Mechanisms of Wound Healing: Studies with the Bilayered Cellular Matrix, OrCel TM Melvin Silberklang, Ph.D. Chief Scientific Officer and Vice President, R&D Ortec International, Inc.

Clinical Application of OrCel TM

Description OrCel TM (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, non- porous 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 1 st U.S. Clinical trial of CCS in burn surgery 2001: FDA PMA approval for OrCel TM

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

Collagen Sponges Lamination with Collagen Gel Inoculation with Cells OrCel Culture 9 – 14 Days Cryopreservation (P1) Cryopreservation (P3) Cryopreservation (P1) Cryopreservation (P3/P4) Keratinocyte Cell Line Expansion Fibroblast Cell Line Expansion Enzymatic Detachment of Epidermal and Dermal Layers Neonatal Foreskin OrCel™ Manufacturing Process

Scanning EM of Collagen Sponge (cross-section)

Blood Tests, Donor’s MotherTests, Donor’s Cells (K & F Cell Lines) ALTSterility 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 RPRKaryotype Isoenzyme Analysis (species ID) Cell Growth and Morphology Safety Testing of Donor and of Allogeneic Cells

P-63

Bcl-2

Clinical Rationale: u Co-Cultured Keratinocytes and Fibroblasts Supply Optimal Composition of Growth Factors/Cytokines u Cell-deposited Biomatrix Creates Optimal Scaffold for Wound Area Cell Migration and Proliferation u Product Resorbs Rapidly In Vivo ( days) OrCel TM Wound Healing Rationale: Facilitated Tissue Regeneration

Biological Profiles OrCel™ cells are in growth phase OrCel™ cells demonstrate high viability OrCel™ cells are highly productive for wound- healing cytokines and growth factors Co-cultured, compartmentalized cells produce more extracellular factors than either keratinocytes or fibroblasts alone

OrCel TM Cell Composition by Flow Cytometry 7.6% 92.3% Cells Fluorescence (FITC – pan-cytokeratin) Ungated

OrCel ™ Histology

OrCel TM Histology (Ki67 Immunohistochemistry) Confidential

Immunohistochemical Screening for HLA-DR Antigen in Foreskin Tissue vs. OrCel TM 10X 20X 10X 20X

GMCSFPGE2VEGF Rate of Secretion into Culture Medium, pg/cm 2 /24 hours Fibroblast-Only OrCel™ Keratinocyte-Only OrCel™ Co-Cultured OrCel™ bFGFKGF-1 Rate of Secretion into Culture Medium, pg/cm 2 /24 hours Fibroblast-Only OrCel™ Keratinocyte-Only OrCel™ Co-Cultured OrCel™ Cytokine Production by Fibroblasts, Keratinocytes and Co-Cultured Cells

Confidential ABCD OrCel

Immunodetection of VEGF in OrCel- Treated Nude Mice

Comparative Histological Profiles ApligrafOrCel

Output, OrCel TM and Apligraf bFGFGM-CSFHGFKGF-1VEGFIL-1aMMP-9PGE-2 Cytokine Productivity, pg/cm 2 /day OrCelApligraf

Effect of Fenestration on Cytokine Expression Day 1Day 2Day 1Day 2Day 1Day 2 bFGFKGF-1Il-1a Cytokine Per Unit Area, pg/cm 2 /day WholeFenes Day 1Day 2Day 1Day 2Day 1Day 2 bFGFKGF-1Il-1a Cytokine Per Unit Area, pg/cm 2 /day WholeFenes.

Effect of Fenestration on Cytokine Expression Cytokine Production by Whole vs. Fenestrated OrCel Day 1Day 2Day 1Day 2Day 1Day 2 GM-CSFMCSFVEGF Cytokine Per Unit Area, pg/cm 2 /day WholeFenes. Cytokine Production by Whole vs. Fenestrated Apligraf Day 1Day 2Day 1Day 2Day 1Day 2 GM-CSFMCSFVEGF Cytokine Per Unit Area, pg/cm 2 /day WholeFenes.

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 Cell Count

Cryo-OrCel vs. OrCel TM bFGFGM-CSFIL-1aKGF-1MCSFVEGF Cytokine Productivity, pg/cm 2 /day Fresh OrCelCryo OrCel

Cryo-ORCEL vs. Fresh ORCEL Equivalent keratinocyte and fibroblast cell density Equivalent cell viability Equivalent or better metabolic activity Essentially equivalent cytokine expression

Days to 100% Healing (Median) OrCel Standard of Care DONOR SITES Pivotal: 2/01 Based on Photography 22 Days 15 Days PMA Claims Accelerated healing Less Scarring Reduced time to recropping

Cryo-OrCel TM Preliminary Results Weeks Venous Leg Ulcers Percentage of Patients Achieving 100% Wound Closure Pilot: 8/00  Cry0-OrCel  Standard of Care %

OrCel ™ Heals Wounds Faster Diabetic Foot Ulcers % of Patients Achieving 100% Wound Closure By 12 Weeks Ortec Standard of Care 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