Massachusetts Institute of Technology
Technolog
Harvard Medical School
Schoo
Brigham and Women’s Hospital
VA Boston Healthcare System
2.79J/3.96J/20.441/HST522J
BIOMATERIALS--TISSUE INTERACTIONS:
BIOMATERIALS
“T l ” ffor U
“Tools
Understanding
d
di the
h M
Molecular,
l l C
Cellular,
ll l
and Physiological Bases of the Tissue Response to
Implants
p
M. Spector
Spector,, Ph.D.
BIOMATERIALS-TISSUE
INTERACTIONS
Tissue* + Biomaterial**
Cell
+ Matrix**
* Structure comprising cells of the same type
** Solid surface
CELL-MATRIX INTERACTIONS
In Tissue
Tissu
Cell + Extracellular Matrix
In Tissue Engineering Scaffolds
Cell + Biomaterial Scaffold
CONCEPTS FOR UNDERSTANDING
BIOMATERIALS-TISSUE INTERACTIONS
•
•
•
•
•
Control Volume
Unit Cell Processes
Types of Tissues
Tissue
Tissue Formation and Remodeling In Vitro
W
Wound
dH
Healing
li In
I Vi
Vivo
Articular Cartilage
Extracellular
Extracellular
Matrix
Cell
Figures by MIT OpenCourseWare.
4 mm
10 μm
Chondrocytes (P2 Canine) in a
Type I Collagen-GAG Scaffold
40min
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
2
1
3
“Control Volume”
4
B. Kinner
5
Zaleskas, J. M., et al. "Contractile forces generated by
articular chondrocytes in collagen-glycosaminoglycan
matrices." Biomaterials 25 no. 7-8 (2004):1299-1308.
6
UNIT CELL PROCESSES
Concept of a “Control Volume” around a Cell
Soluble
Regulator
A
Cell
Matrix
Cell + Matrix
Product + Soluble
(Regulator)
Regulator B
(Regulator)
Product
“Control Volume”
UNIT CELL PROCESSES
Concept of a “Control Volume” around a Cell
Soluble
Regulator
Autocrine factor
Cell
Matrix
Cell +
+ Matrix
Product + Soluble
(Regulator)
(Regulator)
Regulator
Product
“Control Volume”
Another Cell; Circulation;
Paracrine
Endocrine
UNIT CELL PROCESSES
Concept of a “Control Volume” around a Cell
Soluble
Mechanical
Regulator
Loading (Strain); Subject 2.785
A
Cell
Cell ++ Matrix
Matrix
Product + Soluble
(Regulator)
Regulator B
(Regulator)
Product
“Control Volume”
CONCEPTS FOR UNDERSTANDING
BIOMATERIALS-TISSUE INTERACTIONS
•
•
•
•
•
Control Volume
Unit Cell Processes
Types of Tissues
Tissue
Tissue Formation and Remodeling In Vitro
W
Wound
dH
Healing
li In
I Vi
Vivo
UNIT CELL PROCESSES
•
•
•
•
•
•
Mitosis
Migration
Synthesis
C
Contraction
i
Endocytosis
Exocytosis
UNIT CELL PROCESSES
•
•
•
•
•
•
•
•
Mitosis
Migration
Synthesis
C
Contraction
i
Endocytosis
Exocytosis
?
?
UNIT CELL PROCESSES
•
•
•
•
•
•
•
•
Mitosis
Migration
Synthesis
C
Contraction
i
Endocytosis
Exocytosis
Apoptosis
Differentiation
COLLAGEN-GAG MATRICES: MODEL BIOMATERIALS
(ANALOGS OF EXTRACELLULAR MATRIX)
Investigation of cell interactions (UCPs) in vitro
• Type I (bovine and porcine)
• Type
T
II ((porcine)
i )
• Chondroitin 6-sulfate
1mm
• Freeze-dried
• Dehydrothermally cross
cross-linked
linked
• Additional cross-linking
See IV Yannas, et al. PNAS, 1989
500μm
–
CELL –MATRIX
INTERACTIONS WITH
COLLAGEN-GAG MATRICES IN VITRO
• Can provide insights into interrelationships
among cell processes.
