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Algae-Derived Omega-7 Accelerates Wound Healing
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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 Methods Significance of the Study Conclusions Preliminary Results Diabetes affects approximately 170 million people worldwide, including 20.8 million in the USA, and by 2030 these numbers are projected to double. • 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. • Wound healing progresses in phases: Preliminary Results 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. • We have identified an omega-7-rich algae that is appropriate for large scale production. • This algae can be effectively opened by exposure to an appropriate electric field. • Omega-7 can be recovered using column chromatography. • Omega-7 extracts improve blood flow in wounded sheep. • Omega-7 accelerates wound closure and minimizes scar 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. 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 Omega-7 treated wound epithelialized rapidly and smoothly. 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. Coagulation fibrin plug formation, release of growth factors, cytokines, hypoxia Phase s Time Main Cell Types Specific Events Inflammation cell recruitment and chemotaxis, wound debridement Migration/ proliferation collagen deposition, angiogenesis, ECM deposition, contraction Remodeling scar formation and revision, ECM degradation, further contraction Hours Days Weeks to months Platele ts Neutrophi ls, monocytes Macrophage s Keratinocy tes, fibroblast s, endothelia l cells Myofibroblas ts Platelet aggregation, release of fibrinogen and other pro- inflammatory mediators Selectins slow down neutrophils, diapedesis through integrin binding Hemidesmosome breakdown, keratinocyte migration Crosstalk between MMPs, VEGF signaling, NOS activation, EPC production and migration, ECM production 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. •The essential fatty acid palmitoleic acid (Omega-7) promotes the formation of new blood vessels and collagen deposition at the site of injury. • Currently, Omega-7 is primarily derived from Sea Buckthorn, a cold weather plant that is harvested once a year. • We have identified an algae that produces large quantities of Omega-7 and can be harvested daily. Migrating keratinocytes EPCs homing to wound site Circulatory system VEGF eNOS activation ↑ NO . Endothelial Progenitor Cell (EPC) mobilization into the circulation Healthy Wound Diabetic Wound collage n deposit ion Neovasculogenes is and wound healing MP MF MP – macrophage MF – myofibroblast Impaired neovasculogenesis and wound healing VEGF Impaired eNOS activation Limite d NO . Limited EPC mobilization into the circulation Limited EPC homing to wound site Bone marrow 4 / 5 o f s t o c k 95L bioreactor 12L media 12L stock 15L bioreactor 24L algae 24L media 42L media 48L algae D a y 1 Day 3 D a y 5 D a y 8 Stock culture Exposure to an Electric Field Recover Omega-7 Oil Concentrate 100X M a t r i g e l 3 D c u l t u r e keratinocytes f i b r o b l a s t s In vitro 3D cell culture 1 Matrigel 3D culture keratinoc ytes fibroblas ts Diabetic mouse Control mouse 2 In vivo mouse studies 3 Large animal studies 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) 7 days post surgery 14 days post surgery 18 days post surgery Contro l Omega- 7 Fatty Acid Algae Oil Extract 16:0 16:1 (n-7) 18:1 (n-9) 18:2 (n-6) 18:3 (n-3) other 30.9 33.5 14.6 5.3 5.7 10.0 Blood flow measured in donor and grafted side by dopler 350 – 300 – 250 – 200 – 150 – 100 – 50 – 0 – 7 d 18 d Graft Treatment Graft Control Donor Treatment Donor Control Post Op 7 days Post Op 18 days Control Omega-7
description
Sheep: 672M. Control. Omega-7. 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. Time. Control. Phases. Main Cell Types. Omega-7. - PowerPoint PPT Presentation
Transcript of Algae-Derived Omega-7 Accelerates Wound Healing
Algae-Derived Omega-7 Accelerates Wound HealingR. Connelly, M. Montoya, D. Schmid, R. Pearsall, M. Werst, R. Hebner
University of Texas at Austin, Center for Electromechanics and OpenAlgae LLC
MethodsSignificance of the Study
Conclusions
Preliminary Results
Diabetes affects approximately 170 million people worldwide, including 20.8 million in the USA, and by 2030 these numbers are projected to double.
• 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.
• Wound healing progresses in phases:
Preliminary Results
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.
• We have identified an omega-7-rich algae that is appropriate for large scale production.
• This algae can be effectively opened by exposure to an appropriate electric field.
• Omega-7 can be recovered using column chromatography.
• Omega-7 extracts improve blood flow in wounded sheep.
• Omega-7 accelerates wound closure and minimizes scar formation.
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.
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 Omega-7 treated wound epithelialized rapidly and smoothly.
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.
