Mechanisms of Wound Healing: Studies with the Bilayered Cellular Matrix, OrCelTM Melvin Silberklang, Ph.D. Chief Scientific Officer and Vice President, R&D Ortec International, Inc. Clinical Application of OrCelTM Description • OrCelTM (Bilayered Cellular Matrix) is a preformed bovine collagen sponge matrix, gel-coated on one side, in which normal human allogeneic skin cells are cultured: • dermal fibroblasts within the porous sponge • epidermal keratinocytes on the gel-coated, nonporous side of the sponge. Development: from Epidermolysis Bullosa to Wound Healing • 1986: collagen sponge matrix-cultured autologous keratinocyte technology adapted to treat junctional Epidermolysis Bullosa (M. Carter, et al.) • 1989: first use of allogeneic bilayered composite cultured skin (CCS) in recessive dystrophic Epidermolysis Bullosa hand surgery (M. Eisenberg & D. Llewelyn) • 1990: optimized CCS in normal volunteers • 1991: Ortec founded to commercialize CCS • 1994: FDA approval to launch 1st U.S. Clinical trial of CCS in burn surgery • 2001: FDA PMA approval for OrCelTM FDA Approvals for OrCel™ • Humanitarian Use Device Exemption (HDE) for the treatment of surgical wounds and donor sites associated with mitten hand deformities in patients with Recessive Dystrophic Epidermolysis Bullosa (RDEB) • Split thickness Donor Sites in Burn Patients OrCel™ Manufacturing Process Neonatal Foreskin Enzymatic Detachment of Epidermal and Dermal Layers Cryopreservation (P1) Cryopreservation (P1) Keratinocyte Cell Line Expansion Fibroblast Cell Line Expansion Cryopreservation (P3) Cryopreservation (P3/P4) Collagen Sponges Lamination with Collagen Gel Inoculation with Cells Culture 9 – 14 Days OrCel Scanning EM of Collagen Sponge (cross-section) Safety Testing of Donor and of Allogeneic Cells Blood Tests, Donor’s Mother Tests, Donor’s Cells (K & F Cell Lines) ALT Sterility Cytomegalovirus (Ab) Mycoplasma Epstein Barr Virus (Capsid Ab) In Vitro Viral Assay Hepatitis B (Core Ab & SAg) Epstein-Barr Virus (PCR) Hepatitis C (Ab) Hepatitis B (PCR) Herpes Simplex 1&2 (Ab) HIV Antigen (P24) Human Herpes 6 (Ab) HIV 1&2 (PCR) HIV 1 (Ab) HTLV 1&2 (PCR) HIV 2 (Ab) h-Herpes 6 (PCR) HTLV 1&2 (Ab) In Vivo Tumorigenicity RPR Karyotype Isoenzyme Analysis (species ID) Cell Growth and Morphology P-63 Bcl-2 OrCelTM Wound Healing Rationale: Facilitated Tissue Regeneration Clinical Rationale: Co-Cultured Keratinocytes and Fibroblasts Supply Optimal Composition of Growth Factors/Cytokines Cell-deposited Biomatrix Creates Optimal Scaffold for Wound Area Cell Migration and Proliferation Product Resorbs Rapidly In Vivo (7 - 14 days) Biological Profiles • OrCel™ cells are in growth phase • OrCel™ cells demonstrate high viability • OrCel™ cells are highly productive for woundhealing cytokines and growth factors • Co-cultured, compartmentalized cells produce more extracellular factors than either keratinocytes or fibroblasts alone OrCelTM Cell Composition by Flow Cytometry Ungated Cells 7.6% 92.3% Fluorescence (FITC – pan-cytokeratin) OrCel™ Histology OrCelTM Histology (Ki67 Immunohistochemistry) Confidential Immunohistochemical Screening for HLA-DR Antigen in Foreskin Tissue vs. OrCelTM 10X 10X 20X 20X /24 hours Fibroblast-Only OrCel™ Keratinocyte-Only OrCel™ Co-Cultured OrCel™ 2 