In Vitro Mineralisation of Human Bone Marrow Cells Cultured on Bonelike®

Bonelike is a CaO-P2O5 based glass-reinforced hydroxyapatite (HA) designed to mimic the inorganic composition of the bone tissue. This work evaluates the response of human bone marrow cells to Bonelike  concerning cell proliferation and osteoblast differentiation. HA was used as control material. Results showed that Bonelike  allowed the proliferation of bone marrow cells and their complete differentiation, as evidenced by t he formation of cell-mediated mineralisation. In comparison with HA, Bonelike  had a positive effect on the expression of alkaline phosphatase and also on the formation of a mineralised matrix, two osteobla st markers. Introduction Bonelike is a synthetic hydroxyapatite (HA) that is sintered in the pr esence of CaO-P 2O5-based glasses using a patented process [1]. This synthetic bone graft w as designed to improve the mechanical properties of calcium phosphate ceramics and mimic the inorganic composition of bone tissue. The physicochemical and mechanical behaviour of Bonelike  hav been extensively reported in literature [1-3]. Previous in vitro biological studies showed that glass-reinforced HA composites al low the proliferation of MG63 osteoblast-like cells and human bone marrow ce lls and the expression of osteoblast markers [4-6]. Also, in vivo studies performed in a rabbit model demonstrated that Bonelike composites induced earlier new bone formation around implants than HA [7]. Recently, a composite prepared by the addition (4 wt %) of a glass with the composition of 65P2O5-15CaO-10CaF 2-10Na2O (in % mol) to HA was subject of clinical trials in implantol ogy and maxillofacial surgery [8]. This study demonstrated extensive new bone formation around implanted granules and continuous replacement by new bone. Osteoblasts a re the cells responsible for the formation of the bone tissue at the bone/material interface and the present work evaluates the response of human bone marrow cells to Bonelike  composite, with the same chemical composition, concerning cell proliferation and osteoblast differentiation. HA was used as control material. Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 821-824 doi:10.4028/www.scientific.net/KEM.254-256.821 © 2004 Trans Tech Publications Ltd, Switzerland All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications Ltd, www.scientific.net. (Semanticscholar.org-13/03/20,19:47:23) Materials and methods For the preparation of Bonelike , a P2O5-based glass with the chemical composition of 65P 2O515CaO-10CaF 2-10Na2O, in mol %, was prepared from reagent grade chemicals by conve ntional techniques. The glass was then milled and added to HA powder in a prop ortion of 4.0 wt % using methanol as solvent medium. Disc samples were then prepared by uniax ial pressing at 288 MPa and sintered at 1300 oC for 1h followed by natural cooling inside the fur nace. Detailed description of Bonelike preparation was previously reported [1]. For cell culture studie s, iscs were polished down to 1 μm, ultrasonically cleaned and sterilised before testing. HA sam ples were also prepared and used as control material. Human bone marrow cells (first subculture) were cultured on the sur face of Bonelike and HA material samples for 28 days in α-MEM, 10 % foetal bovine serum, 50 μg/ml gentamicin and 2.5 μg/ml fungizone, and supplemented with 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate and 10 nM dexamethasone, experimental conditions that favour the complete expression of the osteoblast phenotype [9]. As reference control, cells were cultured i n parallel on standard tissue culture plastic plates. Material samples were also incubated i n the absence of cells in the same experimental conditions as those of the seeded materials. Control cul ures and seeded materials were characterised at days 1, 3, 7, 14, 21 and 28 for cell viability/ proliferation (MTT assay), total protein content and alkaline phosphatase (ALP) activity, using procedures previously described in detail [6,7,10,11]. Control and seeded materials were observed by scanning electron microscopy (SEM). The in vitro biological studies were performed at the Faculdade de Medicina Dentária da Universidade do Porto. Results and discussion Biochemical evaluation of the seeded Bonelike  showed that human bone marrow cells proliferated during approximately three weeks and attained a stationary pha se afterwards, as shown by the results concerning the MTT reduction and total protein content (Figs. 1 A and B). Cells expressed ALP, especially during the second and third weeks of c ulture (Fig. 1C). Seeded HA presented a similar behaviour concerning cell growth but lower (20 to 30 %) ALP a ctivity than that measured on seeded Bonelike .