– How do mitosis and synthesis interrelate?
– How do mitosis and synthesis relate to
contraction?
– How does migration relate to contraction?
• Can provide insights into cell behavior in vivo.
• Can provide insights into scaffold composition
and structure for improved
p
p
performance in
regenerative medicine.
Chondrocytes (Passage 2 Canine) in a
Type I Collagen-GAG
Collagen GAG Matrix
Live cell imaging
for a period of 5
hours.
J. Cheng
CELL –MATRIX INTERACTIONS
•
•
•
•
Mitosis
Migration
S th i
Synthesis
Contraction
Chondrocyte (P2 Canine) in a Type I Collagen-GAG Matrix:
Mitosis
Photo removed due to
copyright restrictions.
J. Cheng
Effects of Cross-Linking on Chondrocyte Proliferation
DNA Content
in Collagen-GAG
Matrices
60
Mean ± SEM
DN
NA (ug
g)
50
DHT
UV
GTA
EDAC
40
30
20
Inc. cross
cross-linking
10
0
00
10
10
Day
20
20
30
30
CR Lee, et al., Biomat. 2001;22:3145
CELL ±MATRIX INTERACTIONS
‡
‡
‡
‡
Mitosis
Migration
Synthesis
Contraction
Fibroblasts Migrate Away from Soft Substrates
NIH 3T3 cells are plated on polyacrylamide substrates with a transition in flexibility. The
soft side is marked with fluorescent beads (to the left). Cells turn to avoid the soft
substrate as they approach the boundary from the stiff side, by retracting the leading
lamellipodium that crossed the boundary.
Courtesy of Yu-Li Wang. Used with permission.
C.-M. Lo, et al., Biophys. J. 79:144 (2000)
Fibroblasts Migrate Toward Stiff Substrates
NIH 3T3 cells are plated on polyacrylamide substrates with a transition in flexibility. The
soft side is marked with fluorescent beads (to the left). Cells turn toward and enter the
stiff side as they approach the boundary from the soft side, by expanding protrusions
toward the boundary into a leading lamellipodium.
Courtesy of Yu-Li Wang. Used with permission.
C.-M. Lo, et al., Biophys. J. 79:144 (2000)
Fibroblasts Migrate Toward Stretching Forces
NIH 3T3 cells are plated on polyacrylamide substrates. Pulling forces are exerted by
inserting a blunted needle in the substrate near the trailing end of the cell and dragging
the needle away from the cell. Cells switch the direction of migration by expanding
secondary protrusions toward the needle into a leading lamellipodium.
Courtesy of Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
C.-M. Lo, et al., Biophys. J. 79:144 (2000)
Fibroblasts Migrate Away from Compressing Forces
NIH 3T3 cells are plated on polyacrylamide substrates. Pushing forces are exerted by
inserting a blunted needle in the substrate near the leading edge of an approaching cell
and moving the needle toward the cell. Cells switch the direction of migration by
retracting the leading lamellipodium.
Courtesy of Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
C.-M. Lo, et al., Biophys. J. 79:144 (2000)
Migration of Epithelial Cells In Vitro in a Wound Healing Assay
100 μm
© 2001 L. C. Santy and J. E. Casanova. License CC BY-NC-SA.
Published by The Rockefeller
Published
Rockefeller University
University Press.
Press
http://dx.doi.org/10.1083/jcb.200104019
Monolayers were ““wounded” by scraping.
LC Santy, J. Cell Biol. 154:599 (2001)
Chondrocytes (P2 Canine) in a Type I CollagenGAG Matrix: Migration and Contraction
40min
J. Zaleskas, et al.,
Biomat. 25:1299 (2004)
2
1
3
4
5
B. Kinner
6
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
Diagram removed due to copyright restrictions.
Fig. 2 in
Fi
i MMad
dri,
i Kid
KidneyIInt. 41 (1992):
(1992) 562.
