Collagen/hydroxyapatite nanostructured layers with controlled release of different antimicrobial agents: the release kinetics, antimicrobial activity and cytocompatibility

IntroductionThe infection of implanted endoprostheses represents a serious problem with respect to orthopaedic and trauma surgery. One of the ways in which to increase the efficacy of therapy consists of the application of a... [ view full abstract ]

Introduction

The infection of implanted endoprostheses represents a serious problem with respect to orthopaedic and trauma surgery. One of the ways in which to increase the efficacy of therapy consists of the application of a local antibiotic delivery system. The local delivery of antibiotics maximises target tissue concentration while minimising the risk of systemic toxicity. The aim of this study is to compare the release kinetics, antimicrobial activity and cytocompatibility of collagen/hydroxyapatite (COL-CaP) nanostructured carriers loaded by three different antibiotics.

Methods

Nanostructured layers based on collagen (type I, VUP Medical, CZ) and 0, 5 or 15wt% of hydroxyapatite (HA) nanoparticles (avg. 150nm, Sigma Aldrich) were prepared employing electrospinning and subsequently cross-linked by EDC/NHS (Sigma Aldrich). Cross-linked electrospun mats were subsequently impregnated with i) 10wt% of vancomycin (Mylan S.A.S), ii) gentamicin (Sigma Aldrich) or iii) combination of vancomycin/gentamicin (1/1, w/w) respectively. The in vitro release rates of antibiotics were characterized by HPLC. The antimicrobial effects of the layers were determined using agar diffusion testing against four different clinical isolates. The in vitro biological evaluation was conducted using SAOS-2 cells in direct contact with the layers or 24h infusions (MTS/LDH tests).

Results

The maximum concentration of the released antibiotics was assessed for COL-CaP-VANCOMYCIN layers 700mg/l immediately after 3hours, for COL-CaP-GENTAMICIN layers 1020mg/l after 96hours, and for COL-CaP-VANCOMYCIN/GENTAMICIN layers 250mg/l of vancomycin after 96hours and 340mg/l of gentamicin immediately after 3 hours (see Figure 1). Concentrations exceeded the minimum inhibitory concentrations for three weeks. The effect of different amount of HA wasn’t statistically significant. Agar diffusion tests revealed that COL-CaP-VANCOMYCIN layers exhibited the highest antimicrobial activity. Biological evaluation demonstrated that gentamicin impregnated layers dramatically reduced cell metabolic activity and cell amounts and vancomycin impregnated layers were markedly less cytotoxic. The positive effect of HA content on cells was demonstrated, HA induced reduction of cytotoxicity of all antibiotics-impregnated layers.

Discussion

Our results suggest that the local application of high-dose vancomycin via drug delivery carriers provides a safe therapeutic osteomyelitis treatment method that prevents the development of bacterial resistance.

Acknowledgments

This study was supported by the Technology Agency of the Czech Republic (project no. TA04010330).