesis (EC(50) = 88 pmol/L) induced by 500 ng/ml of fibroblast growth factor-2. This inhibition correlates with decreased endothelial cell invasion. DIVAA efficiently detects differences in anti-angiogenic potencies of thrombospondin-1 peptides (25 micro mol/L) and demonstrates a partial inhibition of angiogenesis ( approximately 40%) in a matrix metalloprotease (MMP)-2-deficient mouse compared with that in wild-type animals. Zymography of angioreactors from MMP-deficient and control animals reveals quantitative changes in MMP expression. These results support DIVAA as an assay to compare potencies of angiogenic factors or inhibitors, and for profiling molecular markers of angiogenesis in vivo.