Background

Septic arthritis and implant-related infections are serious complications in orthopaedic surgery with tremendous consequences for the patient. A wide range of bacteria can be causal, with Staphylococcus aureus being the most frequent microbial agent. Hyaluronic acid (HA) hydrogels have been studied for their potential to deliver antibacterial agents to the infection site. We aim to develop a novel antibiotic hydrogel based on HA to ensure locally controlled antibiotic release. Therefore, it is essential to examine whether antibiotics and HA hydrogels exert toxic effects on osteoblasts. Additionally, it is vital to examine how HA hydrogels affect osteoblast cultures when loaded with different antibiotic concentrations.

Methods

We used primary osteoblast cultures derived from human femoral bone. A novel cross-linked biocompatible hydrogel based on HA was developed and combined with vancomycin or cefuroxime. Release kinetics of each antibiotic from the hydrogel was examined by spectrometry. Different concentrations of each antibiotic were applied to osteoblast cultures, and cell metabolism, cytotoxicity, and cell proliferation were studied by measuring Alkaline Phosphatase (ALP), Lactate Dehydrogenase (LDH) and CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assays, respectively. Osteoblast cultures maintained in the presence of antibiotic-free medium were used as controls. Subsequently, cultures were maintained on HA-hydrogels loaded with different concentrations of vancomycin and cefuroxime. Cell metabolism and cell proliferation were studied by measuring ALP and LDH and by using MTS assays. A 7-day long time-lapse analysis was carried out to examine the potential impact on cell activity and growth rate.

Results

After a period of 24h 20% of vancomycin and almost 70% of cefuroxime were released from the HA hydrogel. Vancomycin concentrations of up to 10 times the minimal inhibitory concentration (MIC) did not affect osteoblast cultures, as measured by ALP- and LDH-activity and by MTS metabolization. Cefuroxime concentrations of up to 30x MIC stimulated ALP activity and reduced LDH activity, whereas concentrations of up to 20x MIC did not affect cell proliferation. HA hydrogel's presence initially hampered cell proliferation; however, the osteoblasts continued to grow at a slower rate. HA hydrogels, combined with cefuroxime concentrations of up to 30x MIC, did not affect cell activity and proliferation compared to osteoblast cultures mixed with HA hydrogels without the presence of antibiotics. HA hydrogels, combined with vancomycin concentrations of up to 30x MIC, did not affect cell activity and proliferation compared to osteoblast cultures mixed with HA hydrogels without the presence of antibiotics.

Conclusion

This study demonstrates that vancomycin and cefuroxime at concentrations considerably higher than the MIC for common bacterial strains had no toxic effects on osteoblasts. Additionally, the HA hydrogel did not inhibit osteoblast proliferation. Moreover, high concentrations of vancomycin need to be loaded into hydrogels since considerable proportions of this antibiotic will not be initially released, whereas cefuroxime shows a rapid release.