A rapid method for the generation of uniform acellular bone explants: a technical note.

BACKGROUND: Bone graft studies lack standardized controls. We aim to present a quick and reliable method for the intra-operative generation of acellular bone explants. METHODS: Therefore, ovine cancellous bone explants from the iliac crest were prepared and used to test several methods for the induction of cell death. Over night heat inactivation was used as positive treatment control, methods to be investigated included UV light, or X- ray exposure, incubation in a hypotonic solution (salt-free water) and a short cycle of repeated freezing and thawing. RESULTS: Viability of treated and 2 days cultured bone explants was investigated by lactate dehydrogenase assay. Non-treated cultured control explants maintained around 50% osteocyte viability, while osteocyte survival after the positive treatment control was abolished. The most dramatic loss in cell viability, together with a low standard deviation, was a repeated cycle of freezing and thawing. CONCLUSIONS: To summarize, we present a freeze-thaw method for the creation of acellular bone explants, which is easy to perform, not time-consuming and provides consistent results.

TGFbeta3 and loading increases osteocyte survival in human cancellous bone cultured ex vivo.

The goal of this study was to assess the effect of the addition of TGFbeta(3), alone or in combination with loading, on the survival of osteocytes in 3D human explant cancellous bone during long-term culture in an ex vivo loading bioreactor. Human cancellous bone explants were cultured for up to 14 days with or without TGFbeta(3) (15 ng ml(-1)) and with or without loading (300 cycles, at 1 Hz, producing 4000 microstrain). Bone core response was visualized using undecalcified histology with morphological methods after embedding with Technovit 9100 New resin. Histological examination revealed normal gross level bone structure with or without the application of load or the addition of TGFbeta(3). The viability of the osteocytes within the bone was assessed by lactate dehydrogenase (LDH) activity. We demonstrate that this ex vivo loading bioreactor is able to maintain a high percentage (over 50%) of viable osteocytes throughout the bone explants after 14 days in ex vivo culture. Further to this, the combination of daily loading and TGFbeta(3) administration produced superior osteocyte survival at the core centres when compared to loading or TGFbeta alone.

Establishing a 3D ex vivo culture system for investigations of bone metabolism and biomaterial interactions.

A 3D load-providing culture system is presented, which enables human cancellous bone explants to be studied ex vivo. Bone integrity and activity was demonstrated during 2 to 3 weeks of culture. Viable osteocytes and bone specific markers could be identified. Culture conditions were improved using a serum free medium containing TGF-beta3. Therefore this system gives the opportunity to study bone biology, hormonal effects as well as biomaterial interactions ex vivo prior to animal studies.