Algae-Derived Omega-7 Accelerates Wound Healing

Report
Algae-Derived Omega-7 Accelerates Wound Healing
R. Connelly, M. Montoya, D. Schmid, R. Pearsall, M. Werst, R. Hebner
University of Texas at Austin, Center for Electromechanics and OpenAlgae LLC
Significance of the Study
Methods
42L media
4
/
5
o
15L bioreactor
f
s
Stock culture
t
o
c
k
24L media
12L media
12L stock
95L bioreactor
D
a
y
1
48L algae
24L algae
Day 3
D
a
y
5
D
keratinocytes
a
fibroblasts
f
y
i
8 Concentrate
b
100X
r 1
o
b
l
a
s
t
s
• Wound healing progresses in phases:
Time
Phases
Main Cell Types
Specific Events
Platelets
Platelet aggregation, release
of fibrinogen and other proinflammatory mediators
Coagulation
fibrin plug formation,
release of growth factors,
cytokines, hypoxia
Recover Omega-7 Oil
Hours
Neutrophils,
monocytes
Inflammation
cell recruitment and
chemotaxis, wound
debridement
Exposure to an Electric Field
Selectins slow down
neutrophils, diapedesis
through integrin binding
keratinocytes
Matrigel
M
3D culture
a
Diabetic mouse
Control
mouse
t
r
i
In vitro 3D cell culture
2 In vivo mouse studies
3 Large animal studies
g
e
l
3
D
c
Treat with omega-7
prior to wound formation, then analyze:
u
l
t
• Collagen deposition
(western blot, microscopy)
u
• VEGF activation
r (western blot, microscopy)
• NOS activatione (ROS formation DCF fluorescence)
• Cellular proliferation (XTT assay)
• Wound closure (SOC measurements)
• Scar formation (SOC measurements)
Macrophages
Hemidesmosome breakdown,
keratinocyte migration
Days
Migration/
proliferation
collagen deposition,
angiogenesis, ECM
deposition, contraction
Keratinocytes,
fibroblasts,
endothelial
cells
Remodeling
scar formation and
revision, ECM degradation,
further contraction
Weeks to
months
Phenotypic switch to
myofibroblasts from fibroblasts
• One of the cardinal features of wound healing is the
formation of new small blood vessels at the site of injury.
Healthy Wound
Diabetic Wound
Migrating keratinocytes
VEGF
Preliminary Results
Crosstalk between MMPs, VEGF
signaling, NOS activation, EPC
production and migration, ECM
production
Myofibroblasts
Blood flow measured in donor and grafted side by dopler
Fatty Acid
Algae Oil Extract
VEGF
300 –
16:0
30.9
16:1 (n-7)
33.5
250 –
18:1 (n-9)
14.6
200 –
18:2 (n-6)
5.3
150 –
18:3 (n-3)
5.7
other
10.0
Neovasculogenesis
and wound healing
Impaired
neovasculogenesis
and wound healing
EPCs homing
to wound site
Control
• We have identified an omega-7rich algae that is appropriate for
large scale production.
Donor Treatment
100 –
Donor Control
Omega-7
Limited
EPC homing
to wound site
Circulatory
system
Conclusions
Graft Control
MF
MP
350 –
50 –
0–
7d
18 d
Figure 2. Blood flow in control and omega-7 treated sheep.
Sheep were given 10 mm surface wounds, then were treated
with omega-7 or nothing. Blood flow is increased in omega-7
treated sheep, suggesting increased neovasculargenesis at the
wound site.
↑ NO.
eNOS activation
Impaired
eNOS activation
Limited
NO.
• This algae can be effectively
opened by exposure to an
appropriate electric field.
• Omega-7 can be recovered using
column chromatography.
7 days post surgery
Endothelial Progenitor Cell (EPC)
mobilization into the circulation
Figure 4. Ultrasound assessment of skin graft healing 7 and
18 days post surgery. Untreated control wound between the
mesh was epithelialized slowly and irregularly. The Omega7 treated wound epithelialized rapidly and smoothly.
