Balancing mechanical stability and biological performance through a dual-setting approach in granular silicate-CPCs.

Abstract

Granular bone substitute systems are widely used to treat complex and irregular defects due to their adaptability and prefabricated nature, which eliminates in situ setting-related instability. However, their limited mechanical stability makes their performance highly dependent on intraoperative handling and increases the risk of impaired tissue ingrowth. In this study, granular brushite- and monetite-based calcium phosphate cements (CPCs) were developed and systematically investigated to improve mechanical performance and cytocompatibility through a dual-setting approach. Silicate-based precursors, specifically tetraethyl orthosilicate (TEOS) and 1,8-bis(triethoxysilyl)octane (BTESO), were incorporated into the CPC formulation prior to granulation to establish a reinforcing secondary network within the cement matrix. Structural and mechanical properties were assessed, including compression-induced changes, which were analysed using micro-computed tomography (μCT). The biocompatibility of the CPCs was further investigated using WST assays, cell counting, DNA quantification, and scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX). The incorporation of TEOS significantly increased mechanical stability and cytocompatibility compared to conventional formulations. TEOS-modified granules exhibited less than one-third of the compression-induced deformation observed for the control, which showed a height reduction of more than 5 mm. μCT analysis further revealed a shift in the intergranular pore-size peak from approximately 350 to 250 μm after compression in the control, whereas no apparent shift was observed for TEOS-modified granules. Granule size itself affected intergranular pore geometry, with larger granules showing higher metabolic activity. TEOS-modified granules showed the most favourable overall biological response, whereas the addition of BTESO provided no further mechanical or biological benefit. Furthermore, a cryosectioning approach enabled fluorescence imaging of osteoblast-like cells on CPCs with high autofluorescence, allowing visualisation of cell morphology on the material surface. Overall, the dual-setting approach, particularly using TEOS, improved resistance to compression while preserving intergranular pore architecture and maintaining favourable cytocompatibility, thereby addressing a key limitation of granular CPC-based bone substitute systems. In parallel, cryosectioning enables reliable fluorescence-based visualisation of cells on highly autofluorescent materials, offering a practical approach for the biological assessment of such systems.

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