McKay Orthopaedic Research Laboratory

Mechanotransduction in Stem Cells for Cartilage

Tissue Engineering

Ryan LiCase Western Reserve University

Advisor: Dr. Robert Mauck

SUNFEST Final Presentation

August 10, 2007

Articular Cartilage Overview

• Biochemical Composition:– Extracellular matrix

– Collagen Type II (10%-30% wet wt)

– Sulfated proteoglycans (3%-10% wet weight)

– Chondrocytes

• Function:– Compressive

Properties

– Tensile Properties

– Fluid Flow

Hyaluronan Aggrecan

Interstitial fluidCollagen fibril

Attached aggrecan

Cartilage Degeneration

ModulusHydraulic PermeabilityModulusHydraulic Permeability

Matrix DeformationFluid PressurizationMatrix DeformationFluid Pressurization

Load-Bearing CapacityLoad-Bearing Capacity

Proteoglycan LossCollagen DamageProteoglycan LossCollagen Damage

Tissue Engineering Paradigm

Cells• Chondrocytes• Mesenchymal

Stem Cells (MSCs)

Scaffolds•Polymer foams•Polymer meshes•Hydrogels

ExtracellularEnvironment•Mechanical Signaling•Chemical growth factors

“a field…that seeks to develop functional cell, tissue, and organ substitutes to repair, replace or enhance biological function...” [NIH]

Mesenchymal Stem Cells

Scaffold Design

Agarosehttp://www.biologie.uni-hamburg.de/b-online

Hyaluronic Acid

Agarose

•Thermo-crosslinkable hydrogelmade of polysaccharides (D- and L- galactose)•Cellular encapsulation•Mechanical Properties

•Not biodegradable•Immunogenic

Hyaluronic Acid (HA)

•Photo-crosslinkablepolysaccharide hydrogel

•Natural extracellular matrix component – biodegradable

•Successful cell encapsulation

Physiologic Loading of Cartilage

3

12

•Compressive Forces

•Sliding Forces

Mechanical conditioning has been shown to improve

chondrocyte mechanical properties

•Compressive Forces

•Sliding Forces

Mechanical conditioning has been shown to improve

chondrocyte mechanical properties

MechanotransductionExtracellular Signal

MAP Kinase activation / Signal transduction

Gene Expression Changes

Matrix Composition Biochemical Change

Mechanical Properties (Equilibrium Modulus, Dynamic Modulus, etc.)

Chondrogenesis in 3-D Culture

*p<0.05 vs. day 0, **p<0.05 (day 70)*p<0.05 vs. day 0, **p<0.05 (day 70)

•MSC-laden constructs increase in mechanical properties with time•Young’s Modulus of MSC-laden constructs << that of chondrocyte-laden constructs

•MSC-laden constructs increase in mechanical properties with time•Young’s Modulus of MSC-laden constructs << that of chondrocyte-laden constructs

Day 0Day 0Day 14Day 14Day 42Day 42Day 70Day 70

002020404060608080

100100120120140140160160

CHONDCHOND MSCMSC

Youn

g’s

Mod

ulus

(kPa

)Yo

ung’

s M

odul

us (k

Pa)

****

********

n=3-6/group

Mauck+ 2006Mauck+ 2006

Target Gene Expression Results

0

2

4

6

8

10

CHOND d14 d28 d42 d56

00.5

11.5

22.5

3

CHOND d14 d28 d42 d56

0.00E+005.00E-061.00E-051.50E-052.00E-052.50E-053.00E-05

CHOND d14 d28 d42 d56

0.00E+00

1.00E-05

2.00E-05

3.00E-05

4.00E-05

CHOND d14 d28 d42 d56

Xylosyltransferase-1(XT-1)

GalNAc4,6S-disulfotransferase

(GalNAc)

Chondroitin-4-sulfotransferase-1

(C4st-1)

Chondroitin-4-sulfotransferase-2

(C4st-2)

Hypothesis and Rationale:

Hypothesis: Compressive loading of MSC-seeded scaffolds leads to upregulation

of matrix biosynthetic genes in the short term and increases in mechanical

properties in the long term.