6000 Rate of Secretion into Culture Medium, pg/cm Rate of Secretion into Culture Medium, pg/cm 2 /24 hours Cytokine Production by Fibroblasts, Keratinocytes and Co-Cultured Cells 4000 2000 0 50 Fibroblast-Only OrCel™ Keratinocyte-Only OrCel™ Co-Cultured OrCel™ 25 0 GMCSF PGE2 VEGF bFGF KGF-1 OrCel C o m p a r i s o n o f C C S C y t o k i n e P r o d u c t i o n w i t h t h a t o f A c u t e H u m a n S k i n W o u n d s ( U r e e t a l . , 1 9 9 8 ; V o g t e t a l . , 1 9 9 8 ) 2/day 1 0 0 0 0 C C S P r o d u c t i o n OrCel H u m a n W o u n d s 1 0 0 0 1 0 0 (NoDat) (NoDat) (NoDat) (NoDat) (NoDat) 1 bGM FGF -CSF IL-1a IKL-1b GMF-1 -CTSF GTGF-a FTG-b1 F-b2T NVF-a EGF CytokineOutpPerUnitArea,pg/cm 1 0 ABCD Confidential Immunodetection of VEGF in OrCelTreated Nude Mice Comparative Histological Profiles Apligraf OrCel Output, OrCelTM and Apligraf 100000 Cytokine Productivity, pg/cm2/day OrCel Apligraf 10000 1000 100 10 bFGF GM-CSF HGF KGF-1 VEGF IL-1a MMP-9 PGE-2 1000 Whole Fenes. 100 10 Day 1 Day 2 bFGF Day 1 Day 2 KGF-1 Day 1 Day 2 Il-1a Cytokine Per Unit Area, pg/cm2/day Cytokine Per Unit Area, pg/cm2/day Effect of Fenestration on Cytokine Expression 1000 Whole Fenes. 100 10 Day 1 Day 2 bFGF Day 1 Day 2 KGF-1 Day 1 Day 2 Il-1a Cytokine Production by Whole vs. Fenestrated OrCel 10000 Whole Fenes. 1000 100 10 Day 1 Day 2 GM-CSF Day 1 Day 2 MCSF Day 1 Day 2 VEGF Cytokine Per Unit Area, pg/cm2/day Cytokine Per Unit Area, pg/cm2/day Effect of Fenestration on Cytokine Expression Cytokine Production by Whole vs. Fenestrated Apligraf 10000 Whole Fenes. 1000 100 10 1 Day 1 Day 2 GM-CSF Day 1 Day 2 MCSF Day 1 Day 2 VEGF Challenges in Cryopreservation of Tissue Engineered Products • Optimization of Cryoprotectants and Freeze Cycle -- Minimizing Ice Crystal Damage to Cells and Matrix • Shipping and Storage at End-User Sites -Storage Condition and Shelf Life • Development of Thawing Procedure -Minimizing Ice Crystal Damage • Post-thaw Rinse-out of Cryoprotectants -Practicality, User-Friendliness Retention of Functionality after Cryopreservation 120 Relative Performance 100 80 60 40 20 0 Cell Count % Viability Cell Count Pre-Cryo Post-Thaw Post-Thaw + 48 Hr Met. Activity Cryo-OrCel vs. OrCelTM 10000 Cytokine Productivity, pg/cm2/day Fresh OrCel Cryo OrCel 1000 100 10 bFGF GM-CSF IL-1a KGF-1 MCSF VEGF Cryo-ORCEL vs. Fresh ORCEL • Equivalent keratinocyte and fibroblast cell density • Equivalent cell viability • Equivalent or better metabolic activity • Essentially equivalent cytokine expression DONOR SITES 22 Days 22 20 18 16 14 12 10 8 6 4 Pivotal: 2/01 Based on Photography 2 0 OrCel Standard of Care 15 Days PMA Claims •Accelerated healing •Less Scarring •Reduced time to recropping Cryo-OrCelTM Preliminary Results % Venous Leg Ulcers 80 Cry0-OrCel Standard of 71% 70 Care 60 47% 50 37% 40 26% 30 20 12% 10 0% 0 4 Pilot: 8/00 8 24 Weeks OrCel ™ Heals Wounds Faster Diabetic Foot Ulcers 60 47% 50 Ortec Standard of Care 40 30 23% 20 10 0 Ortec Standard Of Care ACKNOWLEDGEMENTS Lee McDonald Avi Lasdun Sandy Lerman Stephanie Knight Dan Dwyer Sunny Luke Rachel Tewari Tina Huang Nameeta Chimanji Lara Silberklang Melissa Steiner Flora Elequin Celina Choi Caroline Tang Steven Peltier Liza Moore Kathy Tygum Nitya Ray Hsin-Chien Tai Alla Laufer Ying Song