Nacre Initiâtes Biomineralization by Human Osteoblasts Maintained in Vitro
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Calcif Tissue Int (1992) 51:363-369 Calcified Tissue International 1992 Springer-Verlag New York Inc. Nacre Initiâtes Biomineralization by Human Osteoblasts Maintained In Vitro Caroline Silve,1 Evelyne Lopez,2 Bernadette Vidal,2 David C. Smith,3 Serge Camprasse,4 Georges Camprasse,4 and Gérard Couly5 'CNRS URA 583 and 2Service de Stomatologie. Hôpital Necker Enfants Malades, 149 rue de Sevrés, 75015 Paris, France; 3CNRS URA 90, Laboratoire de Physiologie Générale et Comparée and 4Laboratoire de Minéralogie, Muséum National d'Histoire Naturelle, Paris, France; and 'Cabinet Dentaire, Chelles, France Received November 22, 1991. and in revised form February 19. 1992 Summary. When nacreous shell produced by the marine ability to specifically stimulate new bone formation would be oyster Pinctada maxima, used as a biomaterial in oral sur- bénéficiai. gery, is implanted in human bone, new bone formation oc- Currently, the factors controlling thèse steps in vivo are curs, resulting in a tight welding of the bone to the nacre [16]. poorly understood. The availability of an in vitro model of Thèse findings are consistent with the possibility that nacre normal bone formation would be of considérable use in adjacent to bone can locally stimulate osteogenic activity. studying thèse questions. When osteoblasts are maintained To test this hypothesis, we have evaluated the effect of the in vitro, however, bone formation does not occur spontane- simultaneous présence of bone and nacre on human osteo• ously. As a conséquence, virtually ail studies of in vitro min• blasts in vitro. Nacre chips (1 mm3) were placed at ~1 mm eralization have used chemical inducers, especially p-glyc• distance from a similarly sized bone chip on a layer of first erophosphate, to stimulate bone mineralization. p-glycero• passage human osteoblasts. None of the chemical inducers phosphate clearly modulâtes alkaline phosphatase activity generally required to obtain bone mineralization in vitro (in and minerai déposition by cultured osteoblasts [3-13]. The particular, p-glycerophosphate) was added to the cultures. physiological relevance of the initiation of mineralization by Mineralized sections of the cultures were evaluated by light P-glycerophosphate, however, is not clear. Mineralization in and électron microscopy, contact microradiography, and the présence of p-glycerophosphate but in the absence of Laser Raman Spectroscopy. The results demonstrated that osteoblasts has been demonstrated [14], and cellular toxicity nacre has strong osteogenic effects on human osteoblasts has been observed in the présence of p-glycerophosphate when placed in proximity to bone in vitro. New bone forma• [14, 15]. In addition, when p-glycerophosphate is added to tion occurred by both appositional growth on the existing bone explants, the sites at which mineralization occur can- bone and by the formation of mineralized nodules within the not be controlled and bone formation occurs at ectopic sites matrix adjacent to the bone explant. Electron microscopic in the periosteum, not by appositional growth on existing évaluation of thèse sites demonstrated findings typical of bony surfaces [15]. Thus, the identification of substances those described in the course of bone formation in vivo, and that induce controlled appositional growth of bone remains a no évidence of toxicity was observed. In addition, under the worthwhile goal. In particular, materials that could be used conditions of culture used, nacre can also promote the for• as implants and that have intrinsic osteogenic properties in mation by osteoblasts of a structure with characteristics sim- the local milieu would be very useful. ilar to nacre (e.g., lamellar organic matrix mineralized with Camprasse et al. [16] have previously shown that when aragonite, as demonstrated by Laser Raman Spectroscopy). nacreous shell produced by the marine oyster Pinctada ma• Given the unusual capacity of nacre in the présence of hu• xima, used as a biomaterial in oral surgery, is implanted in man osteoblasts in vitro to induce différent types of miner• human bone, new bone formation occurs, resulting in a tight alization, this model should provide insights into the spécifie welding of the bone to the nacre. Thèse findings are consis• interactions between matrix proteins and minerais that con- tent with the possibility that nacre adjacent to bone can lo• trol the type of minerai produced in the course of biominer• cally stimulate osteogenic activity. To test this hypothesis, alization. we have evaluated the effect of the simultaneous présence of bone and nacre on human osteoblasts in vitro. The results demonstrate that biomineralization is strongly stimulated at sites where nacre is in close proximity to bone. Interestingly, The formation of bone by osteoblasts is the resuit of a séries the biominerals deposited adjacent to the preexisting bone of events that include the