Impact of Vancomycin Treatment on Human Mesenchymal Stromal Cells During Osteogenic Differentiation

Abstract

Category: Basic Sciences/Biologics Introduction/Purpose: Vancomycin is often delivered locally for surgical site infection prophylaxis. Recent reports of possible osteotoxicity have led to uncertainty concerning vancomycin’s safety in the setting of arthrodesis and bone healing. Bone formation during arthrodesis takes place as recruited human mesenchymal stromal cells (hMSCs) proliferate and differentiate into mature osteoblasts. The purpose of this research was to determine the impact of vancomycin treatment on hMSCs during osteogenic differentiation. Methods: Human MSCs were cultured in MSC growth media to an appropriate confluence. Cells were cultured for 24 hours to facilitate adherence, after which the media was aspirated and replaced with osteogenic differentiation media (Lonza, Switzerland). Osteogenic differentiation media was supplemented with vancomycin powder to yield solutions with concentrations of 0, 50, 500 & 5000 µg/mL. Fresh vancomycin powder was added with every media change. MSCs viability and proliferation were assessed via live/dead staining with 1 µM calcein-AM and 0.5 µM ethidium homodimer-1 (EthD-1) after 1, 3, and 7 days of differentiation and vancomycin treatment. Mineralization of differentiated cells was assessed via staining with 40 mM alizarin red (ARS; pH 4.1) after 21 days. Semi-quantification of the degree of mineralization was performed by measuring absorbance values at 405 nm using a microplate reader. Microscopy was used for qualitative evaluation. Results: Cell viability decreased with increasing vancomycin concentrations. Impairment of hMSC proliferation was also observed with increasing concentrations of vancomycin. MSCs treated with 5000 µg/mL vancomycin demonstrated significantly less cell growth compared to all other treatment groups (P=0.0001). Absorbance measurements from each well stained with alizarin red was used for semi-quantification of the degree of mineralization. As vancomycin concentrations were increased, absorbance levels decreased (Figure). This reduction in mineralization was also demonstrated qualitatively; with alizarin red less apparent in the wells with increasing vancomycin concentrations (Figure). Conclusion: Local vancomycin is utilized for prevention of infection, often in procedures that necessitate the formation of new bone. Bone healing requires migration, proliferation and differentiation of hMSCs. This work demonstrates impaired viability and function of hMSCs following vancomycin as well as decreased osteoblastic mineralization. Future work will require in vivo studies aimed at determining relative nonunion rates in the setting of vancomycin prophylaxis. Still, the results of this study suggest that vancomycin may be toxic to hMSCs and caution should be exercised by providers when considering vancomycin in foot and ankle patients requiring bony healing following fracture or arthrodesis.