IMPROVING PERFUSION OF
SYNTHETIC SKIN
Jordan S. Pober and Jeffrey S.
Schechner
CLINICAL PROBLEM
• Patients with impaired angiogenesis (e.g.,
the elderly, diabetics) develop non-healing
wounds
• Avascular skin substitutes (Apligraf) may
promote healing but fail as long term grafts
• Graft failure is ischemic, not immunological
HYPOTHESIS
• Incorporating endothelial cells into
synthetic skin will promote perfusion
• The effect will be most obvious in a setting
of impaired angiogenesis
EXPERIMENTAL SYSTEM
• Synthetic skin prepared from decellularized
human dermis seeded with human
keratinocytes +/- human endothelial cells
• Orthotopic skin graft on immunodeficient
(C.B-17 SCID/bg) mice +/- rapamycin
• Assess vascularity by histology
Vascularized Synthetic Skin
(orthotopic implant)
Human
Neonatal
Foreskin
Human
Umbilical
Vein
Human
Adult
Cadaver Skin
Human
Umbilical
Cord Blood
Human
Adult
Peripheral Blood
EPC
Cultured
Keratinocytes
Decellularized
Dermis
Simple
Synthetic Skin
Control
SCID Mouse
Recipient
Moderately
Vascularized Skin Graft
(Mouse EC-Lined
Vessels Only)
Cultured
EC
+/- Bcl-2
Transduction
EC-Seeded
Synthetic Skin
Rapamycin-Treated
SCID Mouse
Recipient
Control
SCID Mouse
Recipient
Rapamycin-Treated
SCID Mouse
Recipient
Poorly
Vascularized Skin Graft
(Necrosis)
Well Vascularized
Skin Graft
(Mouse and Human
EC-Lined Vessels)
Well Vascularized
Skin Graft
(Human EC-Lined
Vessels Only)
A
B
C
D
E
F
Figure 1 Culture of human EC derived from cord blood or adult peripheral blood (a). Colonies of differentiated cells were noted
as early as 7-10 days after isolation from CB and 21-35 days after isolation from AB using the conditions of isolation and culture
described in the Materials and Methods(b). Colonies of differentiated EC differentiated from CB-EPC continued to proliferate,
establishing a cellular monolayer capable of serial passage and expansion. AB-EC exhibited similar behavior (c,d).
Immunofluorescence microscopy of monolayers of cells derived from AB-EPC revealed the presence of VE-cadherin at the cell
junctions, and cytoplasmic granules of vWF (e,f), indicative of differentiated EC. Similar cells were derived from CB-EPC. Each
cell type was cultured on at least five separate isolations with similar results.
A
B
C
D
E
F
Figure 5 Histologic analysis of microvessels in human skin equivalents harvested 21 days after
transplantation. Note the paucity of vessels in grafts constructed without EC-seeding (a), compared
with well vascularized grafts seeded with AB-EC (b) or CB-EC (c). In human skin equivalents seeded
with CB-EC both mouse EC-lined (d) and human EC-lined (e) vessels were present. The observed
microvessels were coated by cells expressing smooth muscle cell-specific alpha actin, indicative of
vessel maturation. All images are representative of the results seen in AB-EC or CB-EC-seeded
human skin equivalents from at least four separate experiments with each cell type (except actin
staining, which was only examined in two experiments).
3500
3500
3000
3000
3000
2500
2500
2500
*
*
2000
1500
1000
500
Vessels/mm2
3500
Vessels/mm2
Vessels/mm2
HUMAN EC-LINED VESSELS
2000
1500
1000
500
0
HUVEC
*
HUVEC
Rapamycin
#
HUVEC Bcl-2
Rapamycin
1500
1000
500
0
HUVEC Bcl-2
2000
0
CB
CB Bcl-2
#
CB Rapamycin
#
CB Bcl-2
Rapamycin
AB EPC
AB EPC Bcl-2
#
AB EPC
Rapamycin
#
AB EPC Bcl-2
Rapamycin
#
2000
2000
1500
1500
1500
1000
500
Vessels/mm2
2000
Vessels/mm2
Vessels/mm2
MOUSE EC-LINED VESSELS
1000
500
0
0
HUVEC
HUVEC Bcl-2
HUVEC
Rapamycin
HUVEC Bcl-2
Rapamycin
1000
500
0
CB
CB Bcl-2
CB Rapamycin
CB Bcl-2
Rapamycin
AB EPC
AB EPC Bcl-2
AB EPC
Rapamycin
AB EPC Bcl-2
Rapamycin
New Questions
• Will the inclusion of EC accelerate and/or
increase perfusion of synthetic skin?
• What is the best source of EC?
• Will Bcl-2 or other transduced genes
improve EC performance in synthetic skin?
• Will endothelialized skin provoke rejection?
• CAN PERFUSION BE QUANTIFIED?
Two Photon Excited Florescence
Two photons can interact
simultaneously with a molecule
adding their energies to produce
an excitation equal to the sum of
their individual energies.
i.e. 2 red photons can = 1 blue
photon
1 photon
excitation
Fluorescence
Increasing Wavelength
Increasing Energy
2 photon
excitation
Two Photon Excitation is Spatially Localized
Relative Fluorescence
Because two photons arriving at the same
time are required for excitation the emission
depends on the square of the intensity, rather
then being linearly proportional.
F  I2
FI
0
0.1
0.2
0.3
0.4
0.5
0
5
10
15
20
Power at focus (mW)
At “normal” imaging intensities, excitation is
only appreciable at the focal point.
25
Single photon
excitation
(488 nm)
Two photon
excitation
(900 nm)
Advantages of Multi-photon Excitation
In addition to limiting photobleaching and photodamage
to the image plane, multi-photon excitation has several
other advantages:
• Near-IR light scatters less than blue light in many
biological samples
• More efficient light collection
• Can excite dyes in their UV absorption bands
– Can use wide range of useful UV dyes
– Good for multicolor imaging
– Important for visualizing intrinsic fluorescence
Multiphoton Excitation and Nonlinear
Scattering of Intrinsic molecules
Heart
Arteriole
Lung
Images courtesy of www.drbio.cornell.edu
In Situ Imaging of Deep Structures
15 mm
Microangiography, in situ, from wild-type mouse
cortex
608 nm Quantum-dots
~800 mm
Blood flow ~0.6 mm/s
Levene et. al., J. Neurophys. 2003
>2 mm
>3 mm(Fluorescein-Dextran)
Microangiography, in situ, from wild-type mouse
dermis
550 nm Quantum-dots
Larson et. al., Science 2003
Blood flow ~0.01 mm/s
Future Directions
•
•
•
•
Use MPM to assess neovascularization
Quantify perfusion
Improve imaging depth for clinical evaluation of healing
Develop imaging system amenable to clinical environment
– Fiber-coupled imaging system
– Catheter version of multiphoton microscope (~1 mm diameter)
• Other collaborations include
– NADH imaging of astrocyte metabolism in epileptic tissue with
Anne Williamson and Dennis Spencer
– Intrinsic signal imaging in lung for asthma and emphysema with
Robert Homer and Jack Elias
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