Survival and resorptive activity of chick osteoclasts in culture

Previous studies have shown that osteoclasts obtained from chopped bones resorb surrogate calcified tissue substrata in vitro. These cultures contained all bone and marrow cell types pooled together. We have now parted the marrow from the bone and cultured the cells from the two fractions separately: on both resorbable substrates and on plastic in order to test their longevity in culture and ability to resorb following trypsinisation. Marrow-fraction, bone-fraction or whole bone derived cells were harvested from prehatch chick long bone shafts after removing the periosteum; seeded on sperm whale dentine (SWD) slices or plastic dishes and cultured continuously, or trypsinised and reseeded on to fresh substrata at weekly or half-weekly intervals. Observations were made by light microscopy and SEM. Many multinucleate cells were observed in the marrow fraction immediately after settling, deriving presumably from poorly adherent osteoclasts, next to bone, which had not been resorbing at the time of harvesting. By three days in culture on plastic, multinucleate cells were very large both in terms of plan extent and nuclear number: cell fusion occurred between osteoclasts and between osteoclasts and small, round uninuclear cells. SWD was extensively resorbed. The adherence of the osteoclasts was greater (a) to plastic upon trypsinisation than that of the other cells; and (b) to SWD than to plastic, particularly if the cells were resorbing. Trypsinised cells regained their resorptive capacity after seeding on to new SWD, but only for 1 or 2 treatments. Bone derived cells were similar to the marrow cultures, except for the much higher proportion of other bone cell types. Trypsinisation caused a higher proportional loss of multinucleate cells from both SWD and plastic. Resorption was still occurring at 6 weeks in all cultures. A wide diversity existed in the shapes, depths, plan areas and volumes of the resorption pits.