Short-time pre-washing of brushite-forming calcium phosphate cement improves its in vitro cytocompatibility

Abstract

A pre-washing protocol was developed for resorbable, brushite-forming calcium phosphate cements (CPCs) to avoid harmful in vitro effects on cells. CPC discs (JectOS+, Kasios; self-developed CPC) were pre-washed with repeated changes of phosphate-buffered saline (PBS; 24h total). Unwashed or PBS-pre-washed discs were incubated in culture medium (5% fetal calf serum; up to 10days) and then tested for their influence on pH/calcium/phosphate levels in H₂O extracts. Effects on pH/calcium/phosphate levels in culture supernatants, and morphology, adherence, number, and viability of ATDC5 cells and adipose-tissue derived stem cells were analyzed in co-culture. Pre-washing did not alter CPC surface morphology or Ca/P ratio (scanning electron microscopy; energy-dispersive X-ray spectroscopy). However, acidic pH of unwashed JectOS+ and self-developed CPC (5.82; 5.11), and high concentrations of Ca (2.17; 2.40mM) and PO₄ (38.15; 49.28mM) in H₂O extracts were significantly counteracted by PBS-pre-washing (pH: 7.92; 7.92; Ca: 0.64; 1.11mM; PO₄: 5.39-5.97mM). Also, PBS-pre-washing led to physiological pH (approx. 7.5) and PO₄ levels (max. 5mM), and sub-medium Ca levels (0.5-1mM) in supernatants and normalized cell morphology, adherence, number, and viability. This CPC pre-washing protocol improves in vitro co-culture conditions without influencing its structure or chemical composition.

Keywords: Biocompatibility; Brushite-forming calcium phosphate cement; Cell line; Pre-washing.