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.