Mechanisms of Wound Healing: Studies with the Bilayered Cellular Matrix, OrCel TM Melvin...

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, 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 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

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/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

Scanning EM of Collagen Sponge (cross-section)

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

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

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

OrCelTM Cell Composition by Flow Cytometry

7.6%

92.3%

Cel

ls

Fluorescence

(FITC – pan-cytokeratin)

Ungated

OrCel™ Histology

OrCelTM Histology (Ki67 Immunohistochemistry)OrCelTM Histology (Ki67 Immunohistochemistry)

ConfidentialConfidential

Immunohistochemical Screening for HLA-DR Antigen in Foreskin Tissue vs. OrCelTM

10X 20X

10X 20X

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

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

Immunodetection of VEGF in OrCel-Treated Nude Mice

Comparative Histological ProfilesComparative Histological Profiles

Apligraf OrCel

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

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


bFGF KGF-1 Il-1a

Cyt

oki

ne

Pe

r U

nit

Are

a,

pg

/cm

2 /d

ay

Whole Fenes.

Effect of Fenestration on Cytokine Expression

Cytokine Production by Whole vs. Fenestrated OrCel

10

100

1000

10000


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


GM-CSF MCSF VEGF

Cyt

oki

ne

Pe

r U

nit

Are

a,

pg

/cm

2 /d

ay

Whole Fenes.

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 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

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

Cryo-ORCEL vs. Fresh ORCEL

• Equivalent keratinocyte and fibroblast cell density

• Equivalent cell viability• Equivalent or better metabolic activity• Essentially equivalent cytokine expression

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

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

%

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

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

Mechanisms of Wound Healing: Studies with the Bilayered Cellular Matrix, OrCel TM Melvin...

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