SKIN TISSUE ENGINEERING:

From the biomaterials

development to in vivo tests

SKIN IS A COMPLEX TISSUE…AN ORGAN

http://trekity.com/skin-care-routine/

D. Markeson, et al., J Tissue Eng Regen Med, 2013

MAIN NORMAL SKIN COMPONENTS

Adapted from Kern et al., 2011

ADULT NORMAL WOUND HEALING EVENTS

Adapted from M. T. Cerqueira, R. P. Pirraco, R. L. Reis, A. P. Marques, Advances in Wound Care, 2016, 5(4): 164-175. doi:10.1089/wound.2014.0607

Gurtner, et. al, Wound Repair and Regeneration, Nature 453, 314-321

Repair

Regeneration

Scar

Myofibroblast presence - Contribution to wound contraction

Non-functioning mass of fibrotic tissue

Adaptation of normal skin to tissue damage

No Scar

Restoration of normal structure and function of the injured tissue

Functional tissue

Regenerative potential in Adult mammals is limited

Wound Healing

REGENERATION VS REPAIR

COMPARISON OF FETAL SCARLESS HEALING AND

HYPERTROPHIC SCAR FEATURES

S. Sanon et al., Skin Tissue Engineering and Regenerative Medicine, 2016, Pages 19–47

Worldwide Wound Management

MAIN PROBLEMATIC IN SKIN HOMEOSTASIS

Source: MedMarket Diligence, LLC, Report #S190

R. Harries et al., Skin Tissue Engineering and Regenerative Medicine, 2016, Pages 125–143

PRODUCT

CLINICALLY

AVAILABLE

SINCE

DESCRIPTION INDICATION

Integra Integra Life Sciences 1984 Polysiloxane membrane and complex

matrix (collagen and glycosaminoglycans) Burns

Transcyte Advanced Tissue

Sciences 1997

Silicone membrane and ECM from neonatal human fibroblast cells cultured on

a nylon mesh DFU

Alloderm Life Cell 2001 Decellularized human cadaver dermis Soft Tissue

Repair

PRODUCT

CLINICALLY

AVAILABLE

SINCE

DESCRIPTION INDICATION

Epicel Genzyme Biosurgery 1987 Autologous keratinocytes and xenogenic proliferation arrested

mouse fibroblasts in petroleum gauze dressing Full thickness burns taking >30%

of body area

Epidex Modex Therapeutics 1999 Cultured epidermal skin equivalent derived from keratinocyte

precursors of human hair follicles Chronic Leg ulcers

Dermagraft Advanced Biohealing Inc 1999 Allogeneic neonatal fibroblasts seeded in a polyglycolic acid (Dexon)

or polyglactin-9-10Vicryl scaffold. Full thickness DFU; Epidermolysis

Bullosa

Transcyte Advanced Biohealing Inc 1999 Human allogeneic fibroblasts cultured on a nylon mesh pre-coated

with collagen Burns; Transparent dressing

Laserskin and Hyalograft Fidia Farmaceutical 2000 Autologous fibroblasts and keratinocytes cultured on a hyaluronic

acid laser perforated membrane DFU and Chronic

Wounds

Orcel Ortec International 2001 Type 1 Bovine collagen seeded with allogeneic neonatal fibroblasts

and keratinocytes Donor sites for autografting, DFU;

Epidermolysis Bullosa

Apligraft /Graftskin Organogenesis 2001 Allogeneic neonatal foreskin keratinocytes and fibroblasts seeded in

a type 1 bovine collagen

DFU; Venous leg ulcers; Partial thickness burns; Epidermolysis

Bullosa

Bioseed-S BioTissue Technologies 2001 Autologous keratinocytes resuspended in a fibrin sealant Venous leg ulcers

Myskin Altrika 2008 Autologous keratinocytes grown on a silicone layer with irradiated

murine fibroblasts Non-healing wounds; DFU, Burns,

Pressure ulcers

Permaderm Regenicin

Pre-market approval

Autologous keratinocytes and fibroblasts seeded on collagen biomaterial Burns; Chronic Wounds

