Transcript Clinical Experience with the use of OrCelTM in Recessive

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
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(NoDat)
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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