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Thesis summary

I presented my thesis on June 25th, 2003 at the prestigious "Grande Galerie de l'Evolution" auditorium, of the Natural History Museum (MNHN) of Paris, France. The jury decided to rate this work with the most honorific grade: "Mention très honorable avec les félicitations du Jury”

EXAM COMITTEE

• Denis ALLEMAND, Professor University of Nice-Sophia Antipolis, France
• João COIMBRA, Professor University of Porto, Portugal
• Xavier BOURRAT, Professor University of Bordeaux, France
• Dominique DOUMENC, Professor MNHN, Paris, France
• Christian MILET, Maître de Conference MNHN, Paris, France
• Cédric POIGNEAU, President of SIERA S.A., a biotechnology company financing the work of this Ph.D.

KEY WORDS
Nacre, organic matrix, proteins, osteogenesis, biomineralization.

SUMMARY
Nacre or mother of pearl is a biomineral composite of calcium carbonate crystallised in the aragonite polymorph, associated with an organic matrix. The organic matrix is a complex mixture of biopolymers, essentially proteins and polysaccharides, the actual nature and functions of which remain unelucidated. Previous in vivo studies have shown that nacre from the inner shell layer of the pearl oyster Pinctada maxima causes osteoinduction and osteogenesis in mammals. These properties, shared with bone, are supported by diffusible factors, present in the nacre organic matrix. The study of the organic matrix from this biomineral represents therefore a major interest for biomedical applications and for the understanding of the mechanisms governing the biomineralization process.

The aim of this work was to demonstrate that the nacre biological activity on the osteoinduction and osteogenesis processes is triggered by the water-soluble matrix and to characterize the underlying molecular components. To do this, we attempted to characterize the molecular composition of the water-soluble matrix.

An advance in our research concerns the method of extraction used to isolate the organic matrix from the mineral. No demineralization step was performed. The organic matrix released by this patented process exhibits paradoxical features. It is highly hydrophobic and rich in glycine and alanine (more than 65% of the total amino acid composition). After a separation step using size-exclusion chromatography, a large number of protein units were detected using an antibody raised against a specific fraction of the nacre water-soluble matrix (SE4). These units are common to the four size-exclusion isolated fractions. This approach enabled us to purify the most immunoreactive protein, P18, and to obtain a partial N-terminal sequence. The overall results led us to propose a hypothetical model for the representation of self-assembly properties of the protein units in the nacre water-soluble matrix. Furthermore, convergent data seems to confirm the existence of a molecular relationship between the nacre water-soluble matrix and factors present in bone matrix.

Finally, our in vitro experiments on the different mammalian cell types involved in bone regeneration proved that the water-soluble matrix isolated from Pinctada maxima nacre contains the signals responsible for the biological activity of the whole nacre. Indeed, this matrix acts in particular on bone cell differentiation, up until the final step of mineralization. The biological factors creating these effects are present in the SE4 fraction, isolated by size-exclusion chromatography.