562
Schematic of the modulation of microvascular
endothelial cell phenotype during angiogenesis.
Madri, Kidney Int. 41:562 (1992)
CELL –MATRIX INTERACTIONS
•
•
•
•
Mitosis
Migration
Synthesis
Contraction
nmol/h
hour/μ
hour/
μg DNA
Effects of Cross-Linking on Chondrocyte Biosynthesis
in Collagen-GAG- Matrices
01
0.15
Protein Synthesis;
p
Proline Incorporation
DHT
UV
GTA
EDC
0.1
0.03
0.02
0.05
0.01
0
0
2
7
15
Day
29
Proteoglycan Synthesis;
Sulfate Incorporation
2
7
15
29
Day
CR Lee, et al., Biomat. 2001;22:3145
CELL –MATRIX INTERACTIONS
•
•
•
•
Mitosis
Migration
Synthesis
Contraction
α-smooth muscle actin-fusion peptide (SMA-FP) inhibits the tension exerted by lung
fibroblasts on silicone substrates. After washing our of the FP, cells contract again.
See video at
http://jcb.rupress.org/content/suppl/2002/05/03/jcb.200201049.DC1/1.html
Hinz B, et al., J Cell Biol 157:657 (2002)
Chondrocytes (P2 Canine) in a Type I
Collagen-GAG
g
Matrix: Contraction
J. Zaleskas, et al.,
Biomat. 25:1299 (2004)
40 min
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
B Kinner
40 min
40 min
300 min
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
J. Zaleskas, et al.,
Biomat. 25:1299 (2004)
Non-Seeded:
8 days
Non-Seeded:
8 days
Cell-Seeded:
- 88 days
Cell-Seeded:
days
10mm
10
10mm
Non-Seeded and Cell-Seeded
Collagen-GAG Scaffolds
S. Vickers
Courtesy of Scott Vickers. Used with permission.
21 days
Adult
canine
articular
chondrocytes
(passage
3)3)
contract
a type
I I
Adult
canine
articular
chondrocytes
(passage
contract
a type
collagen-GAG
matrix,
reflected
inContraction
thethe
decrease
inin
diameter
collagen-GAG
g Cell
matrix,
, reflected
in
decrease
diameter
Cell-Mediated
Mediated
%O
Origina
al Diam
meter
100
Mean ± SEM
Highly x-link.,
high modulus
90
80
DHT
UV
GTA
EDAC
70
60
50
40
Little x-link.,
low modulus
30
0
10
Day
20
30
CR Lee, et al., Biomat. 2001;22:3145
Human Articular Chondrocytes in Monolayer Culture
IH - Green: α-smooth muscle actin; Orange: type II collagen
Chondrocytes
express the gene
for α-smooth
muscle actin and
this enables them
to contract
Courtesy of John Wiley and Sons, Inc. Used with permission.
Source: Kinner, B., and M. Spector. J Orthop Res 19 (2001): 233-241.
B. Kinner, et al. JOR 2001;19:233
α-Smooth Muscle Actin Immunohistochemistry
of Human Articular Cartilage
Neg. control
Courtesy of John Wiley and Sons, Inc. Used with permission. Fig 1a and b in Kim, A. C.,
and M. Spector. "Distribution of Chondrocytes Containing Alpha-smooth Muscle Actin in
Human Articular Cartilage." J Orthop Res 18 (2000): 749.
Kim and Spector, JOR 2000;18:749
MUSCULOSKELETAL CELLS THAT CAN EXPRESS
a
a-SMOOTH MUSCLE ACTIN AND CAN CONTRACT
Articular chondrocyte
Osteoblast
Meniscus fibroblast and fibrochondrocyte
Intervertebral disc fibroblast and
fibrochondrocyte
• Ligament fibroblast
• Tendon fibroblast
• Synovial cell
M. Spector,
Mesenchymal
• esenchymal
stem
cellcell
Mesenchymal
stem
Wound Repair Regen.
