Massach
usetts
Massachu
setts Ins
Institute of Technolo
Technology
Harva
Harvarrd Me
Medical
dical School
hool
Brig
ham
m an
Brigha
and Women
men’s Ho
Hospital
VA Bo
thca
are Sy
Boston Heal
Healthc
System
2.782J/3.961J/BEH.451J/HST524J
TISSUE ENGINEERING
III. Growth Factors and Genes
M. Spector, Ph.D.
DIFFUSIBLE REGULATORS OF CELL
FUNCTION
Cytokines are polypeptides (proteins) that regulate
many cell functions. They act on a target cell by
binding to specific high-affinity receptors.
Cytokines that act on the same cell that
produced them are called autocrine factors;
those that act on other cells are called paracrine
paracrin
factors; those that act systemically (through the
vascular system) are referred to as endocrine
crin
factors. Molecules that switch on (i.e., regulate)
mitosis are referred to as growth factors.
Page 1
DIFFUSIBLE REGULATORS OF CELL
FUNCTION
Eicosanoids are chemically related signaling lipid
molecules made primarily from arachidonic acid
(fatty acid). Eicosanoids include prostaglandins,
leukotrienes, thromboxanes, and lipoxins.
Prostaglandins are continuously synthesized in
membranes from precursors (20-carbon fatty acid
chains that contain at least 3 double bonds, e.g.,
arachidonic acid) cleaved from membrane
phospholipids by phospholipases, membrane-bound
enzymes. They are continuously released by the cell,
and are degraded by enzymes in the extracellular
fluids. The subscript of PGE2 refers to the 2 double
bonds outside the ring structure.
DIFFUSIBLE REGULATORS OF CELL
FUNCTION
Cytoki
Cytokines
Interleukins
ILIL-1
ILIL-6
Tumor Necrosis Factor
Factor (TNF)
Platelet Derived Growth
Growth Factor (PDGF)
(PDGF)
InsulinInsulin-like Growth
Growth Factor (IGF)
(IGF)
IGF1
and
IGF2
IGF
IGF
Fibroblast Growth
Growth Factor (FGF)
(FGF)
basic FGF (FGF(FGF-2)
Transforming Growth
Growth Factor (TGF)
TGFTGF-β
Page 2
BONE MORPHOGENETIC PROTEINS
• Induces bone formation in nonosseous tissue
• Sources: bone matrix, tooth matrix,
osteosarcoma, epithelia
• Heterodimers of BMP more active than
homodimers (BMP-2/7 is 20 times more active
than BMP-2)
BMP / TGF ACTIVITY
Osteogenin (BMP-3)
Chemotaxis/
Migration
MONOCYTE
IL-1
PDGF
FGF
TGF-β1
MESENCHYMAL
CELL
Differentiation
ENDOTHELIAL CELL
Migration/
Proliferation of
Blood Vessels
CHONDROBLAST
Page 3
OSTEOBLAST
4x
Photos removed due to copyright restrictions.
Photos removed due to copyright restrictions.
Page 4
GENE-SUPPLEMENTED
COLLAGEN-GAG MATRICES
• Bolus delivery of growth factors
factors to a defect does not allow
for a prol
l
onged
effect.
pro
• Transfer of the gene for
for a selected cytokine to the cells
involved in the reparative process may maintain
therapeutic leve
levels through
through the later phases of the repai
repair
process.
• Prolonged release is necessary when
– target cells do not appear
appear at the implant site until days
or weeks
weeks postoperative
– there is a premat
premature loss
loss of expression
expression in transfected
transfected
cells
– transfected cells migrate away
away from the defect site.
P2 Canine Chondrocyte-Seeded Type II Collagen (CD x-linked), 4w
* Cells in the collagen scaffold
transfected with the gene for
IGF-I using Geneporter
+IGF-I gene*
Samuel RE, Delivery of plasmid DNA to
articular chondrocytes via novel
collagen-glycosaminoglycan matrices.
Human Gene Therapy 2002;13:791-802
Courtesy of Mary Ann Liebert, Inc. Used with permission.
Page 5
GENE-SUPPLEMENTED
COLLAGEN-GAG MATRICES
• Plasmid DNA added to pre-fabricated
collagen-GAG matrices can transfect seeded
chondrocytes.
• Conditions under which the DNA is
incorporated into the matrices will affect
retention and prolonged release
• Cross-linking method will affect transfection
rates
Collagen-GAG Matrix to which
Plasmid DNA has been added
SEM
Plasmid DNA
100 µm
TEM
200 nm
Without DNA Added
200 nm
Courtesy of Mary Ann Liebert, Inc. Used with permission.
Page 6
R. Samuel, et al.
Retention of DNA in Collagen Matrices
Luciferase gene --> DNA + matrix + adult canine chondrocytes
0.8
none pH 7.5
Re sidual Fraction of DNA
0.7
none pH 2.5
DHT pH 7.5
DHT pH 2.5
• 250 µg
DNA loaded/matrix
UV pH 7.5
UV pH 2.5
• Different
pH and cross-linking
EDAC pH 7.5
EDAC pH 2.5
conditions
0.6
0.5
0.4
0.3
0.2
0.1
0
0
100
200
R. Samuel, et al.
300
400
500
600
28 days
Time (hours)
In Situ Transfection of Chondrocytes
Based on Luciferase Activity
Normalized RFU
1600
1400
1200
1000
No cross-linking
A
B
C
800
600
400
200
0
pH X
pH Y
28 days post-seeding
R. Samuel, et al.
Page 7
700
DISCUSSION
• Plasmid DNA could be bound to prefabricated collagen-GAG matrices
• Small percentage of DNA was tightly
bound, higher in matrices prepared at a
certain pH
• Higher level of transfection in matrices
prepared at other pHs
DISCUSSION
• Selected collagen-GAG matrices could be
formulated to provide for the prolonged (greater
than 1 month) release of plasmid DNA.
• A significant percentage (20-40%) of the DNA
added to the matrices resist passive release into the
leaching buffer. For comparison, in a prior study
(Shea
h., 1999) investigating release of
Shea, et al.,
al., Nature Biotec
Biotech.,
plasmid DNA from copolymers of D,L-lactide and
glycolide, less than 10% of the DNA remained in
the synthetic polymer construct after 28 days in
leaching studies.
Page 8