Staphylococcus aureus is a human pathogen involved in implant-related infections. In these diseases, biofilm production is the key pathogenic event, and it increases antibiotic resistance of the organism. Because this phenomenon, local delivery of antibiotics could allows reaching high concentrations in the infected tissue without the secondary effects linked to systemic administration. Here we report the use of a ceramic biomaterial (SBA-15) as a carrier of antibiotics in order to deliver them directly in the infected tissue. SBA-15 discs were loaded with vancomycin, rifampin and a combination of both according to the protocol described by Molina-Manso et al. Loaded discs were introduced in a 0.5 McFarland suspension of S. aureus 15981 and incubated during 6 and 24 hours in order to develop a biofilm. After incubation, samples were sonicated during 5 minutes and 1:10 serial dilutions were performed in order to count viable bacteria. All experiments were performed in triplicate.Background
Material and methods
SBA-15 is a siliceous mesoporous ordered material with hexagonal arrangement of 9-nm tubular pores connected by micropores, high pore volume and abundance of silanol groups. This functionalised material could thus tailor the release kinetics of specific biomolecules to the clinical needs. Non-functionalized SBA-15 and its C8- or C3-alkyl-derivatives were coated with parathyroid hormone–related protein (PTHrP)(107–111) to assess their relative effects on osteoblastic cell growth and function. SBA-15 was functionalized with either octyl or propyl trimethoxysilane (C8 or C3 precursor, respectively) in ACN for 24h and then were coated (or not) by dipping in 10 nM PTHrP (107–111) solution for 24 h at 4°C. After air drying, biomaterials were transferred to culture dishes. MC3T3-E1 cells were cultured in differentiation medium with SBA-15, C3-SBA-15 and C8-SBA-15, loaded or not with the peptide. Cell viability and proliferation were evaluated by trypan blue exclusion and a proliferation kit (Promega), respectively. Alkaline phosphatase (ALP) activity and collagen secretion were determined by colorimetric methods. Gene expression was analyzed by real-time PCR. Mineralization was assessed by alizarin red staining. PTHrP(107–111)-coated SBA-15 increased cell proliferation (50%), cell viability (20%), and ALP activity (15%) over control values within 2–4 days. At day 2, collagen secretion increased (20%), and also the gene expression of ALP, PTHrP, and VEGF, which normalized at day 8, in these cells. An increase (by 30–40%) in all of these parameters was induced by peptide-coated C3-SBA-15 at day 4. Similar stimulatory effects were also observed with PTHrP(107–111)-coated C8-SBA-15 but only at day 8. At day 10, collagen secretion slightly increased (10–15%), and also mineralization (30–40%) with both functionalized materials coated with the PTHrP peptide. In conclusion, PTHrP(107–111)-coated SBA-15 stimulates osteoblastic function in vitro; an effect delayed by C3- or C8-functionalization. These data further support the clinical impact of this bioceramic as functionalized implants in vivo.
