Using the trabecular bone bioreactor (ZETOS) developed in our laboratories we have investigated the formation of bone using the fluorescent bone seeking markers calcein and alizarin red. And the association of bone formation with the increase in stiffness with mechanical loading.

10 mm diameter bone cores 5 mm thick were obtained from the distal radius /ulna of cows obtained at the slaughter house. by precision cutting with diamond saws and keyhole cutters (our pattern) in sterile 7–10°C phosphate buffered saline (PBS) and cultured in a variation of DMEM containing fructose HI GEM.

Results: Loading the bone 30x 4,000μ per day resulted in an increase of stiffness of 35%, by day 30 while the non loaded controls decreased in stiffness. Calcein was added at day 27 to the circulating medium for 4 hours and then fresh medium was circulated. On day 30 alazarin red was circulated through the trabecular bone. The bones were subsequently fixed and embedded in resin and sectioned by classical histological techniques. The difference in distance between the two dyes indicated the amount of bone formation. The mechanically loaded bones showed significant evidence of formation and also significant numbers of active osteoclasts indicating high bone turnover. No evidence of necrosis or cartilage formation was found. Formation in unloaded bones was much reduced and on many areas no active osteoblasts could be observed. This is the first demonstration of bone formation ex vivo after 30 days of culture.

We gratefully acknowledge support by the German Arthrose Foundation (DAH) and the AO in Davos, CH. DJ is a recipient of a Fork award from the AO