•
•
•
•
9:119:11-18 (2001)
POSSIBLE ROLES FOR a-SMOOTH MUSCLE
ACTIN-ENABLED CONTRACTION
Musculoskeletal Connective Tissue Cells
• Tissue engineering Contracture of scaffolds
• Healing
Closure of wounds
((skin wounds and bone fractures))
• Disease processes Contracture (Dupuytren’s)
• Tissue formation Modeling of ECM architecture
and remodeling
(e.g., crimp in ligament/tendon?)
CONCEPTS FOR UNDERSTANDING
BIOMATERIALS-TISSUE INTERACTIONS
•
•
•
•
•
Control Volume
Unit Cell Processes
Types of Tissues
Tissue
Tissue Formation and Remodeling In Vitro
W
Wound
dH
Healing
li In
I Vi
Vivo
TYPES OF TISSUES
Which Tissues Can Regenerate Spontaneously?
Yes
No
Connective Tissues
• Bone
• Articular Cartilage,
Ligament, Intervertebral
Disc, Others
Epithelia (e.g., epidermis)
√
√
√
Muscle
√
• Cardiac, Skeletal
• Smooth
Nerve
√
√
BIOMATERIALS-TISSUE
INTERACTIONS
C ll + Matrix
Cell
M t i
Connective
Tissue
Epithelia
M l
Muscle
Nerve
UNIT CELL PROCESSES
Concept of a “Control Volume” around a Cell
Soluble
Mechanical
Regulator
Loading (Strain)
A
Cell
Cell +
+ Matrix
Matrix
(Regulator)
(Regulator)
Product + Soluble
Product
Regulator B
“Control Volume”
BIOMATERIALS-TISSUE
INTERACTIONS
Cell + Matrix
Connective
Tissue
Epithelia
Muscle
N
Nerve
Adhesion
Protein
Collagen
Biomaterial
BIOMATERIALS-TISSUE
INTERACTIONS
Cell + Matrix
Connective
Tissue
Epithelia
Muscle
Nerve
Adhesion
Protein
Collagen
Biomaterial
Integrin
Chinese hamster ovary cell migration in a wound-healing assay
CHO cells
express the
α5β1 but not
α4β1 integrin
C. Watters,, Cell Biol. Ed.
2:210 (2003)
K.A. Pinco, Mol. Biol.
Cell 13:3203 (2002)
Photos removed due to copyright restrictions.
Figure 3 in Watters, C. Cell Biol. Ed. 2:210 (2003)
Image and links to associated videos at
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC256980/figure/F3/
Cells transfected with plasmid DNAs for α4 and α4/GFP
Migration of fibroblast-like fibrosarcoma cells in a
3-D collagen lattice
K. Wolf, et al., J. Cell
Biol. 160:267 (2003)
Reduction of migration speed and
induction of detached, nonmobile
spherical morphology by adhesion
perturbing
t bi anti–α1
ti 1 iintegrin
t i
antibody, as a consequence of
impaired collagen fibril binding.
