1. 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 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 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. Coagulation fibrin plug formation, release of growth factors, cytokines, hypoxia Coagulation fibrin plug formation, release of growth factors, cytokines, hypoxia PhasesTimeMain Cell TypesSpecific Events Inflammation cell recruitment and chemotaxis, wound debridement Inflammation cell recruitment and chemotaxis, wound debridement Migration/ proliferation collagen deposition, angiogenesis, ECM deposition, contraction Migration/ proliferation collagen deposition, angiogenesis, ECM deposition, contraction Remodeling scar formation and revision, ECM degradation, further contraction Remodeling scar 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. 4/5 of stock4/5 of stock 95L bioreactor 12L media 12L stock 15L bioreactor 24L algae 24L media 42L media 48L algae Day 1Day 1 Day 3Day 5Day 5 Day 8Day 8 Stock culture Exposure to an Electric FieldRecover Omega-7 Oil Concentrate 100X Matrigel3D cultureMatrigel3D culture keratinocytes fibroblastsfibroblasts In vitro 3D cell culture 1 Matrigel 3D 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 ControlOmega-7 Fatty AcidAlgae 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 d18 d Graft Treatment Graft Control Donor Treatment Donor Control Sheep: 672M Post Op 7 days Post Op 18 days ControlOmega-7

3. PhasesTimeMain Cell TypesSpecific Events Inflammation cell recruitment and chemotaxis, wound debridement Coagulation fibrin plug formation, release of growth factors, cytokines, hypoxia Migration/ proliferation collagen deposition, angiogenesis, ECM deposition, contraction Remodeling scar 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. 4/5 of stock 95L bioreactor 12L media 12L stock 15L bioreactor 24L algae 24L media 42L media 48L algae Day 1Day 3Day 5Day 8stock culture Exposure to an Electric FieldRecover Omega-7 Oil concentrate 100X

5. Matrigel 3D culture keratinocytes fibroblasts in vitro 3D cell culture 1 diabetic mousecontrol 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)

6. Blood flow measured in donor and grafted side by dopler Blood flow is greater in treated wound with omega-7 oil

7. 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 controlOmega-7

8. 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 controlOmega-7