sécrétion of matrix proteins, the and nacre had différent morphologie, biréfringent, and spec- spécifie organization of the matrix in a fashion that promûtes trometric features, which resembled those of bone and na• crystal nucleation, and the sécrétion of ions necessary for cre, respectively. minerai growth [1,2]. Better understanding of thèse events is essential to our understanding of bone growth, and would have important clinical implications in situations where the Materials and Methods Primary Osteoblast Cultures Offprint requesls to: C. Silve Human osteoblasts were grown from biopsies of alveolar maxillary 364 C. Silve et al.: Nacre and Biomineralization ln Vitro bone obtained from seven children (aged 1-5 years, mean 2.1 years) tions were also made on sections stained with lead citrate only, as it undergoing necessary surgery as previously described [17] with is known that uranyl acétate can resuit in partial demineralization of slight modifications. Bone biopsies were eut into small chips (1 sections [22]. mm3), incubated for 5 minutes in a lysing solution (10 mM KHC03, 156 mM NH4C1, in H20) to lyse red blood cells, and extensively rinsed with BGJb médium (Eurobio, Les Ulis, France) without cal• cium, supplemented with 100 U/ml penicillin G, 100 p-g/ml strepto- Microradiography mycin, and 10% fetal calf sérum (FCS). Bone chips were then cul- tured in the same médium in 10 cm2 culture dishes (Corning), and Contact microradiographs of each undecalcifïed section before cells were allowed to grow from the bone explants. Cultures were grinding (100 \i.m) were made with a PW 1008 Massiot-Phillips X-ray maintained at 37°C in 95% air/5% C02, and médium was changed apparatus using a K„, copper target; exposures were made at 30 kV every third day until cells reached confluence (approximately 2-3 and 20 mA on Kodak 649-0 film. weeks). Cultures were then trypsinized, filtered through a nylon cloth (35 u.m mesh) to remove bone pièces, and cells were replated at a density of 5 x 104 cells/cm2 in 10 cm2 culture dishes and cultured in 3 ml of BGJb médium (1.2 mM Ca2 + , 2.2 mM P04, pH 7.4) Laser Raman Spectroscopy supplemented as for primary cultures. Ail experiments were per- formed on first passage cells. Spécimens for laser Raman spectroscopy were prepared as de• The osteoblastic phenotype of the cells used for the experiments scribed above for light microscopy, and analyzed using a Raman described was confirmed by (1) the sécrétion of bone gla protein microprobe [23-26] (Société Dilor) as follows: objective x50; green (BGP) in the absence and présence of 10"9 M l,25(OH)2D for 3 days Argon laser excitation at 514.53 nm; laser power 100-200 mW; mul- (8.1 ± 0.8 and 55.2 ± 4.3 ng/100 u.g protein, respectively); (2) alka- tichannel détection in the range 131-1201 cm-1 with two batteries of line phosphatase activity (9.4 ± 0.8 IU/100 |xg protein); (3) produc• 1204 diodes; slit width 100-150 u.m; 20 accumulations of 5 seconds; tion of cAMP in response to 8 x 10~8 M human parathyroid hor• peak position calibration corrected to standard diamond at 1332 mone 1-34 (PTH) (1.2 ± 0.1 and 16.2 ± 2.6 pmol cAMP/10 minutes/ cm-1. 100 p.g protein, respectively without and with PTH); and (4) synthesis of type I collagen, as detected by indirect immunofluores- cence using rabbit anti-human type I collagen antiserum (Institut Pasteur, Lyon, France). Results Effect of Nacre on Osteoblasts In Vitro Evaluation of the Effect of Nacre on Mineralization Fragments of nacre produced by P. maxima were obtained as pre• When bone chips were placed in proximity of similarly sized viously described [16] and sterilized for 30 minutes at 100°C for the nacre chips on a layer of confluent human osteoblasts, the experiments. Fragments of human bone were those previously cul• gap between the adjacent bone and nacre was progressively tured to obtain bone cells, and were not devitalized. To initiate filled over a 4-6 week period by dense tissues extending experiments, 1 mm3 nacre chips were placed at =1 mm distance from the opposing surfaces of both the bone and nacre in ail from a similarly sized bone chip on a layer of first passage human experiments (n = 7) (Fig. 1). Light microscopic observations osteoblasts 48 hours after subculture. Bone fragments and osteo- confirmed that thèse tissues were continuous between the blast lines were obtained from the same individual in ail cases. Cul• opposing surfaces of bone and nacre, and were formed by a tures containing bone chips only or neither bone nor nacre chips were prepared in parallel. Culture médium was as described for matrix containing both fibrous and cellular éléments (Fig. 2). primary cultures; médium was replaced every 4th day. Neither Collagen fibers were oriented along an axis extending from chemical inducers generally required to obtain bone formation in the bone explant towards the nacre (Fig. 2a). Foci with his• vitro (in particular, B-glycerophosphate) nor ascorbic acid was tologie features typical of nodules of mineralization were added to the cultures.