Enzyme-functionalized biomimetic apatites: concept and perspectives in view of innovative medical approaches.

Biomimetic nanocrystalline calcium-deficient apatite compounds are particularly attractive for the setup of bioactive bone-repair scaffolds due to their high similarity to bone mineral in terms of chemical composition, structural and substructural features. As such, along with the increasingly appealing development of moderate temperature engineered routes for sample processing, they have widened the armamentarium of orthopedic and maxillofacial surgeons in the field of bone tissue engineering. This was made possible by exploiting the exceptional surface reactivity of biomimetic apatite nanocrystals, capable of easily exchanging ions or adsorbing (bio)molecules, thus leading to highly-versatile drug delivery systems. In this contribution we focus on the preparation of hybrid materials combining biomimetic nanocrystalline apatites and enzymes (lysozyme and subtilisin). This paper reports physico-chemical data as well as cytotoxicity evaluations towards Cal-72 osteoblast-like cells and finally antimicrobial assessments towards selected strains of interest in bone surgery. Biomimetic apatite/enzyme hybrids could be prepared in varying buffers. They were found to be non-cytotoxic toward osteoblastic cells and the enzymes retained their biological activity (e.g. bond cleavage or antibacterial properties) despite the immobilization and drying processes. Release properties were also examined. Beyond these illustrative examples, the concept of biomimetic apatites functionalized with enzymes is thus shown to be useable in practice, e.g. for antimicrobial purposes, thus widening possible therapeutic perspectives.

Nanocrystalline Apatites: Post-Immersion Acidification and How to Avoid It-Application to Antibacterial Bone Substitutes.

Biomimetic nanocrystalline apatites analogous to bone mineral can be prepared using soft chemistry. Due to their high similarity to bone apatite, as opposed to stoichiometric hydroxyapatite for example, they now represent an appealing class of compounds to produce bioactive ceramics for which drug delivery and ion exchange abilities have been described extensively. However, immersion in aqueous media of dried non-carbonated biomimetic apatite crystals may generate an acidification event, which is often disregarded and not been clarified to-date. Yet, this acidification process could limit their further development if it is not understood and overcome if necessary. This may, for example, alter biological test outcomes, during their evaluation as bone repair materials, due to potentially deleterious effects of the acidic environment on cells, especially in in vitro static conditions. In this study, we explore the origins of this acidification phenomenon based on complementary experimental data and we point out the central role of the hydrated ionic layer present on apatite nanocrystals. We then propose a practical strategy to circumvent this acidification effect using an adequate post-precipitation equilibration step that was optimized. Using this enutralization protocol, we then showed the possibility of performing (micro)biological assessments on such compounds and provide an illustration with the examples of post-equilibrated Cu2+- and Ag+-doped nanocrystalline apatites. We demonstrate their non-cytotoxicity to osteoblast cells and their antibacterial features as tested versus five major pathogens involved in bone infections, therefore pointing to their relevance in the field of antibacterial bone substitutes. The preliminary in vivo implantation of a relevant sample in a rat's calvarial defect confirmed its biocompatibility and the absence of adverse reaction. Understanding and eliminating this technical barrier should help promoting biomimetic apatites as a genuine new class of biomaterial-producing compounds for bone regeneration applications, e.g., with antibacterial features, far from being solely considered as "laboratory curiosities".

Real-Time Detection of Tumor Cells during Capture on a Filter Element Significantly Enhancing Detection Rate.

Circulating tumor cells (CTCs) that enter the bloodstream play an important role in the formation of metastases. The prognostic significance of CTCs as biomarkers obtained from liquid biopsies is intensively investigated and requires accurate methods for quantification. The purpose of this study was the capture of CTCs on an optically accessible surface for real-time quantification. A filtration device was fabricated from a transparent material so that capturing of cells could be observed microscopically. Blood samples were spiked with stained tumor cells and the sample was filtrated using a porous structure with pore sizes of 7.4 µm. The possible removal of lysed erythrocytes and the retention of CTCs were assessed. The filtration process was observed in real-time using fluorescence microscopy, whereby arriving cells were counted in order to determine the number of CTCs present in the blood. Through optimization of the microfluidic channel design, the cell retention rate could be increased by 13% (from 76% ± 7% to 89% ± 5%). Providing the possibility for real-time detection significantly improved quantification efficiency even for the smallest cells evaluated. While end-point evaluation resulted in a detection rate of 63% ± 3% of the spiked cells, real-time evaluation led to an increase of 21% to 84% ± 4%. The established protocol provides an advantageous and efficient method for integration of fully automated sample preparation and CTC quantification into a lab-on-a-chip system.