Aims

1. MRC Mouse NetworkModels for Disorders of Bone and CartilagePresentation on behalf of Bone and Cartilage Consortium:Professor R V ThakkerMay Professor of MedicineUniversity of OxfordFirst meeting of MRC Mouse Network,Harwell, UK13th January 2012

2. Aims 1. To establish mouse models of bone and cartilage disorders e.g. osteoporosis, osteoarthritis, skeletal trauma, chondrocalcinosis, Paget’s disease, and skeletal dysplasia 2. To use these models for: elucidating the underlying biological mechanisms, facilitating translational research, and development of biomarkers 3. To evaluate translation of findings to the human population level by comparison to large scale GWAS data sets (GEFOS and TREAT-OA)

3. Why is this important? • Disorders of bone and mineral homeostasis: • Osteoporosis • Osteoarthritis • Skeletal trauma • Chondrocalcinosis • Gout • Paget’s disease • These disorders are very common, and a major burden on the health services • Effective treatments and prevention strategies are required

4. BASIC SCIENCE A Boyde P Croucher J Edwards N Horwood A Pitsillides T Skerry ENDOCRINOLOGY R Thakker G Williams D Bassett RHEUMATOLOGY M Brown M Feldman S Ralston T Vincent MOUSE GENETICS SBrown R Cox P Potter Consortium Members HUMAN GENETICS RivadeneiraUitterlinden Group ENGINEERING / MATERIAL SCIENCE H Gupta ORTHOPAEDICS J Nanchahal VETERINARY SURGERY A Goodship

5. Bone Phenotyping • Primary Screen • - Performed at MRC Harwell • - Bone & cartilage screen fits within primary screens for other systems • - Development, growth, biochemistry, body composition, whole body X-ray, DEXA – for bone mineral density • Intermediate high resolution secondary screen • - Faxitron – high throughput quantative analysis of bone mineral content • - Micro CT – high throughput quantitative analysis of structure, mineralization and static morphometry • Detailed and bespoke secondary screening • - Back scattered electron scanning electron microscopy (BSE-SEM) • - Quantitative BSE-SEM • - Bone histology & histomorphometry • - Con-focal microscopy • - Mechanical testing • - Nanomechanical (synchroton X-ray scattering) • - Primary bone cell culture • - Longitudinal studies

6. Collection, fixation, storage and distribution of skeletal elements between laboratories & multiple analyses per sample upholds principles of 3Rs and economy From Williams & Bassett

7. Bone Phenotyping • Primary Screen • - Performed at MRC Harwell • - Bone & cartilage screen fits within primary screens for other systems • - Development, growth, biochemistry, body composition, whole body X-ray, DEXA – for bone mineral density • Intermediate high resolution secondary screen • - Faxitron – high throughput quantative analysis of bone mineral content • - Micro CT – high throughput quantitative analysis of structure, mineralization and static morphometry • Detailed and bespoke secondary screening • - Back scattered electron scanning electron microscopy (BSE-SEM) • - Quantitative BSE-SEM • - Bone histology & histomorphometry • - Con-focal microscopy • - Mechanical testing • - Nanomechanical (synchroton X-ray scattering) • - Primary bone cell culture • - Longitudinal studies

8. cell lysates supernatants WT WT Ser1386Pro Ser1386Pro - - Lpk/+ Humerus Ulna WT Lpk: Spondyloepiphyseal Dysplasia Congenita Lpk/+ WT Lpk/+ WT WT Lpk/+ Proliferating chondrocytes • Dominant mutant • Disproportionately shortened humerus and secondary osteoarthritis • Flattened epyphyses and disorganized growth plate • Due to Ser1386Pro Col2a1 mutation ; in C-propeptide domain • ER retention of mutant Col2a1 Hypertrophic chondrocytes Esapa et al, JBMR, 2012, in press Antibody: anti c-Mmyc

