Does Vitamin D3 Prevent the Inhibitory Effect of Vancomycin on Osteoblasts?
Authors: Tsuji Kentaro , Kimura Soichiro , Tateda Kazuhiro , Takahashi Hiroshi 
Published: December 2019

Abstract

Background
The utility of vancomycin powder to prevent surgical site infection, mainly in spinal surgery, has been widely examined, and the local administration of vancomycin powder to wounds has been reported to be effective in preventing surgical site infections after spine surgery. However, in vitro studies have shown that high local concentrations of vancomycin may inhibit osteogenesis, although it remains unclear how these high concentrations influence osteoblasts. No candidate drug has been reported to recover cytotoxicity with high concentrations of vancomycin, but we suggest that vitamin D3, which induces osteoblast proliferation, may be administrated concomitantly with vancomycin in these situations.

Questions/purposes
(1) Does a high concentration of vancomycin reduce viable osteoblast numbers in cell culture compared with controls? (2) Does vitamin D3 administration confer a protective effect on osteoblasts when administered with continuous vancomycin? (3) Does vitamin D3 administration confer a protective effect on osteoblasts when administered with pulsed vancomycin (24 hours of administration)? (4) Does vitamin D3 administration confer alkaline phosphatase, mineralization, and gene expression when administered with pulsed vancomycin?

Methods
MC3T3-E1 cells were cultured at 37° C in an α-minimum essential medium supplemented with 10% fetal bovine serum in a humidified incubator containing 5% CO2. The experimental concentrations of vancomycin (2500 μg/mL, 5000 μg/mL, and 7500 μg/mL) were determined based on previous reports and preliminary experiments. We concomitantly administered vitamin D3 (0.01 nM) to prevent cytotoxicity in osteoblasts, using two different treatments: continuous vancomycin administration (measured at 6 hours, 12 hours, 24 hours, and 72 hours) and pulsed vancomycin for 24 hours (measured at 1 days, 3 days, and 7 days). We analyzed cell numbers and morphologic changes in cells treated with vancomycin or vancomycin plus 0.01 nM vitamin D3. Osteoblast differentiation was assessed with alkaline phosphatase staining, alkaline phosphatase activity, and Alizarin red S staining.

Results
The number of cells was reduced at 6 hours, 24 hours, 48 hours, and 72 hours in response to continuous vancomycin administration at 7500 µg/mL (at 72 hours, control 14.6 × 104 cells/mL ± 0.260 × 104 cells/mL, vancomycin at 0.917 × 104 cells/mL ± 0.288 × 104 cells/mL, mean difference -13.7 × 104 cells/mL ± 0.388 × 104 cells/mL [95% CI -14.5 to -12.9]; p < 0.001). Vitamin D3 did not have a protective effect when vancomycin was administered continuously at 7500 µg/mL (at 72 hours, vancomycin alone 0.917 × 104 cells/mL ± 0.288 × 104 cells/mL, vancomycin + vitamin D3 1.67 × 104 cells/mL ± 0.310 × 104 cells/mL, mean difference 0.75 × 104 cells/mL ± 0.423 × 104 cells/mL [95% CI -0.127 to 1.63]; p = 0.09).
With pulsed administration for only the first 24 hours, the number of cells was reduced at 1 day, 3 days, and 7 days at 7500 μg/mL (at 7 days, control 18.6 × 104 cells/mL ± 1.29 × 104 cells/mL, vancomycin at 3.46 × 104 cells/mL ± 0.292 × 104 cells/mL, mean difference -15.1 × 104 cells/mL ±1.33 × 104 cells/mL [95% CI -17.9 to -12.4]; p < 0.001 for all). However, vitamin D3 had a recovery effect when vancomycin was administered only for 24 hours (cell number with 7500 μg/mL, day 7: vancomycin alone 3.46 × 104 cells/mL ± 0.292 × 104 cells/mL, vancomycin +vitamin D3 10.6 × 104 cells/mL ± 0.900 × 104 cells/mL, mean difference 7.13 × 104 cells/mL ± 0.946 × 104 cells/mL [95% CI 5.16 to 9.09]; p < 0.001).
With the addition of vitamin D3, we observed recovery of alkaline phosphatase staining and Alizarin red staining (evidence of calcification) but no difference in the gene expression of Type I collagen (vancomycin alone 0.319 ± 0.0730, vancomycin + vitamin D3 0.511 ± 0.139, mean difference 0.192 ± 0.157 [95% CI -0.483 to 0.867]; p = 0.345), alkaline phosphatase (vancomycin alone 0.532 ± 0.0210, vancomycin + vitamin D3 0.785 ± 0.0590, mean difference 0.253 ± 0.0620 [95% CI -0.0150 to 0.521]; p = 0.0550), and cathelicidin antimicrobial peptide (vancomycin alone 0.885 ± 0.0520, vancomycin + vitamin D3 1.24 ± 0.125, mean difference 0.355 ± 0.135 [95% CI -0.0200 to 0.730]; p = 0.0580).

Conclusion
We found that 7500 µg/mL of vancomycin is cytotoxic to osteoblasts. Cytotoxicity could be prevented by administering vitamin D3 in combination with vancomycin.

Clinical Relevance
The high concentrations of vancomycin routinely used clinically raises concerns related to osteoblast cytotoxicity, which may contribute to pseudoarthrosis after spinal surgery. Thus, vitamin D3, which is frequently used to treat osteoporosis, may have efficacy as a concomitantly administered drug by inducing the proliferation of osteoblasts. These results indicate that a combination therapy of vancomycin and vitamin D3 may prevent adverse events such as osteoblast cytotoxicity.