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
Prosthetic joint infections (PJI) occur infrequently, but due to its increased clinical use represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus and coagulase-negative staphylococci are the most common infecting agents associated with PJI. A possible therapeutic approach could be the local antibiotic by fluoride-TiO2 nanostructured anodic layers in order to prevent surface colonisation during the early moments after surgery. Here we describe the first results of this model using two common antibiotics. Fluoride-TiO2 nanostructured anodic layers on Ti6Al4V alloy were produced as described previously by Arenas et al (2013). Discs shaped pieces of Ti6Al4V alloy were loaded with a solution of 150 mg antibiotic (vancomycin or gentamicin)/20 ml sterile distilled water. Samples were immersed in this solution during 24 hours at room temperature with agitation, and then were dried during 48 hours at 20°C. Antibiotic release was studied by introducing both discs in sterile PBS and samples were taken at different times. Samples were then frozen at −80°C until HPLC measurements and biological activity tests using Bacillus subtilis ATCC 6051 (vancomycin) and Escherichia coli ATCC 25922 (gentamicin) were performed.Introduction
Methods
Parathytorid hormone-related protein (107–111) loaded onto biopolymer-coated nanocrystalline hydroxyapatite (HAGlu) improves the bone repair in a cavitary defect in rat tibiae. Biopolymer-coated nanocrystalline hydroxyapatite (HAGlu) made as macroporous foams are promising candidates as scaffolds for bone tissue engineering applications. They exhibit optimal features, promoting internalization, proliferation and differentiation of osteoprogenitors, with an adequate cell colonization over the entire scaffold surface. Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Its 107–111 epitope (osteostatin) exhibits osteogenic properties at least in part by directly acting on osteoblasts. The main aim of this study was to evaluate whether osteostatin loading into HAGlu scaffolds might improve their bone regeneration capacity.Summary Statement
Introduction
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.