Coagulationfibrin plug formation,
release of growth factors, cytokines, hypoxia
PhasesTime Main Cell Types Specific Events
Inflammationcell recruitment and chemotaxis, wound
debridement
Migration/proliferation
collagen deposition, angiogenesis, ECM
deposition, contraction
Remodelingscar formation and
revision, ECM degradation, further contraction
Hours
Days
Weeks to months
Platelets
Neutrophils, monocytes
Macrophages
Keratinocytes, fibroblasts, endothelial
cells
Myofibroblasts
Platelet aggregation, release of fibrinogen and other pro-
inflammatory mediators
Selectins slow down neutrophils, diapedesis through integrin binding
Hemidesmosome breakdown, keratinocyte migration
Crosstalk between MMPs, VEGF signaling, NOS activation, EPC
production and migration, ECM production
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.
•The essential fatty acid palmitoleic acid (Omega-7) promotes the formation of new blood vessels and collagen deposition at the site of injury.
• Currently, Omega-7 is primarily derived from Sea Buckthorn, a cold weather plant that is harvested once a year.
• We have identified an algae that produces large quantities of Omega-7 and can be harvested daily.
Migrating keratinocytes
EPCs homingto wound site
Circulatorysystem
VEGF
eNOS activation↑ NO.Endothelial Progenitor Cell (EPC)mobilization into the circulation
Healthy Wound Diabetic Wound
collagendeposition
Neovasculogenesisand wound healing
MP MF
MP – macrophage MF – myofibroblast
Impairedneovasculogenesisand wound healing
VEGF
ImpairedeNOS activation
LimitedNO.
Limited EPC mobilization into the circulation
LimitedEPC homingto wound site
Bone marrow
4/5 of stock
95L bioreactor
12L media12L stock
15L bioreactor24L algae
24L media
42L media
48L algae
Day 1
Day 3 Day 5
Day 8
Stock culture
Exposure to an Electric FieldRecover Omega-7 Oil
Concentrate100X
Matrigel3D culture
keratinocytes
fibroblasts
In vitro 3D cell culture1
Matrigel3D culture
keratinocytes
fibroblasts
Diabetic mouse Control mouse
2 In vivo mouse studies 3 Large animal studies
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)
7 days post surgery
14 days post surgery
18 days post surgery
Control Omega-7
Fatty Acid Algae Oil Extract
16:0
16:1 (n-7)
18:1 (n-9)
18:2 (n-6)
18:3 (n-3)
other
30.9
33.5
14.6
5.3
5.7
10.0
Blood flow measured in donor and grafted side by dopler350 –
300 –
250 –
200 –
150 –
100 –
50 –
0 –7 d 18 d
Graft Treatment
Graft Control
Donor Treatment
Donor Control
Post
Op
7 da
ysPo
st O
p 18
day
s
Control Omega-7
migratingkeratinocytes
EPCs homingto wound site
circulatorysystem
VEGF
eNOS activation↑ NO.Endothelial Progenitor Cell (EPC)mobilization into the circulation
Healthy Wound Diabetic Wound
collagendeposition
neovasculogenesisand wound healing
MPMF
MP – macrophage MF – myofibroblast
impairedneovasculogenesisand wound healing
VEGF
impairedeNOS activation
limitedNO.
limited EPC mobilization into the circulation
limitedEPC homingto wound site
bone marrow
PhasesTime Main Cell Types Specific Events
Inflammationcell recruitment and chemotaxis, wound
debridement
Coagulationfibrin plug formation, release of growth factors, cytokines,
hypoxia
Migration/proliferationcollagen deposition,
angiogenesis, ECM deposition, contraction
Remodelingscar formation and revision,
ECM degradation, further contraction
hours
days
weeks to months
platelets
neutrophils, monocytes
macrophages
keratinocytes, fibroblasts, endothelial
cells
myofibroblasts
platelet aggregation, release of fibrinogen and other pro-inflammatory
mediators
selectins slow down neutrophils, diapedesis through integrin binding
hemidesmosome breakdown, keratinocyte
migration
crosstalk between MMPs, VEGF signaling, NOS
activation, EPC production and migration, ECM
production
phenotypic switch to myofibroblasts from
fibroblasts
4/5 of
stock
95L bioreactor
12L media
12L stock
15L bioreactor
24L algae
24L media
42L media
48L algae
Day 1 Day 3 Day 5 Day 8stock culture
Exposure to an Electric FieldRecover Omega-7 Oil
concentrate100X
Matrigel3D culture
diabetic mouse control mouse
2in vivo mouse studies
3large animal studies
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)
0
50
100
150
200
250
300
350
7d 18d
graf t control
graf t treatment
donor control
donor treatmenat
Blood flow measured in donor and grafted side by dopler
• Blood flow is greater in treated wound with omega-7 oil
no remarkable difference
-epithelization of treated wound was faster treated wound: almost 100％ untreated wound: 78％
-no remarkable difference -treated wound ; almost 100％ -untreated wound ; 97％
Donor side
7 days post surgery
14 days post surgery
18 days post surgery
control Omega-7
Sheep: 672M
Untreated wound between mesh was epthelized irregularly. Treated wound between mesh was epithelized equally.
The wound between the mesh in untreated part was not epithelized.Treated wound epithelized partially.
Post Op 7 days
Post Op 18 days
control Omega-7
