Mechanisms of Wound Healing: Studies with the Bilayered Cellular Matrix, OrCelTM

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Mechanisms of Wound Healing Studies with the
Bilayered Cellular Matrix, OrCelTM

• Melvin Silberklang, Ph.D.
• Chief Scientific Officer and
• Vice President, RD
• Ortec International, Inc.

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Clinical Application of OrCelTM
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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,
  non-porous side of the sponge.

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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

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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

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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
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Scanning EM of Collagen Sponge (cross-section)

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Safety Testing of Donor and of Allogeneic Cells
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P-63
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Bcl-2
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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)

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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

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OrCelTM Cell Composition by Flow Cytometry
Ungated
7.6
92.3
Cells
Fluorescence (FITC pan-cytokeratin)
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OrCel Histology
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OrCelTM Histology (Ki67 Immunohistochemistry)
Confidential
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Immunohistochemical Screening for HLA-DR Antigen
in Foreskin Tissue vs. OrCelTM
20X
10X
10X
20X
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Cytokine Production by Fibroblasts, Keratinocytes
and Co-Cultured Cells
/24 hours
6000
/24 hours
Fibroblast-Only OrCel
Fibroblast-Only OrCel
Keratinocyte-Only OrCel
50
Keratinocyte-Only OrCel
2
Co-Cultured OrCel
2
Co-Cultured OrCel
4000
Rate of Secretion into Culture Medium, pg/cm
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Rate of Secretion into Culture Medium, pg/cm
2000
0
0
GMCSF
PGE2
VEGF
bFGF
KGF-1
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OrCel
OrCel
ABCD
Confidential
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(No Transcript)
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Immunodetection of VEGF in OrCel- Treated Nude
Mice
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Comparative Histological Profiles
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Output, OrCelTM and Apligraf
100000
OrCel
Apligraf
10000
Cytokine Productivity, pg/cm2/day
1000
100
10
bFGF
GM-CSF
HGF
KGF-1
VEGF
IL-1a
MMP-9
PGE-2
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Effect of Fenestration on Cytokine Expression
1000
1000
Whole
Fenes.
Whole
Fenes.
100
Cytokine Per Unit Area, pg/cm2/day
100
Cytokine Per Unit Area, pg/cm2/day
10
10
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
bFGF
KGF-1
Il-1a
bFGF
KGF-1
Il-1a
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Effect of Fenestration on Cytokine Expression
Cytokine Production by Whole vs. Fenestrated OrCel
Cytokine Production by Whole vs. Fenestrated
Apligraf
10000
10000
Whole
Fenes.
Whole
Fenes.
1000
1000
Cytokine Per Unit Area, pg/cm2/day
Cytokine Per Unit Area, pg/cm2/day
100
100
10
1
10
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
GM-CSF
MCSF
VEGF
GM-CSF
MCSF
VEGF
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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

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Retention of Functionality after Cryopreservation
Cell Count
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Cryo-OrCel vs. OrCelTM
10000
Fresh OrCel
Cryo OrCel
1000
Cytokine Productivity, pg/cm2/day
100
10
bFGF
GM-CSF
IL-1a
KGF-1
MCSF
VEGF
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Cryo-ORCEL vs. Fresh ORCEL

• Equivalent keratinocyte and fibroblast cell
  density
• Equivalent cell viability
• Equivalent or better metabolic activity
• Essentially equivalent cytokine expression

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DONOR SITES


? OrCel Standard of Care
22 Days
Days to 100 Healing (Median)
?
15 Days

• PMA Claims
• Accelerated healing
• Less Scarring
• Reduced time to recropping

Pivotal 2/01 Based on Photography
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Venous Leg Ulcers
Cryo-OrCelTM Preliminary Results

Percentage of Patients Achieving 100 Wound
Closure

• Cry0-OrCel
• Standard of Care

Weeks
Pilot 8/00
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Diabetic Foot Ulcers
OrCel Heals Wounds Faster
of Patients Achieving 100 Wound Closure By
12 Weeks

• ? Ortec
• ? Standard of Care

Ortec
Standard Of Care
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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