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International audience; Macromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein-protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines. Establishing cellular models that preserve native genomic regulation of the target protein is instrumental to investigate protein localization and dynamics using fluorescence imaging but also to affinity purify associated protein complexes using anti-GFP antibodies or nanobodies.

The flesh of the Pinctada radiata pearl oyster from coastal Tunisia is considered as a high source of n-3 and n-6 and its shell nacre layer is a promising osteogenic biomaterial. Fatty acid (FA) analysis showed that the major components found in total FA (TFA) were 14:0, 16:0, and 18:0 saturated FA (SFA); 16:1, 18:1, and 20:1 monoenoic FA; 20:4n-6 (ARA), 22:5n-3 (DPA). Characteristically high levels of 20:5n-3 (EPA) and 22:6n-3 (DHA) (6.53-89.75 mg/100 g TFA) polyunsaturated FA (PUFA) were found, respectively, in the TFA of nacre and flesh. Evaluated the effects in vitro of lipids extracted from nacre (Ln) and from flesh (Lc) of P. radiata on growth and the differentiation of osteoblasts. Cytotoxicity tests (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide [MTT] and lactic acid dehydrogenase c [LDH]) demonstrated that both extracts are nontoxic. Alizarin Red staining was used in an osteoblast differentiation model using the osteoblast MC3T3-E1 cell line. It showed that the FA of both extracts induced osteoblast differentiation leading to mineralization. Reverse transcription-polymerase chain reaction (RT-PCR) showed a significantly higher expression of osteocalcin (Bglap) and runt-related transcription (Runx2) in MC3T3-E1 cells in the presence of Ln. No difference of osteopontin (Spp1) and Collagen type I (Col1a1) genes compared to the control was observed. In conclusion, these results supported, obtained from our in vitro experimental model used, the interest/potential of lipids extracted from nacre and P. radiata flesh to stimulate bone formation.

Nacre is able to induce bone-forming cells mineralization, and gains widely interest in bone regeneration. While, the osteoinductive compounds are not yet identified. ESM (Ethanol Soluble Matrix), a nacre extract from powder of Pinctada margaritifera pearl oyster shell, has been firstly proven having the capacity to induce mineralization and to restore mineralization defect in vitro. It is suitable to treat ESM as a source of osteoinductive compounds. Herein, we develop a new method for separating and purifying nacre extracts by an ionic approach. At first, cationic ESM (ESMc) and anionic ESM (ESMa) were achieved with ion-exchange resin. Then, ESM was separated and collected on cation exchange HPLC. Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectrometry (EDS) was used to reveal the concentrated elements in ESM fractions. A coupled cell models were used to test the ESM fractions. Alizarin Red staining was performed and quantified to evaluate the mineralization level. ESMc and 2 HPLC fractions stimulated the mineralization in both cells. EDS demonstrated the abundant presence of calcium and chloride in the osteogenic fractions. To validate, pure CaCl2 was tested and proven having an osteogenic effect in both cells, but less stable than ESM. The mineralization nodules induced by ESM fractions and CaCl2 differed in both cells. In conclusion, a new method was developed for separating and purifying nacre extracts by an ionic approach. By which, the osteoinductive compounds in ESM were proven cationic, and calcium in ESM was demonstrated to play a role in inducing the cell mineralization.

International audience

International audience; Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds. (C) 2015 Elsevier Inc. All rights reserved.