2.4% 2.6% CONTROL n= % VANCOMYCIN n= % infection rate in spinal fusion. Vanco effectively reduces infection.

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1 Disclosures Vancomycin is Toxic to Mesenchymal Stem Cells in Vitro: A Pilot Study Alfred Kuo Research support from MTF and StemRD No other disclosures Stacey Chu, BA; Nita Chen, BA; Alexis B.C. Dang, MD; Alfred C. Kuo, MD, PhD; Alan B.C. Dang, MD 2.4% infection rate in spinal fusion 2.4% INFECTION IN SPINAL FUSION n=72,534 Smith, et al Vanco effectively reduces infection 2.6% CONTROL n= % VANCOMYCIN n=911 Sweet, et al

2 Vanco effectively reduces infection Why we care about a 1.2% difference 1.2% INFECTION instrumented spine cases with vanco n= 663 Vancomycin seems to decrease infection from 2.4% to around 1%. That doesn t seem like much, but with the number of spinal fusions performed in the US each year and the average cost to treat a spine infection, the real difference is about $1 billion. Molinari, et al Vancomycin is effective and cheap. Each dose of vancomycin is only $5. Side effects? We know vancomycin is good at stopping infection, but are there negative effects? Does vancomycin affect human mesenchymal stem cells (MSCs) (MSCs can grow up to be osteoblasts) 2

3 Background Data Hypothesis Surgeons use either 1 gram or 2 grams of vancomycin in the spine. There is no standard at the moment. Elevated doses of vancomycin are toxic to hmscs in vitro in comparison to a control group. When a surgeon puts 2 grams of vancomycin, the actual drug levels in the wound can vary: In the surgical drain, there can be as much as 200X greater concentration of vanco in serum concentrations than is recommended for endocarditis and necrotizing fasciitis. Harvesting hmscs Patient Data and Culture Conditions Discarded bone marrow obtained during preparation of the femoral canal during elective hip replacement surgery Plastic adhesion used to select out MSCs Cells allowed to reach confluence in a 75cm 2 culture flask 10% FBS used as culture media Aggregated Patient Data Pt1 55 yo male Pt2 61 yo male Pt3 62 yo male Pt4 71 yo male Pt5 60 yo male Pt 7 65 yo male Pt 8 65 yo male Pt 9 75 yo male Culture Conditions Different doses of vancomycin: o mcg/ml o 6400 mcg/ml o 4800 mcg/ml o 3200 mcg/ml o 1600 mcg/ml o 400 mcg/ml o 0 mcg/ml 3

4 Protocol 71 yo male Control 6400 µg/ml Treatment with varying dosages of vancomycin (between 0 and mcg/ml) for 24 hours Viability Assay: Live-Dead Stain Green = Live Red = Dead Viability Assay: Presto Blue ANOVA with Tukey Post-tests Post-hoc power analysis: we can detect 20% or greater cell death VANCOMYCIN CONCENTRATION (mcg/ml) CELL DEATH 0 0% (control definition) P-VALUE (compared to control) % P = % P = % P < % P < % P < % P < yo male Results VANCOMYCIN CONCENTRATION (mcg/ml) CELL DEATH 0 0% (control definition) P-VALUE (compared to control) % P = % P = % P = % P < % P < % P <

5 All 9 patients Patient Patient Variability NOT predicted by diagnosis Osteoarthritis v. Osteonecrosis NOT predicted by age Discussion Clinical Recommendations Statistically significant differences were seen in all tested conditions that our study was adequately powered to detect. The doses used in our study included doses that were higher those reported in the Sweet, et al. study Patient to patient variability exists Some patient cells seem to be more sensitive to vancomycin toxicity Consequence of early MSC death is unknown Though MSCs may differentiate into osteoblasts and fibroblasts This study does not establish a contraindication to the use of vancomycin. This study does not suggest that pseudarthroses can be blamed on vancomycin 5

6 Conclusions Many Thanks To: No consensus on topical vancomycin Best available clinical literature supports using up to 2 grams in the wound No clinical literature against vancomycin use Basic science suggests that high doses may be harmful So if you do use it, consider the risks of going beyond 2g Nita Chen, BA UCSF/VAMC Research Team Hubert Kim, MD, PhD Alfred Kuo, MD, PhD Xuhui Liu, MD Brian Feeley, MD Alexis BC Dang, MD Alan BC Dang, MD Alizarin Red Qualitative Assessment of Calcium Production Alizarin Red S staining showed similar qualitative calcium production in ITS v. FBS groups. There appeared to be less calcium production at the highest dose of vancomycin used (12800 µg/ml) for both types of media 6

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