9. Bone Phenotyping • Primary Screen • - Performed at MRC Harwell • - Bone & cartilage screen fits within primary screens for other systems • - Development, growth, biochemistry, body composition, whole body X-ray, DEXA – for bone mineral density • Intermediate high resolution secondary screen • - Faxitron– high throughput quantative analysis of bone mineral content • - Micro CT – high throughput quantitative analysis of structure, mineralization and static morphometry • Detailed and bespoke secondary screening • - Back scattered electron scanning electron microscopy (BSE-SEM) • - Quantitative BSE-SEM • - Bone histology & histomorphometry • - Con-focal microscopy • - Mechanical testing • - Nanomechanical (synchroton X-ray scattering) • - Primary bone cell culture • - Longitudinal studies

10. Faxitron: deiodinase mutants have increased bone mineral Bassett et al, 2010, PNAS

11. Bone Phenotyping • Primary Screen • - Performed at MRC Harwell • - Bone & cartilage screen fits within primary screens for other systems • - Development, growth, biochemistry, body composition, whole body X-ray, DEXA – for bone mineral density • Intermediate high resolution secondary screen • - Faxitron – high throughput quantative analysis of bone mineral content • - Micro CT – high throughput quantitative analysis of structure, mineralization and static morphometry • Detailed and bespoke secondary screening • - Back scattered electron scanning electron microscopy (BSE-SEM) • - Quantitative BSE-SEM • - Bone histology & histomorphometry • - Con-focal microscopy • - Mechanical testing • - Nanomechanical (synchroton X-ray scattering) • - Primary bone cell culture • - Longitudinal studies

12. Adult D2KO and D1/D2KO mice have brittle bones Stiffness Maximum load Yield load Fracture load D2KO WT 16w * * * ** Biomechanical characteristics and strength properties determined by destructive 3 point bend testing Bassett et al, 2010, PNAS

13. Bone Phenotyping • Primary Screen • - Performed at MRC Harwell • - Bone & cartilage screen fits within primary screens for other systems • - Development, growth, biochemistry, body composition, whole body X-ray, DEXA – for bone mineral density • Intermediate high resolution secondary screen • - Faxitron – high throughput quantative analysis of bone mineral content • - Micro CT – high throughput quantitative analysis of structure, mineralization and static morphometry • Detailed and bespoke secondary screening • - Back scattered electron scanning electron microscopy (BSE-SEM) • - Quantitative BSE-SEM • - Bone histology & histomorphometry • - Con-focal microscopy • - Mechanical testing • - Nanomechanical (synchroton X-ray scattering) • - Primary bone cell culture • - Longitudinal studies

14. Wild type Hpr 1 week ii. Hpr i. Wild-type Collagen fibril b 4 weeks l L 7 weeks Mineral coating B C D d D T = 0 s T = 240 s 10 weeks Karunaratne et al, JBMR, 2012, in press 16 weeks

15. 1 week old mice scapula Mineral particles are randomly orientated due to the low muscle forces and less activity levels in 1 week old mice Disruption of muscle stress-mediated mineral orientation at the nanoscale in the flat scapular bones of rahictic mice (HPR) 10 weeks old wild type mice scapula WT 10 weeks old Hpr mice scapula HPR Due to the disturbed local mechanical and structural environment mineral particles are randomly orientated Mineral particle orientated along the muscle force direction Karunaratne et al, in preparation

16. Summary – Phenotyping by Bone & Cartilage Consortium • Primary Screen • - Performed at MRC Harwell • - Bone & cartilage screen fits within primary screens for other systems • - Development, growth, biochemistry, body composition, whole body X-ray, DEXA – for bone mineral density • Intermediate high resolution secondary screen • - Faxitron – high throughput quantative analysis of bone mineral content • - Micro CT – high throughput quantitative analysis of structure, mineralization and static morphometry • Detailed and bespoke secondary screening • - Back scattered electron scanning electron microscopy (BSE-SEM) • - Quantitative BSE-SEM • - Bone histology & histomorphometry • - Con-focal microscopy • - Mechanical testing • - Nanomechanical (synchroton X-ray scattering) • - Primary bone cell culture • - Longitudinal studies