Rationale:

•Mechanical signals are relevant developmentally

•Changes in gene expression and chondrogenicexpression in chondrocytes after mechanical signaling

•Target genes shown to be mechanically sensitive in chondrocytes

3D Hydrogel Culture3D Hydrogel Culture

2% Agarose (@45oC) or 2% Hyaluronic Acid:20 x 106 cells/ml

2% Agarose (@45oC) or 2% Hyaluronic Acid:20 x 106 cells/ml

Disks: 2.25 X Ø 5.0 mm

Disks: 2.25 X Ø 5.0 mm Chondrogenic Medium

(CM-/CM+) (+ TGF-β3)Chondrogenic Medium

(CM-/CM+) (+ TGF-β3)

Free Swelling CultureFree Swelling Culture

Bovine MSC Harvest and Expansion

Dynamic Compression BioreactorDynamic Compression Bioreactor

Dynamic Compression

Bioreactor (10% deformation,

sinusoidal waveform)

Dynamic Compression

Bioreactor (10% deformation,

sinusoidal waveform)

LoadingPlate

LoadingPlate

Constructsin Petri

Dish

Constructsin Petri

Dish

-160.0

-140.0

-120.0

-100.0

-80.0

-60.0

-40.0

-20.0

0.040.0 41.0 42.0 43.0 44.0 45.0 46.0

-160.0

-140.0

-120.0

-100.0

-80.0

-60.0

-40.0

-20.0

0.040.0 41.0 42.0 43.0 44.0 45.0 46.0

-160.0

-140.0

-120.0

-100.0

-80.0

-60.0

-40.0

-20.0

0.040.0 41.0 42.0 43.0 44.0 45.0 46.0

-160.0

-140.0

-120.0

-100.0

-80.0

-60.0

-40.0

-20.0

0.040.0 41.0 42.0 43.0 44.0 45.0 46.0

(mic

rons

)(m

icro

ns)

(mic

rons

)(m

icro

ns)

(sec)(sec)

(sec)(sec)

1.0 Hz1.0 Hz

3.0 Hz3.0 Hz

Bioreactor Displacement

Bioreactor Displacement

0.33 Hz0.33 Hz

(sec)(sec)

(mic

rons

)(m

icro

ns)

Methods

Stress-Relaxation Displacement Curve

0

50100

150200

250

0 300 600 900 1200Time (sec)

Disp

lace

men

t (m

icro

ns)

Stress-Relaxation Force Response

0102030405060

0 300 600 900 1200Time (sec)

Forc

e (g

ram

s)

•Mechanical Properties – Equilibrium Modulus

•Creep testing•Stress relaxation

•Biochemistry:•GAG content•Collagen Content•DNA content

•Gene Expression –•TRIZOL extraction•cDNA synthesis•RT-PCR

•Histology

RX2: Encapsulation of MSCs in HyaluronicAcid Scaffold Promotes Cell Growth and

Chondrogenesis

05

101520253035404550

CM CM+ CM- CM+ CM-

d0 d7 d21

Agarose

Hyaluronic Acid

00.20.40.60.8

11.21.41.61.8

HA Agarose

% W

et W

eigh

t

CM-CM+

MTT Assay3-(4, 5-Dimethylthiazol-2-yl)-2, 5– diphenyltetrazolium bromide) –

GAG Quantification on day 21

RX3, RX5: Short Term Gene Expression in MSC-laden HA Constructs

Dynamic Loading @ 1 Hz(CM+/CM-)

3 hours

3 hours

6 hours

24 hours

Free Swelling Culture (CM+/CM-)

