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

One of the mechanisms which controls bone growth, repair remodeling and absorption is mechanical loading. There exists no long-term in vitro model to study bone cells together with their matrix, nor a model that can apply quantitative mechanical forces of physiological amplitudes and frequencies. The analysis of the mechanical properties of bone (Young’s modulus and visco-elastic moduli) on small pieces of bone is also difficult with present devices. We have built a device that can maintain full viability and physiological response of bone for a period of several weeks and integrates all three functions.

10mm diameter bone cores 5 mm thick were obtained from the trabecular bone of the distal ulna of a 24 months old cow 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: The results of these studies have shown that perfusion of trabecular bone can maintain all cells and maintain bone structure for at least 72 days. In conventional methods for bone organ cultures, small bones, such as rat calvaria, quickly start to resorb bone and degenerate. In our perfusion system we see no evidence of change.. Initial experiments have indicated that there are 2 visco-elastic moduli of bone with different time constants, that the elastic modulus of trabecular bone varies is site dependant and that loading to 0.4% compression raises prostaglandin E2 and insulin-like growth factor 1 within a few hours. Mechanical stiffness of bone is increased by 35% when loaded for 20 days at 4,000μ, and decreases by 25% when not loaded. PTH at 10-10M increases stiffness over the load effect and 10-6M PTH decreases stiffness even in the presence of loading. Active osteoclasts are seen during the whole culture period indicating that the stem cells are present and functional.

We gratefully acknowledge support by the German Arthrose Foundation (DAH) and the AO in Davos, CH.