Graft Treatment
Figure 1. Fatty Acid composition of the
algal extract. The primary fatty acids
present in this algae are palmitic and
palmitoleic (Omega-7) fatty acids.
collagen
deposition
Limited EPC mobilization
into the circulation
Bone marrow
MP – macrophage
Omega-7
Post Op 7 days
• Impaired wound healing is a major clinical problem in
diabetic patients, affecting about 15 percent of them and is
the leading cause of lower limb amputations.
Control
Post Op 18 days
Diabetes affects approximately 170 million people
worldwide, including 20.8 million in the USA, and by 2030
these numbers are projected to double.
Preliminary Results
MF – myofibroblast
•The essential fatty acid palmitoleic acid (Omega-7) promotes
the formation of new blood vessels and collagen deposition at
the site of injury.
14 days post surgery
• Currently, Omega-7 is primarily derived from Sea Buckthorn,
a cold weather plant that is harvested once a year.
18 days post surgery
• We have identified an algae that produces large
quantities of Omega-7 and can be harvested daily.
Figure 3. Wound closure. The untreated control wound
failed to fully close by 18 days post surgery and scar tissue
was apparent. The Omega-7 treated wound closed 97% by
day 14, and completely by day 18 post surgery. The
epithelium was smooth with minor scarring.
• Omega-7 extracts improve blood
flow in wounded sheep.
• Omega-7 accelerates wound
closure and minimizes scar
formation.
Healthy Wound
Diabetic Wound
migrating
keratinocytes
VEGF
VEGF
collagen
deposition
MF
MP
neovasculogenesis
and wound healing
impaired
neovasculogenesis
and wound healing
EPCs homing
to wound site
limited
EPC homing
to wound site
circulatory
system
Endothelial Progenitor Cell (EPC)
mobilization into the circulation
↑ NO.
eNOS activation
bone marrow
MP – macrophage
MF – myofibroblast
impaired
eNOS activation
limited
NO.
limited EPC mobilization
into the circulation
Time
Phases
Main Cell Types
Coagulation
fibrin plug formation, release
of growth factors, cytokines,
hypoxia
hours
Inflammation
cell recruitment and
chemotaxis, wound
debridement
platelets
neutrophils,
monocytes
Specific Events
platelet aggregation,
release of fibrinogen and
other pro-inflammatory
mediators
selectins slow down
neutrophils, diapedesis
through integrin binding
macrophages
hemidesmosome
breakdown, keratinocyte
migration
days
Migration/
proliferation
collagen deposition,
angiogenesis, ECM deposition,
contraction
keratinocytes,
fibroblasts,
endothelial
cells
crosstalk between MMPs,
VEGF signaling, NOS
activation, EPC production
and migration, ECM
production
myofibroblasts
weeks to
months
Remodeling
scar formation and revision,
ECM degradation, further
contraction
phenotypic switch to
myofibroblasts from
fibroblasts
42L media
4/5
of
stock
24L media
48L algae
12L media
24L algae
12L stock
15L bioreactor
95L bioreactor
stock culture
Day 1
Day 3
Day 5
Day 8
concentrate
100X
Recover Omega-7 Oil
Exposure to an Electric Field
Matrigel
3D culture
diabetic mouse
control mouse
in vivo mouse studies
2
Treat with omega-7 prior to wound formation, then analyze:
• collagen deposition (western blot, microscopy)
• VEGF activation (western blot, microscopy)
• NOS activation (ROS formation DCF fluorescence)
• cellular proliferation (XTT assay)
• wound closure (SOC measurements)
• scar formation (SOC measurements)
large animal studies
3
Blood flow measured in donor and grafted side by dopler
350
300
250
graft control
200
graft treatment
donor control
150
donor treatmenat
100
50
0
7d
18d
• Blood flow is greater in treated wound with omega-7 oil
control
Omega-7
Donor side
7 days post surgery
no remarkable diffe
14 days post surgery
-epithelization of
faster
treated wound
untreated wou
18 days post surgery
-no remar
-treated
-untrea
Sheep: 672M
control
Omega-7
Post Op 7 days
The wound between the mesh in untreated part was not epithelized.
Treated wound epithelized partially.
Post Op 18 days
Untreated wound between mesh was epthelized irregularly. Treated wound between mesh was epithelized equally.

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