TRIZOL extraction, cDNAsynthesis,

RT-PCR on all samples

RX3, RX5: Matrix Biosynthesis Genes are Upregulated in Response to Short-Term

LoadingFree Swelling Constructs

05

10152025

3035

0 hr 3 hr 6 hr 24 hr

CM-

CM+

1 Hz Dynamic Loading

05

101520253035

0 hr 3 hr 6 hr 24 hr

CM-

CM+

Free Swelling Constructs

01

2345

67

0 hr 3 hr 6 hr 24 hr

CM-CM+

1 Hz Dynamic Loading

01

23

45

67

0 hr 3 hr 6 hr 24 hr

CM-CM+

Mechanical upregulationof GalNAcis enhanced

in absence of TGF-b3

C4st-1 upregulation is

enhanced in presence of TGF-

b3

RX3, RX5: C4st-2, XT-1 are Upregulated in Response to Short-Term Loading

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 hr FS 0 hr DL 24 hr FS 24 hr DL

CM-

CM+

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 hr FS 0 hr DL 24 hr FS 24 hr DL

CM-

CM+ XT-1C4st-2

Question: Does increased gene expression translate to long-term

improvements in mechanical properties?

RX1: Long Term Mechanical Loading

Free Swelling Culture (CM+) Free Swelling Culture (CM+/CM-)

Dynamic Loading (3 hrs/day)

3 Hz

1 Hz

0.33 Hz

0 days 21 days 42 days

Agarose and HA Hydrogels

Dynamic Loading Causes Decrease in Equilibrium Moduli in Agarose and HA

Constructs

Equilibrium Moduli - Agarose

020

406080

100120

FS 3 Hz 1 Hz 0.33 Hz

d0 d21 d42

kPa

CM-

CM+

Equilibrium Moduli - Hyaluronic Acid

010

203040

5060

d0 d21 FS 3 Hz 1 Hz 0.33 Hz

d42

kPa

CM-

CM+

Matrix Biosynthesis Genes are Upregulatedin Response to Long Term Loading

Agarose Long Term

0

0.5

1

1.5

2

2.5

3

3.5

d0 d21 d42 FS d42 1 Hz

CM-

CM+

HA Long Term

0

0.5

1

1.5

2

2.5

3

3.5

d0 d21 d42 FS d42 DL

CM-

CM+

Agarose Long Term

0

0.2

0.4

0.6

0.8

1

1.2

1.4

d0 d21 d42 FS d42 1 Hz

CM-

CM+

HA Long Term

00.20.40.60.8

11.21.4

d0 d21 d42 FS d42 DL

CM-

CM+

C4st-1

C4st-2

Matrix Biosynthesis Genes are Upregulatedin Response to Long Term Loading

Agarose Long Term

0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

d0 d21 d42 FS d42 1 Hz

CM-

CM+

HA Long Term

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

d0 d21 d42 FS d42 DL

CM-

CM+

Agarose Long Term

00.10.20.30.40.50.60.70.80.9

1

d0 d21 d42 FS d42 1 Hz

CM-

CM+

HA Long Term

00.10.20.30.40.50.60.70.80.9

1

d0 d21 d42 FS d42 DL

CM-

CM+

GalNAc

XT-1

Discussion

• Hyaluronic Acid is a viable alternative scaffold promoting cell proliferation and chondrogenesis

• Matrix biosynthetic genes C4st-1, 2, XT-1, and GalNAc are mechanically sensitive in MSCs

• Dynamic loading causes upregulation of biosynthetic genes

• Increases in gene expression levels do not translate to mechanical and biochemical improvements in the long term

Thank YouDr. Robert Mauck

Alice HuangIsaac Erickson

Dr. Meira Yeger-McKeeverBrendon Baker

Nandan NerurkarBurdick Lab

Ashwin NathanAshley Stein

NSFSUNFEST

McKay Orthopaedic Research Laboratory

Dynamic Loading Decreases GAG Biosynthesis in Agarose Constructs

Agarose GAG Content

0

0.5

1

1.5

2

2.5

3

3.5

FS 3 Hz 1 Hz 0.33 Hz

d0 d21 d42

% W

et W

t

CM-

CM+

HA Hydrogels Show Negligible Changes in Biochemical Composition in Response to

Dynamic Loading

Hyaluronic Acid GAG Content

0

0.5

1

1.5

2

2.5

3

3.5

d0 d21 FS 3 Hz 1 Hz 0.33 Hz

d42

% W

et W

t CM-

CM+