dr. a. mobasheri nc3 rs and bbsrc symposium 1 2 april 2009 final version

School of Veterinary Medicine and Science

A Tissue Engineered Model of Osteoarthritis

Challenges and Opportunities for Applying the 3Rs in Osteoarthritis Research

Ali Mobasheri

Tissue EngineeringSymposium 1-2 April 2009


Articular Cartilage

• Mechanically unique connective tissue designed to:– withstand and

distribute load– act as an elastic shock

absorber– provide a wear

resistant surface to articulating joints


• Avascular, aneural and alymphatic• Contains a single cell type: the chondrocyte• Derived from mesenchymal progenitor cells

Articular Cartilage


The Chondrocyte

Nucleus

Cytoplasm

ECM

Synthesizes a mechanically resilient extracellular matrix of collagens and aggregating proteoglycans


Major Constituents of Articular Cartilage Matrix

Collagen IX

Collagen II

Fibronectin

COMP

Aggrecan

Hyaluronan

Chondrocyte

Thrombospondin

Decorin

BiglycanFibromodulin


Articular Cartilage Degradation

• Despite its durability, cartilage has a very limited self maintaining capability

• Cartilage is vulnerable to mechanical injury and prone to structural damage and degradation


Osteoarthritis (OA)• Most common form of arthritis

characterised by progressive deterioration and loss of articular cartilage

• Affects load-bearing synovial joints causing pain, inflammation and loss of mobility

• Associated with ageing and predicted to increase as the ageing population grows


Risk Factors for OA

• Age• Lifestyle/occupation• Joint trauma• Joint instability• Genetics• Metabolic/endocrine disease• Obesity


General Features of OA

Synovial space (as assessed by radiography)

Cartilage anabolism and impaired cartilage repair

Synovial inflammation and hyperplasia

Proteolytic activity (collagenases, gelatinases)

Cartilage degeneration


Cartilage Fibrillation and Loss of Proteoglycans in OA

Human femoral head

H&E

Safranin O

Safranin-O stains glycosaminoglycans red


Molecular Changes in OA

SYNOVIAL MEMBRANE

SYNOVIALFLUID

ARTICULAR CARTILAGE

SUBCHONDRAL BONE

chondrocyte

IL-1β TNF-α

Pro-MMP ROS

IL-1β TNF-α

PGE2

IL-1β TNF-α

Hyaluronate degradation

Pro-MMP

MMP

ECM DEGRADATION

Plasmin Plasminogen

Serine proteases

Cysteine proteases

↓ ECM synthesis

Substance P

Pain

TRAUMA + INFLAMMATION

TRAUMA

Apoptosis

MMP

Adapted from Goodrich (2006) TRAUMA


Rationale for Studying OA

• OA affects 1 in 6 adults• 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• 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 Osteoarthritis

There are many animal models of osteoarthritis


Animal Models of OA

INJECTION INTO THE JOINT

Papain Injection Monoiodoacetate Injection

Surgical Lesion Model Collagenase InjectionLPS Injection

Other related models: adjuvant injection for modelling pannus formation and joint inflammation in rheumatoid arthritis


ACTMedial Meniscectomy

Anterior Cruciate

Transection (ACT)

Meniscal Transection

SURGICAL CREATION OF

JOINT INSTABILITY

Ligament Transection

Carpal Chip Fragmentation

ACT

Canine Groove Model

Animal Models of OA


SURGICAL REPLICATION

OF JOINT TRAUMA

Ligament Transection

Carpal Chip Fragmentation

ACT

Canine Groove Model

Animal Models of OA

Ovine Groove Model

Surgical Lesion Model


Animal Models of OA

Disadvantages:• Ethical issues• Invasive nature of techniques• Anaesthetics always required• Animals always sacrificed• Models are expensive and laborious to

establish• Endpoint data frequently difficult to relate

to humans


Alternative Models

• Researchers must constantly evaluate the relevance of their animal models to OA in humans– Do animal models accurately represent the clinical

disease of OA in humans?– Can animal models of OA be replaced with

alternatives?– Would these alternatives be suitable for basic

research, drug discovery and safety testing?– Can tissue engineering offer viable alternative

models to study OA?


