Two-photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage-, IL-4 activated macrophage- and osteoclast-based in vitro degradation of beta-tricalcium phosphate bone substitute material.

Two-photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two-photon laser scanning microscopy. Beta-tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin-4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta-tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL-4 activated macrophages. An average of ~32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL-4 activated macrophages. For the first time by applying two-photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design.