In order to repair bone defects, biomaterials are widely used. Among them, porous hydroxyapatite bioceramics have interesting intrinsic properties: they are osteoconductive and bioactive.
However, in most of cases, due to the implant size or the infectious risks, the integration of the part and therefore bone regeneration can fail.
Our objective is to develop a 3rd generation biomaterial which allows increasing osseointegration while limiting the infectious risk. To achieve this, dual functionalization is mandatory. Two types of molecules are being studied, cytokines and vancomycin, which respectively promote the development and differentiation of the patient's cells on the surface of the material and prevent the risks of infection associated with the surgical procedure. Beyond simple support, our bioceramic must respect the release kinetics specific to the modes of action and targets of our two active molecules.
For this purpose, micro-macroporous hydroxyapatite ceramics are elaborated by additive manufacturing (microextrusion). The porosity is particularly important, as it not only allows cell colonization but also control the functionalization and delivery of active molecules. Macropores, whose size is dictated by the extrusion process (paste formulation, printing program, nozzle size, etc.) are used as reservoirs for an hydrogel containing vancomycin. The microporosity, controlled by the sintering cycles, is necessary for cell adhesion but also for the adsorption of cytokines on the surface of our architectures.
In this study, we would know is the microporosity has an impact on the release kinetics of vancomycin. For this purpose, 3 different sintering cycles are applied on our hydroxyapatite bone substitutes 1200°C, 1140°C and 1100°C for 1h. Then they are functionalized with hydrogels containing vancomycin and be placed for 15 days in a perfusion bioreactor mimicking bone physiological conditions: low blood flow (1 ml/min) and a temperature of 37°C.
Samples of the medium is taken twice a day. The concentration of vancomycin released will be determined by High Performance Liquid Chromatography, coupled with a mass spectrometer (HPLC-MS). Indeed, this technique allows separating, identifying and quantifying many organic substances present at very low concentrations in solution (up to 18µg/L) and thus to establish with precision the antibiotic release profile of our functionalized substitute. To ensure the effectiveness of the model, bacteriology experiments are performed on a strain of Staphylococcus aureus. The aim is, on the one hand, to know the minimal concentration of antibiotics having an impact on the bacterial growth and, on the other hand, to determine if the antibiotic is still efficient. Meanwhile, to ensure the vancomycin’s concentration is not toxic, a test is performed on ubiquitous bone cells, such as human endothelial cells, pre-osteoblast and monocyte. In this experiment cells are incubated at the highest vancomycin’s concentration measured by the HPLC and viability and metabolic activity of each cell type is quantified.