Comparative Biochemistry and Physiology Part B 132 (2002) 217–229 1096-4959/02/$ - see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S1096-4959 Ž 01 . 00524-3 Bioactivity of nacre water-soluble organic matrix from the bivalve mollusk Pinctada maxima in three mammalian cell types: fibroblasts, bone marrow stromal cells and osteoblasts  Lucilia Pereira Mouries, Maria-Jose Almeida, Christian Milet, Sophie Berland, ` ´ Evelyne Lopez* Laboratoire de Physiologie Generale et Comparee, Museum National d’Histoire Naturelle, 7 rue Cuvier, 75231-Paris Cedex 05, ´´ ´ ´ France Received 27 January 2001; received in revised form 17 September 2001; accepted 8 October 2001 Abstract In vivo and in vitro studies provide strong evidence of the osteogenic activity of nacre obtained from Pinctada maxima. The in vitro studies indicate that diffusible factors from nacre are involved in cell stimulation. The water- soluble matrix (WSM) was extracted from nacre by a non-decalcifying process, and four fractions (SE –SE ) were 1 4 separated by SE-HPLC. Those fractions were tested in vitro on MRC5 fibroblasts. Alkaline phosphatase (ALP) activity was measured as a marker of osteoblastic differentiation. The anti-apoptotic protein Bcl-2 was also immunodetected in cultured osteoblasts from rat calvaria. WSM and fraction SE increased ALP activity. BMP-2 had the same effect on the 4 cells as WSM and SE . WSM greatly increased the amount of Bcl-2 in the cytoplasm and nucleus of osteoblasts. These 4 in vitro studies support our initial hypothesis that nacre organic matrix (WSM) of a bivalve mollusk contains signal- molecules that can stimulate the osteogenic pathway in mammalian cells that are targets for bone induction.  2002 Elsevier Science Inc. All rights reserved. Keywords: Alkaline phosphatase; Bcl-2; Bone marrow cells; Differentiation; MRC5 fibroblasts; Nacre; Osteoblasts; Water-soluble matrix 1. Introduction Nacre (mother-of-pearl) is a calcified structure that forms the lustrous inner layer of some mol- luskan shells. It is composed of calcium carbonate crystals organized in multiple layers of thin tablets of aragonite, surrounded by an organic matrix  This paper was submitted as part of the proceedings of the 20th Conference of European Comparative Endocrinolo- gists, organised under the auspices of the European Society of Comparative Endocrinology, held in Faro, Portugal, September 5–9, 2000. *Corresponding author. Tel.: q33-1-40-79-36-22; fax: q 33-1-40-79-56-20. E-mail address: lopez@mnhn.fr (E. Lopez). (mostly protein and polysaccharide), which repre- sents approximately 1–5 wt.% of nacre composi- tion (Shaffer et al., 1997; Weiss et al., 2000). The ¨ organic matrix is commonly classified in soluble and insoluble (Crenshaw, 1972; Weiner and Hood, 1975; Krampitz et al., 1983; Weiner et al., 1983). This biogenic composite has been recently con- sidered as a new biomaterial, for bone repair, that can induce osteogenesis. As a matter of fact, our previous in vivo and in vitro studies (Lopez et al., 1992; Silve et al., 1992; Le Faou et al., 1995, 1996; Atlan et al., 1997; Delattre et al., 1997; Lamghari et al., 1999), as reviewed by Westbroek and Marin (1998), suggested that the nacre matrix contains chemical signals that can be released in a