AC

EL

LU

LA

R

CE

LL

UL

AR

SKIN SUBSTITUTES AND THE CLINICAL APPLICABILITY

SKIN REGENERATION: THE GOAL TO ATTAIN

M. T. Cerqueira, et al., unpublished data

Totipotency All somatic cell types,

germ cells, Placenta

Fertilized Egg

Pluripotency All somatic Cell Types,

Germ Cells

Blastocyst – Inner Cell Mass

Multipotency Lineage-restricted cell

types

Unipotency

Neural Stem Cell

Mesenchymal Stem Cells

Epidermal Stem Cells

Single Cell Type

Eckfeldt et al, Nat Rev Molecular Cell Biology, doi:10.1038/nrm1713

TYPES OF STEM CELLS

PROMISE OF STEM CELLS AS THERAPY FOR SKIN REGENERATION

M. T. Cerqueira, R. P. Pirraco, R. L. Reis, A. P. Marques, Advances in Wound Care, 2016, 5(4): 164-175. doi:10.1089/wound.2014.0607

M. T. Cerqueira, A. P. Marques, R. L. Reis, Using Stem Cells in Skin Regeneration: Possibilities and Reality. Stem Cells and Development, 21 (2012) 1201-1214, doi: 10.1089/scd.2011.0539

BUILDING BLOCKS : DIFFERENTIATION OF STEM CELLS IN

EPIDERMAL LINEAGE

STEM CELLS AS SECRETOME UNITS DURING HEALING

M. T. Cerqueira, R. P. Pirraco, R. L. Reis, A. P. Marques, Advances in Wound Care, 2016, 5(4): 164-175. doi:10.1089/wound.2014.0607

POSSIBLE STRATEGIES TO MAXIMIZE STEM CELLS EFFECT

M. T. Cerqueira, R. P. Pirraco, R. L. Reis, A. P. Marques, Advances in Wound Care, 2016, 5(4): 164-175. doi:10.1089/wound.2014.0607

SKIN EXTRACELLULAR-MATRIX (ECM)

M. Climov et al., Skin Tissue Engineering and Regenerative Medicine, 2016, Pages 145–161

M. T. Cerqueira, R. L. Reis, A. P. Marques,, , Current Tissue Engineering, 2 (2013) 145

SKIN EXTRACELLULAR MATRIX (ECM)

Glycosaminoglycans

Proteoglycans

Collagens

Non-collagenous glycoproteins (Elastin,

Laminin, Fibronectin)

THE IMPORTANCE OF CELL-ECM INTERACTIONS

DIFFERENT TYPES OF ECM-MIMICKING

M. Climov et al., Skin Tissue Engineering and

Regenerative Medicine, 2016, Pages 145–161

Natural Vs Artificial ECM – Example at 3B’s Research Group

hASCs CS, after 5 days in culture

Phalloidin/DAPI

Trigger local responses towards

the regenerative pathway

Deposited ECM

Intact

- Cell-Cell

- Cell-Matrix

• High water content

• Enhanced

microstructural and

mechanical

performance

• Cell adhesive

features

• Facilitated diffusion

Freezing Freeze-

-drying

Re-

-hydration

Frozen

Hydrogel Hydrogel Sublimated

Hydrogel

Dried

Polymeric Networks Spongy-like

Hydrogel

da Silva LP, … , Reis RL, Marques AP Acta Biomater, 2014

da Silva LP, …, Reis RL, WO/2014/167513, 2014

HYDROPHILIC

DEADHESION

HYDROPHOBIC

CELL ADHESION

Above 32ºC

Below 32ºC

Adapted from http://www.twmu.ac.jp/ABMES/ja/cellsheet

Natural ECM Cell Sheet Engineering

Artificial ECM Spongy-like Hydrogels

Human Adipose-derived Stem Cells

Human Adipose-derived

Microvascular Endothelial Cells

Human Microvascular

Endothelial Cells

Total cells from the

epidermal fraction

Total cells from the

dermal fraction

ONGOING: Human

dermal fibroblasts, ...

