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Background: Local, intrawound use of antibiotic powder, such as vancomycin and tobramycin, in spinal fusion surgery has become an increasingly common prophylactic measure in an attempt to reduce rates of postsurgical infection. However, the effects of localized antibiotic delivery on fusion remain unclear. The objective of this study was to examine the in vivo effects of intraoperative local delivery of 2 antibiotics commonly used in bone-grafting surgery on spinal fusion outcomes in a rat model. Methods: Single-level (L4-L5), bilateral posterolateral intertransverse process lumbar fusion surgery was performed on 60 female Lewis rats (6 to 8 weeks of age) using syngeneic iliac crest allograft mixed with clinical bone-graft substitute and varying concentrations of antibiotics (n = 12 each): (1) control without any antibiotics, (2) low-dose vancomycin (14.3 mg/kg), (3) high-dose vancomycin (71.5 mg/kg), (4) low-dose tobramycin (28.6 mg/kg), and (5) high-dose tobramycin (143 mg/kg). Eight weeks postoperatively, fusion was evaluated via micro-computed tomography ([micro]CT), manual palpation, and histological analysis, with blinding to treatment group. In the [micro]CT analysis, fusion-mass volumes were measured for each rat. Each spine specimen (L4-L5) was rated (manual palpation score) on a scale of 2 to 0 (2 = fused, 1 = partially fused, and 0 = non-fused). Results: The mean fusion-mass volume on [micro]CT (mm3) was as follows: control, 29.3 +/- 6.2; low-dose vancomycin, 26.3 +/- 8.9; high-dose vancomycin, 18.8 +/- 7.9; low-dose tobramycin, 32.7 +/- 9.0; and high-dose tobramycin, 43.8 +/- 11.9 (control versus high-dose vancomycin, p < 0.05; and control versus high-dose tobramycin, p < 0.05). The mean manual palpation score for each group was as follows: control, 1.46 +/- 0.58; low-dose vancomycin, 0.86 +/- 0.87; high-dose vancomycin, 0.68 +/- 0.62; low-dose tobramycin, 1.25 +/- 0.71; and high-dose tobramycin, 1.32 +/- 0.72 (control versus high-dose vancomycin, p < 0.05). The histological analyses demonstrated a similar trend with regard to spinal fusion volume. Conclusions: Intraoperative local application of vancomycin, particularly at a supraphysiological dosage, may have detrimental effects on fusion-mass formation. No inhibitory effect of tobramycin on fusion-mass formation was observed. Clinical Relevance: When spine surgeons decide to use intraoperative intrawound antibiotics in spinal fusion surgery, they should weigh the reduction in surgical site infection against a possible inhibitory effect on fusion. [ABSTRACT FROM AUTHOR]