CO-48 | HEMOFISH: A NEW ANIMAL MODEL FOR STUDY THE ROLE OF FACTOR VIII IN BONE DISEASE

M. Berni¹, L. Caliogna¹, F. Tonelli², C. Aresi², G. Rizzi¹, A. Forlino², G. Pasta¹ | ¹Orthopedics and Traumatology Clinic, IRCCS Policlinico San Matteo Foundation, Pavia, Italy; ²Dep. of Molecular Medicine, Biochemistry Unit, University of Pavia, Italy

Background and Aims. Haemophilia A is a rare hereditary X-linked recessive disease caused by deficient activity of blood coagulation factor VIII. Patients show a significant reduction in bone mineral density with a greater risk of developing pathological fractures or fractures due to mild trauma. Factor VIII is a plasmatic multi-domain glycoprotein involved in the coagulation cascade mainly synthesized by hepatocytes. In recent years, several studies have confirmed that the reduced levels of FVIII are involved in bone mineral density, bone reduction, osteoporosis and several molecular pathways on bone physiology and turnover. Zebrafish is an excellent animal model because approximately 70% of its genes have an ortholog in human genome. It shows a bone structure, metabolism, and turnover very similar to humans. It is an ideal model for studying bone diseases because it is useful for study bone matrix deposition, bone mineralization, osteogenesis, bone metabolism mechanisms and for creating models of bone diseases. The aim is to study how the lack of FVIII can lead to bone disease in a haemophilic zebrafish.
Materials and Methods. this project study the bone physiology and turnover in haemophilic (FVIII-) and wild-type (WT) zebrafish. After amputation of the bony caudal fin, zebrafish can regenerate it and it is possible to study how the fin and rays regenerate. Osteoclasts are multinucleated cells that contain several cytoplasmic granules rich in tartrate-resistant acid phosphatase (TRAP), a metalloprotein enzyme located in intracytoplasmic vesicles expressed during bone matrix degradation. On amputated fins it is possible to study osteoclasts activity with TRAP staining. Micro-computed tomography allows to study zebrafish skeletal morphology and structure with the analysis of the angle that the neural arch formed with the vertebral body.
Results. After 7 days from caudal fin amputation of 16 FVIII- and 16 WT zebrafish, the fin regeneration is significantly reduced in FVIII- group. New ray regeneration growth and bifurcation numberers significantly reduced in the FVIII- group, p <0,001 and p <0,006 respectively. A different osteoclasts activity is observed on 16 FVIII- and 16 WT zebrafish fin amputated with TRAP staining. Micro-computed tomography on 7 FVIII- and 17 WT zebrafish shows an increase about 6% in neural arch angle in FVIII- zebrafish, with a significance of p <0,05.
Conclusions. the experimental data would seem to indicate that the lack of FVIII influences correct bone formation and turnover.