Important criteria for cartilage models for drug discovery

• Ability to monitor synthesis and release of inflammatory mediators– PGE2, NO, pro-inflammatory cytokines

• Markers of cartilage catabolism– Loss of proteoglycans and GAGs– Collagen matrix degradation (neo-epitopes)

• Markers of cartilage anabolism– De novo synthesis of proteoglycans– Synthesis of collagens (i.e. type II collagen)


In vitro ModelsChondrocyte Monolayers

Curcumin 6.25μM Curcumin 12.5μM

Curcumin 25μM Curcumin 50μM

Ideal forToxicity testing


Chondrocytes isolated by overnight collagenase digestion

Cells counted and seeded into pre-gel mix

Alginate beads set by addition of Ca2+

Cultured at 370C, 5% CO2

in DMEM + 10% FCS

Cells released by chelating calcium with EDTA

Downstream applications

In vitro ModelsCulture of chondrocytes in alginate


In vitro ModelsCulture of chondrocytes in alginate

• Advantages: – The preservation of the chondrocyte

phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro


In vitro ModelsThe Explant Model

• Advantages: – Ideal for studies of extracellular

matrix synthesis and degradation

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


The Promise of Stem Cells and Tissue Engineering

• Stem cells• Biomaterials• Bioreactors

– Induction media– Growth factors

• New tissues

• Transplantation


Stromal Cells from Bone Marrow(Mesenchymal Stem Cells)

Mesenchymal Stem Cells from Bone Marrow

In addition to red blood cells, white blood cells and platelets bone marrow contains stromal cells which are also known as mesenchymal stem cells


Mesenchymal Stem Cells

Cultured MesenchymalStem Cells

Fully Differentiated Connective Tissue Cells for Tissue Engineering and

Autologous Transplantation

Myocytes

Cardiomyopathies

Tenocytes

Tendonitis

Osteoblasts

Bone diseases

Chondrocytes

Osteoarthritis


Osteoblasts(Von Kossa, Calcium)

Adipocytes(Oil red, Fat vacuoles)

Chondrocytes(Alcian blue, CSPG)

MSCs

Multipotency of MSCs


Petri dish

CultureMedium

Steel bridge

FilterCells (Chondrocytes + MSCs)

+ Growth Factors

In vitro ModelsHigh-Density Co-Culture System


High Density Co-Cultures

I II III

MSC Chondrocyte

Merge


Cartilage Formationin 3-Dimensional Co-cultures

M

M

C

C

C


PerichondriumCartilage Matrix

EM Evidence for Chondrogenesis

Chondrocyte


Electron Micrographs of High Density Co-cultures

I II


Chondrocyte

Evidence for Collagen Type II

IEM:


Evidence for Cartilage Specific Proteoglycans (CSPGs)

Chondrocyte

IEM


100% MSCs+GF 100% Chondrocytes

50%/50%MSC/Chondrocytes

100% MSCs-GF

200 kDa

100 kDa

60 kDa

Western Blotting:Collagen Type II


200 kDa150 kDa

60 kDa

100% MSCs-GF 100% MSCs+GF 50%/50%MSCs/Chondrocytes

100% Chondrocytes

Western Blotting:CSPGs


Evidence for cell-cell contacts


Conclusions

• There are dynamic interactions between primary chondrocytes and MSCs

• Chondrocytes and MSCs actively interact and communicate in culture

• The interactions are important for the chondrogenic differentiation of MSCs

• This approach may find future applications in cartilage tissue engineering and regenerative medicine


Prospects

• Tissue engineering cartilage using 3-dimensional cultures of chondrocytes and mesenchymal stem cells provides a realistic alternative to using animals

• Other benefits:– Innovative and relatively inexpensive– Uses bone marrow aspirates from fewer animals– Endpoint data may be relevant to the species

used and may be applied to other species (comparative approach)


Acknowledgements

Professor M. ShakibaeiMunich

Dr. Pat Harris (WALTHAM)Dr. David Allaway (WALTHAM)

Collaborators:

Dr. Stephen RichardsonUniversity of Manchester

Prof. Judith HoylandUniversity of Manchester


Funding:

Acknowledgements


Thank you for your attention

dr. a. mobasheri nc3 rs and bbsrc symposium 1 2 april 2009 final version

Documents

mesenchymal

stem cells

articular

cartilage

western blotting

drug discovery

animal models

high density