Mechanotransduction in Stem Cells for Cartilage Tissue ... · Physiologic Loading of Cartilage 3 1...
Transcript of Mechanotransduction in Stem Cells for Cartilage Tissue ... · Physiologic Loading of Cartilage 3 1...
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+