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MRC Mouse Network Models for Disorders of Bone and Cartilage Presentation on behalf of Bone and Cartilage Consortium: Professor R V Thakker May Professor of Medicine University of Oxford First meeting of MRC Mouse Network, Harwell, UK 13 th January 2012. Aims.

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Aims

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


Aims

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)


Why is this important

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


Aims

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


Bone phenotyping

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


Aims

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

From Williams & Bassett


Bone phenotyping1

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


Lpk spondyloepiphyseal dysplasia congenita

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


Bone phenotyping2

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


Faxitron deiodinase mutants have increased bone mineral

Faxitron: deiodinase mutants have increased bone mineral

Bassett et al, 2010, PNAS


Bone phenotyping3

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


Aims

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


Bone phenotyping4

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


Aims

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


Aims

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


Summary phenotyping by bone cartilage consortium

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


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