"Maintenance of cancellous bone explants ex vivo CM. Davies,"
European Cells and Materials Vol. 7. Suppl. 2, 2004 (page 10) ISSN 1473-2262 Maintenance of cancellous bone explants ex vivo CM. Davies, P. Jäggi, RG. Richards AO Research Institute, Davos, Switzerland. INTRODUCTION: The Zetos culture system1 seams; living osteoclasts in Howship’s lacunae has potential to keep cancellous bone tissue viable were also seen. Bone marrow and osteocytes ex vivo2 through the use of unique chambers and a appeared well preserved. Very little necrotic tissue loading device that allows the tissue to remain in was observed. Noncollagenous proteins such as its natural 3D milieu. Bone and marrow cells osteopontin, osteonectin and osteocalcein, as well constantly interact with each other and with as procollagen Type I and RankL were localised migratory cells and the extracellular matrix. This through immunohistochemical labelling of type of niche has never been reproduced in vitro. sections. Tissue engineering methods have been aimed towards recreating this microenvironment in order to produce a possible means to fill bone defects caused by tissue trauma or disease. This culture system may be used as a means of maintaining tissue for re-implantation into patients or with different bone substitutes to evaluate their potential as bone replacements, reducing the Fig. 1: Left image- protein production in osteocytes amount of animal experimentation conducted. and soft tissue 15 days post-explantation; right - osteoid seam from bovine tissue 30 days post- explantation. METHODS: Human femoral heads (Ethic Commission Graubünden (18/02) approval), distal DISCUSSION & CONCLUSIONS: Culture of ovine femora or distal bovine metacarpals were cancellous bone explants within the loaded Zetos excised and cut into 9mm thick sections with the system maintains bone cells viable for at least 15 use of an Exakt 300 band saw. Cores 10mm in days. Conventional organ culture techniques diameter were bored from the sections with a usually suffer from extremes of too much oxygen specially produced fluted, hollow diamond-tipped at the surface and too little oxygen in the centre drill and cut parallel to 5mm with a Leica annular causing tissue necrosis. By allowing the bone cells saw. Bone cores were inserted inside the diffusion to be cultured in its near to natural chambers and left to settle at 37°C for 24 hours. microenvironment this system could overcome the Bone cores were loaded daily in the loading device barriers of homogeneous cell seeding and scaffold for 300 cycles at 1Hz with a walking or jumping composition associated with tissue engineering waveform, giving each core the equivalent of and other bioreactor systems. The system also has 4,000 µstrain (maximum and minimum potential for studying bone remodeling in normal compression of 20 µm and 5 µm respectively). and osteoporotic animal or human bone, bone bio- Both fresh (at explantation) and bone cores from mechanics, and the effects of drugs, hormones or the Zetos culture chambers (day 15) were fixed in growth factors on cancellous bone, making it an 70% ethanol, before being dehydrated through an essential laboratory aid in the future. ethanol series. Samples were embedded in Technovit 9100, polymerised at –20°C for REFERENCES: 1Jones D.B., Broeckmann E., Pohl T., histological and immunohistochemical evaluation3. and Smith E.L. (2003). Development of a Mechanical 3 H-glycine was placed in the media for 24 h Testing and Loading System for Trabecular Bone Studies allowing incorporation into newly synthesised for Long Term Culture. Eur Cell Mater 5: 48-60 2 proteins. Media and tissue proteins were harvested Clarke M.C.H., Savil J., Jones D.B., Nobel B.S., Brown and analysed for radioactivity. S.B. (2003). J. Cell Biol. 160:4 577-587. 3Yang R. et al., (2003) Immunohistochemistry of matrix markers in Technovit 9100 New®-embedded undecalcified bone RESULTS: 3H-glycine was detected in protein sections. Eur Cell Mater (6) 57-71. through SDS-page and Western blot analysis as well as autoradiography of embedded sections, ACKNOWLEDGEMENTS: Funded by the 3R demonstrating cell viability after 15 days in Foundation grant # 78/01. The authors wish to thank to culture. Histological sections showed osteoblasts Dr Thomas Perren for supplying human tissue had laid down collagen fibres to produce osteoid