“UNIT CELL PROCESSES”
Cell + Matrix
Connective
Tissue
Epithelia
Muscle
Nerve
UCP
Mitosis
S nthesis
Synthesis
Migration
Contraction
Endocytosis
Exocytosis
“UNIT CELL PROCESSES”
Cell + Matrix
Connective
Tissue
Epithelia
Muscle
N
Nerve
UCP
Mitosis
Synthesis
y
Product
Cell proliferation
Matrix molecules,,
enzymes, cytokines
Migration Translocation
Contraction Strain
Endocytosis Solubilized
fragments
Exocytosis Regulators
“UNIT
CELL
PROCESSES”
“
C
OC SS S”
Regulator
UCP
Cell + Matrix
Connective
Tissue
Epithelia
Muscle
N
Nerve
Product + Regulator
Mitosis
Synthesis
Migration
Contraction
Endocytosis
Exocytosis
Cytokines
(Growth Factors)
REGULATORS
• Cytokines/Growth Factors
– http://themedicalbiochemistrypage.org/growth
http://themedicalbiochemistrypage.org/growth-factors.html
– (previously:
http://web.indstate.edu/thcme/mwking/growth-http://web.indstate.edu/thcme/mwking/growth
factors.html
• http://www.copewithcytokines.de/
“UNIT
CELL
PROCESSES”
“
C
OC SS S”
Regulator Mechanical Force (Strain)
UCP
Ce + Matrix
Cell
at
Product
oduct + R eegulator
gu ato
Connective Adhesion Mitosis
Tissue
ssue
Protein Synthesis
Epithelia
Collagen Migration
Muscle
Biomaterial Contraction
Endocytosis
Nerve
Integrin
Exocytosis
Cytokines
(Growth Factors)
“UNIT
CELL
PROCESSES”
“
C
OC SS S”
Regulator Mechanical Force (Strain)
UCP
Ce + Matrix
Cell
at
Product
oduct + R eegulator
gu ato
Connective Adhesion Mitosis
Tissue
ssue
Protein Synthesis
Epithelia
Collagen Migration
Muscle
Biomaterial Contraction
Endocytosis
Nerve
Integrin
Exocytosis
Cytokines
(Growth Factors)
“UNIT
CELL
PROCESSES”
“
C
OC SS S”
Regulator (TGF
(TGF--β1)
UCP
Cell + Matrix
Product + Regulator
Matrix strain Cytokines
Mitosis
Connective Adhesion
(contracture/ (Growth Factors)
Synthesis
Tissue
Protein
shrinkage)
g )
Migration
E ith li
Epithelia
Collagen
Muscle
Biomaterial Contraction
Endocytosis
Nerve
Exocytosis
Integrin
“UNIT
CELL
PROCESSES”
“
C
OC SS S”
TGFTGF
G -β1
Contraction
Fibroblast + Collagen
Contracture + Reg.
CONCEPTS FOR UNDERSTANDING
BIOMATERIALS - TISSUE INTERACTIONS
•
•
•
•
•
Control Volume
Unit Cell Processes
Types of Tissues
Tissue
Tissue Formation and Remodeling In Vitro
W
Wound
dH
Healing
li In
I Vi
Vivo
+FGF-2
TISSUE FORMATION
AND REMODELING
IN VITRO
+FGF-2
Canine chondrocytes grown in
a type II collagen-GAG
scaffold for 2 weeks.
(Safranin O stain for GAGs)
N. Veilleux, et al., Osteoart.
& Cart. 2005;13:278
Courtesy of Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
CONCEPTS FOR UNDERSTANDING
BIOMATERIALS-TISSUE INTERACTIONS
•
•
•
•
•
Control Volume
Unit Cell Processes
Types of Tissues
Tissue
Tissue Formation and Remodeling In Vitro
W
Wound
dH
Healing
li In
I Vi
Vivo
WOUND HEALING
Roots of Tissue Engineering
Injury
Inflammation
(Vascularized tissue)
4 Tissue Categories
Connective Tissue
Epithelium
Nerve
Muscle
R
Reparative
ti
Process
Regeneration*
CT: bone
Ep: eepidermis
pidermis
Muscle: smooth
*spontaneous
Repair (Scar)
CT: cartilage
Nerve
Muscle: cardiac, skel.
RESPONSE TO IMPLANTS:
WOUND HEALING
Surgical Implantation
Acute
Inflammation
Vascular Response
Clotting
Phagocytosis
Neovascularization
New Collagen Synthesis
Tissue of Labile and Stable Cells
Tissue of Permanent Cells
Implant Movement
Framework
F
k Framework
F
k
Intact
Destroyed
Regen.
Scarring
(incorp. (fibrous encapsulation;
of implant) synovium)
Chronic Inflammation
Granulation
Tissue
Scarring
S
i
(fibrous encapsulation;
synovium)
Chronic Inflammation
MIT OpenCourseWare
http://ocw.mit.edu
20.441J / 2.79J / 3.96J / HST.522J Biomaterials-Tissue Interactions
Fall 2009
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