Biological and physico-chemical assessment of hydroxyapatite (HA) with different porosity J.-C. Hornez a, * , F. Chai b , F. Monchau a , N. Blanchemain b , M. Descamps a , H.F. Hildebrand b a Laboratoire des Mate ´riaux et Proce ´de ´s (LMP), EA 2443, UVHC, 59600 Maubeuge, France b Groupe de Recherche sur les Biomate ´riaux, EA 1049, Faculte ´ de Me ´decine, 59045 Lille, France Abstract HAwith specific internal porosities was loaded with different antibiotics (ATBs) and then tested on its microbiological effectiveness. The HA purity was controlled with X-ray diffraction, IR and Raman spectrometry. Varying the sintering temperature and/or adding graphite and PMMA as porogenous agents lead to obtained micro- and meso-porosities. The biological tests concerned cell viability, proliferation and morphology (SEM), and the cytochemical staining of actin and vinculin. The micro- and meso-porous HA samples had an internal pore size of 1–10 mm and 10–50 mm, respectively. X-ray diffraction and FTIR confirmed the high purity of the HA. The cell viability tests with L132 cells confirmed the excellent cytocompatibility of HA, the graphite powder and the ATB vancomycine. Proliferation rate was assessed with MC3T3-E1 osteoblasts. All HA samples produced a higher proliferation than the controls; the micro-porous HA inducing the highest cell growth. The ATB impregnated HA also stimulated cell proliferation but in lower extend. Cytochemical staining of osteoblasts revealed a well-developed cytoskeleton with strong stress fibres. Labelling of the focal adhesion contacts with anti-vinculin showed a less developed adhesion process in the cells on the different HA substrates. It was possible to realize a highly pure hydroxyapatite with different but controlled porosities by varying the sintering temperature and/ or addition of a porogenous agents. This purity and the micro-porosity stimulate significantly cell growth. # 2007 Elsevier B.V. All rights reserved. Keywords: Hydroxyapatite; Micro-porosity; Cell response; Drug delivery system 1. Introduction Calcium phosphate compounds and in particular hydro- xyapatite (HA) and beta-tricalcium phosphate (b-TCP) are presently the most principal synthetic materials used for bone substitutes in many surgical fields like the orthopaedic, dental, and plastic surgeries (Rejda et al., 1977; Jarcho, 1981; Hench, 1998). Its chemical composition, quasi identical to that of the mineral phase of bone, confers to it excellent biocompatibility and osteointegration properties. Nevertheless, these materials have to bring the required qualities to allow an optimal biological response (Stanford and Keller, 1991). In this way, the cellular colonisation of bone substitutes depends on the porosity characteristics of the material (Healy et al., 1996), in particular the size and repartition of pores and the number and size of the interconnections between macro-pores. These interconnections form a kind of tunnel system allowing the passage and renewal of biological fluids and the entering of bone cells. They subsequently facilitate bone formation inside the macro-porous material scaffold. In addition, the micro- porosity of a bioceramic is another possible factor to influence the cell response (Bowers et al., 1992), to modify the resorption behaviour (LeGeros et al., 1988), and to generate multiple possibilities of surface functionalization. In order to prevent the increasing frequency of per-operative infections, bioceramics can be loaded with anti-bacterial agents and different antibiotics (ATB), which are released with respect to their chemical characteristics. To assess the feasibility for further ATB loading and other drug delivery systems, a pure HA was elaborated with specific internal material porosities (Zhou et al., 2002) and then tested on its cytological effectiveness (Hornez et al., 2007). The control of the macro- and micro- porosity is subsequently an important factor to improve the efficiency of the implanted material in the patient (LeGeros et al., 1988; Passuti et al., 1989). www.elsevier.com/locate/geneanabioeng Biomolecular Engineering 24 (2007) 505–509 * Corresponding author. Tel.: +33 3 27 53 16 71; fax: +33 3 27 53 16 67. E-mail address: jean-christophe.hornez@univ-valenciennes.fr (J.C. Hornez). 1389-0344/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2007.08.015