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![Page 1: Mechanisms of Wound Healing: Studies with the Bilayered Cellular Matrix, OrCel TM Melvin Silberklang, Ph.D. Chief Scientific Officer and Vice President,](https://reader035.fdocuments.in/reader035/viewer/2022062421/56649cf95503460f949cb33b/html5/thumbnails/1.jpg)
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.
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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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Collagen Sponges Collagen Sponges
Lamination with Collagen GelLamination with Collagen Gel
Inoculation with CellsInoculation with Cells
OrCelOrCel
Culture 9 – 14 DaysCulture 9 – 14 Days
Cryopreservation (P1)Cryopreservation (P1)
Cryopreservation (P3)Cryopreservation (P3)
Cryopreservation (P1)Cryopreservation (P1)
Cryopreservation (P3/P4)Cryopreservation (P3/P4)
Keratinocyte Cell Line Expansion
Keratinocyte Cell Line Expansion
Fibroblast Cell Line Expansion
Fibroblast Cell Line Expansion
Enzymatic Detachment of Epidermal and Dermal Layers Enzymatic Detachment of Epidermal and Dermal Layers
Neonatal ForeskinNeonatal ForeskinNeonatal ForeskinNeonatal Foreskin
OrCel™ Manufacturing Process
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Scanning EM of Collagen Sponge (cross-section)
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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
Safety Testing of Donor and of Allogeneic Cells
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P-63
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Bcl-2
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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)
OrCelTM Wound Healing Rationale:Facilitated Tissue Regeneration
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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
7.6%
92.3%
Cel
ls
Fluorescence
(FITC – pan-cytokeratin)
Ungated
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OrCel™ Histology
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OrCelTM Histology (Ki67 Immunohistochemistry)OrCelTM Histology (Ki67 Immunohistochemistry)
ConfidentialConfidential
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Immunohistochemical Screening for HLA-DR Antigen in Foreskin Tissue vs. OrCelTM
10X 20X
10X 20X
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GMCSF PGE2 VEGF
Rat
e of
Sec
retio
n in
to C
ultu
re M
ediu
m,
pg/c
m2
/24
hour
s
0
2000
4000
6000Fibroblast-Only OrCel™Keratinocyte-Only OrCel™Co-Cultured OrCel™
bFGF KGF-1R
ate
of S
ecre
tion
into
Cul
ture
Med
ium
, pg
/cm
2/2
4 ho
urs
0
25
50Fibroblast-Only OrCel™Keratinocyte-Only OrCel™Co-Cultured OrCel™
Cytokine Production by Fibroblasts,Keratinocytes and Co-Cultured Cells
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Comparison of CCS Cytokine Productionwith that of Acute Human Skin Wounds
(Ure et al., 1998; Vogt et al., 1998)
bFGF
GM-C
SFIL
-1a
IL-1
b
KGF-1
M-C
SF
TGF-a
TGF-b1
TGF-b2
TNF-a
VEGFCyt
okin
e O
utpu
t Per
Uni
t Are
a, p
g/cm
2/d
ay
1
10
100
1000
10000 CCS Production Human Wounds
(No
Da
ta)
(No
Da
ta)
(No
Da
ta)
(No
Da
ta)
(No
Da
ta)
ConfidentialABCD
OrCel
OrCel
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Immunodetection of VEGF in OrCel-Treated Nude Mice
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Comparative Histological ProfilesComparative Histological Profiles
Apligraf OrCel
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Output, OrCelTM and Apligraf
10
100
1000
10000
100000
bFGF GM-CSF HGF KGF-1 VEGF IL-1a MMP-9 PGE-2
Cyt
ok
ine
Pro
du
cti
vit
y, p
g/c
m2/d
ay
OrCel Apligraf
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Effect of Fenestration on Cytokine Expression
10
100
1000
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
bFGF KGF-1 Il-1a
Cyt
oki
ne
Pe
r U
nit
Are
a,
pg
/cm
2 /d
ay
Whole Fenes.
10
100
1000
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
bFGF KGF-1 Il-1a
Cyt
oki
ne
Pe
r U
nit
Are
a,
pg
/cm
2 /d
ay
Whole Fenes.
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Effect of Fenestration on Cytokine Expression
Cytokine Production by Whole vs. Fenestrated OrCel
10
100
1000
10000
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
GM-CSF MCSF VEGF
Cyt
oki
ne
Pe
r U
nit
Are
a,
pg
/cm
2 /d
ay
Whole Fenes.
Cytokine Production by Whole vs. Fenestrated Apligraf
1
10
100
1000
10000
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
GM-CSF MCSF VEGF
Cyt
oki
ne
Pe
r U
nit
Are
a,
pg
/cm
2 /d
ay
Whole Fenes.
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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 CountCell Count % Viability Met. Activity
Re
lati
ve
Pe
rfo
rma
nc
e
0
20
40
60
80
100
120
Pre-Cryo Post-Thaw Post-Thaw + 48 Hr
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Cryo-OrCel vs. OrCelTM
10
100
1000
10000
bFGF GM-CSF IL-1a KGF-1 MCSF VEGF
Cyt
ok
ine
Pro
du
cti
vit
y, p
g/c
m2/d
ay
Fresh OrCel Cryo OrCel
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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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Days to100% Healing
(Median)
OrCel
Standard of Care
DONOR SITESDONOR SITES
Pivotal: 2/01
Based on Photography
0
2
4
6
8
10
12
14
16
18
20
2222 Days
15 Days
PMA Claims
•Accelerated healing
•Less Scarring
•Reduced time to recropping
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Cryo-OrCelTM Preliminary Results Cryo-OrCelTM Preliminary Results
Weeks
Venous Leg UlcersVenous Leg Ulcers
Percentage of Patients
Achieving 100% Wound Closure
Pilot: 8/00
Cry0-OrCel
Standard of Care
%
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OrCel OrCel ™™ Heals Heals Wounds FasterWounds Faster
47%
23%
0
10
20
30
40
50
60
Diabetic Foot Ulcers
% of Patients Achieving 100% Wound Closure
By 12 Weeks
Ortec
Ortec
Standard of Care
Standard Of Care
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ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
Lee McDonaldLee McDonaldAvi LasdunAvi LasdunSandy LermanSandy LermanStephanie KnightStephanie KnightDan DwyerDan Dwyer
Sunny LukeSunny LukeRachel TewariRachel TewariTina HuangTina HuangNameeta ChimanjiNameeta ChimanjiLara SilberklangLara Silberklang
Melissa SteinerMelissa SteinerFlora ElequinFlora Elequin
Celina ChoiCaroline Tang
Steven PeltierLiza MooreKathy Tygum
Nitya RayHsin-Chien TaiAlla LauferYing Song