Authors: Antoci Jr, Valentin*; Adams, Christopher S.; Powell, Dan K; Antoci, Valentin; Hickok, Noreen J; Shapiro, Iriving M; Parvizi, Javad
Title: Cytotoxicity as a Function of Antibiotic Concentration Released from Cement
Addresses: 1015 Walnut St. 501, Philadelphia, PA 19107
Purpose: The study investigated the influence of various concentrations of three commonly used antibiotics, namely vancomycin, ciprofloxacin, and tobramycin on bone cells.
Methods: Cell lines MC3T3-E1 preosteoblast cells, MLO-A5 osteocyte cells, and N1511 prechondrocyte-like cells were cultured in DMEM. Treatment Cells were passaged into 24 well plates, and after 12h, the culture medium was exchanged with fresh medium supplemented with Ciprofloxacin (0-1000 ìg/ml, Cipro), Ofloxacin (0-1000 ìg/ml, Oflox), Tobramycin (0-4000 ìg/ml, Tobra), or Vancomycin (0-8000 ìg/ml, Vanco) every 24h for 3 days. Cell proliferation/toxicity assays: MTT colorimetric assay (Molecular Probes) every 24h was used to measure cell viability and proliferation by normalization to control cultures. Lactate dehydrogenase activity was used to assess direct cell death.
Results: The effect of Oflox on cellular morphology of MLO-A5 cells was first studied. Osteocytes cultured with increasing dosages of Oflox showed a drastic change in morphology, and cell numbers. In the presence of 25 ug/ml of Oflox, cells are abundant and exhibit cuboidal, well-spread cellular morphology. In contrast, at 1mg/ml Oflox, the cells, if present, appeared non-viable with a globular and detached morphology. When exposed to Cipro, osteoblast/osteocyte and chondrocyte cell lines showed marked inhibition of cellular proliferation. Cipro concentrations of 25 ug/ml induce over 25% decrease in osteoblast and chondrocyte numbers. With Vanco much higher dosages were needed to cause inhibitory effects. Vanco concentrations higher than 100-250 ug/ml showed initial inhibition of proliferation, with 8 mg/ml inducing >50% cell loss. Tobra showed similar toxicity to Vanco. Both cell populations appeared less sensitive at lower dosages, with a robust effect observed in osteoblasts between 2-4 mg/ml. The chondrocytes, in contrast, did not show much sensitivity to the Tobra, with a 20% loss in cell numbers observed at 1- 4 mg/ml.
Discussion: Methylmethacrylate cement (PMMA) is widely used in joint replacement surgery as a depot of delivery for antibiotics. Several studies investigating in vitro elution rates of common antibiotics mixed in PMMA or calcium sulfate, have demonstrated a marked variability in elution depending on volume, exchange rate, and physical environment. The antibiotic concentration can reach intermittent systemic toxicity and prolonged local effects. High local concentration of Cipro, and to a lesser extent Vanco and Tobra were seen to have detrimental effects on osteoblastic and chondrocytic cellular proliferation. Further, the morphology of these cells appeared to be influenced by the presence of antibiotics even at lower concentrations. Further studies are needed to determine the optimal concentration of antibiotics being impregnated into PMMA so that effective infection treatment can be achieved without imparting local cellular toxicity. These results are crucial for determining the optimal antibiotic composition of bone cement that would provide an ideal balance between the microbicidal effects and the host cellular toxicity.