THE COMPARATIVE CLINICAL SCIENCE FOUNDATION (CCSF)SECOND COLLABORATIVE WORKSHOP

16 MARCH 2011THE WELLCOME TRUST

Refining in vitro Models of Articular Cartilage Inflammation for Comparative Studies on Biomarkers of Osteoarthritis

Ali Mobasheri, D.Phil.

Musculoskeletal Research Group

School of Veterinary Medicine and Science

The Disease: Osteoarthritis (OA)

A progressive joint disease characterised by:– Articular cartilage

degeneration– Synovial inflammation– Subchondral bone sclerosis– Osteophyte formation (bony

outgrowths)

SynoviumSynovial fluid

Cartilage

Subchondral bone

The Disease: Osteoarthritis (OA)• Most common form of arthritis in humans and

animals • Major cause of pain, inflammation and loss of

mobility

Risk Factors for OA

• Age• Lifestyle and

occupation• Joint trauma or

instability• Genetics• Metabolic and

endocrine disease• Obesity

Normal Osteoarthritis

Weakened andfrayed tendons/ligaments, muscles

Episodically inflamedsynovium

Reduced viscosityof synovial fluid

Fibrillated/destroyedcartilage

Bony outgrowths(osteophytes)

Bone sclerosis

Frayed, cracked meniscus

Thickened capsule

Bone

Capsule

Synovial fluid

SynoviumMeniscus

Cartilage

Tendons/ligaments

Muscle

Rationale for Studying OA• OA affects 1 in 6 adults (almost 5 out of 6 professors in

the audience)• Most OA patients suffer from pain and disability• By 2030 20% of Americans and Europeans will have OA• There are no disease modifying treatments for OA• There are no established biomarkers for OA• Existing drugs (NSAIDs) only treat the symptoms of OA

– reducing pain and inflammation• Therefore OA represents a major opportunity for basic

and clinical research, drug discovery and the development of novel disease modifying agents and therapeutic approaches

Source: National Institute of Arthritis and Musculoskeletal and Skin Diseases,

NIAMS/National Institutes of Health, Bethesda, MD

Animal Models of OA

Procedures:Injection into the joint, surgical replication of joint trauma and creation of joint instability(cruciate transection, meniscal transection, carpal fragmentation)

Advantages:Equine and canine OA are good models of cartilage ageing, load-induced OA

Disadvantages:Ethical issues Invasive nature of techniquesAnaesthetics always requiredAnimals always sacrificedModels are expensive and laborious to

establishEndpoint data can be difficult to relate to

humans

Can we use explant and high-density culture models of

equine and canine cartilage to identify new biomarkers of OA that may identify early disease

and predict progression in humans and animals?

The Question:

Ideal for high-throughput screening, drug testing testing and studies on chondrocyte cell biology

In Vitro Models: Monolayer Cultures of Chondrocytes

Tissue is harvested under sterile conditions and pieces (explants) are placed in a cell culture dish containing growth media

• Advantages of the Cartilage Explant Culture Model: – Cells remain in their

original surrounding and the extracellular matrix around them accurately mimics the in vivo environment

– Ideal for studies of extracellular matrix synthesis and degradation

– Suitable for proteomic work and studying anti-inflammatory drugs and nutraceuticals

3-D Cartilage Explant Culture

Chondrocytes isolated by collagenase digestion

Cells counted and seeded into pre-gel mix and alginate beads set by addition of Ca2+

Beads cultured at 37oC, 5%

CO2

in DMEM + 10% FCS

Cells released by chelating Ca2+ with EDTA

Downstreamapplications

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Transmission electron micrograph of chondrocytes in alginate beads after 7 days of culture

In Vitro Models: 3-D Alginate Cultures of Chondrocytes

3-Dimensional High-Density Chondrocyte Pellet Culture Model

Petri Dish

CultureMedium Steel

Bridge

FilterChondrocytes + Growth Factors

Pellets Maintained at Air-Liquid Interface

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TEM - Chondrocytes in High-Density Culture, after 7 days of culture

Current Research: Identifying and Validating New Biomarkers of Cartilage Degradation