Human keratinocytes

Melanin

Hyaluronic acid

SPONGY-LIKE HYDROGELS

CELL SHEETS Human

Microvascular

Endothelial Cells

Human keratinocytes

Human dermal

fibroblasts

SKIN LINEAGES

Human Adipose-derived

Stem Cells

STEM CELLS

SPONGY-LIKE HYDROGELS AND CELL SHEETS VERSATILITY

CELL SHEET TECHNOLOGY FOR SKIN WOUND HEALING

H &E

Collagen I

Rete Ridges -like Structures

Hair-Folicle Formation

Early Neovascularization

hD

Fb

+ h

DM

EC

s +

hK

Cs

hD

Fb

+ h

KC

s

hD

Fb

+ h

DM

EC

s

Enhanced Re-epithelialization

HUMAN ADIPOSE STEM CELLS

PROMOTED EPIDERMAL

REGENERATION

SKIN CELLS CELL SHEETS IMPACTED

WOUND HEALING, DEPENDING ON THEIR

CELLULAR COMPOSITION

Cell sheets

Cerqueira MT…Marques AP, Acta Biomaterialia, 2014 Cerqueira MT ….Marques AP, Biomacromolecules 2013

Stable Stem Cell-based

Cell Sheet Constructs

GELLAN GUM-BASED SPONGY-LIKE HYDROGELS

90°C

37°C

CaCl2

Ca2+

PBS

Na+

K+

Medium

Na+

K+

Ca2+

Temperature

decrease

Linear repeating tetrasaccharide unit composed of glucose, rhamose

and glucuronic acid.

Oliveira, J. T. et al. Gellan Gum Based Hydrogels For Regenerative Medicine And Tissue Engineering Applications, Its System, And Processing Devices,

WO/2009/101518 ; PCT/IB2009/000258

Da Silva L. P.. et Al. Gellan Gum Spongy-like Hydrogel, Its Prepararion And Biomedical Applications Thereof, WO 2014/167513 ; PCT/IB2014/060563

Hydrogel

Spongy-like Hydrogel

TAILORING SPONGY-LIKE HYDROGELS PROPERTIES

Hydrogel Spongy-like Hydrogel

GG

0.7

5 %

G

G 1

.25

%

0

10

20

30

40

50

GG 0.75 %

GG 1.25 %

Hydrogel Spongy-like

hydrogel

***

Co

mp

res

siv

e M

od

ulu

s (

KP

a)

0

2000

4000

6000

GG 0.75 %

GG 1.25 %

Hydrogel Spongy-like

hydrogel

**

**

Wa

ter

co

nte

nt

(%)

L. P. da Silva,…A.P. Marques, Acta Biomaterialia 10 (2014) 4787-97

Da Silva L. P.. et Al. Gellan Gum Spongy-like Hydrogel, Its Prepararion And Biomedical Applications Thereof, WO 2014/167513 ; PCT/IB2014/060563

SPONGY-LIKE HYDROGELS PROCESSING VERSATILITY

L. P. da Silva,…A.P. Marques, Acta Biomaterialia 10 (2014) 4787-97 Da Silva L. P.. et Al. Gellan Gum Spongy-like Hydrogel, Its Prepararion And Biomedical Applications Thereof, WO 2014/167513 ; PCT/IB2014/060563

CELL ADHESION TO GELLAN GUM SPONGY-LIKE HYDROGELS

Hyd

rog

el

Sp

on

gy-l

ike

Hyd

rog

el

Phalloidin/DAPI

Phalloidin/DAPI

100 µm

100 µm

hASCs hDMECs hKCs0

20

40

60

80

100 ******

% o

f Entrapped C

ells

hDMECs

Pre

-so

akin

g in

fib

ron

ectin

100 µm

Phalloidin/DAPI

hKCs

100 µm

K14/Phalloidin/DAPI

Cell-type dependent

Sp

on

gy-l

ike

Hyd

rog

el

100 µm

Calcein/PI

100 µm

hASCs

Ki-67/DAPI

100 µm

hASCs

100 µm

hASCs

Phalloidin/DAPI

Absence of

serum

Sp

on

gy-l

ike H

yd

rog

el

L. P. da Silva,…A.P. Marques, Acta Biomaterialia 10 (2014) 4787-97 M. T. Cerqueira, …, A.P. Marques, Materials Today 18 (8), 468-469

SPONGY-LIKE HYDROGELS FOR SKIN REGENERATION

Early neovascularization in the presence of

endothelial cells

Vessel density (microvessels/mm2)

CD31 α-SMA

Ctrl 8.5 ± 3.5 7.9 ± 3.5

GG-HA 1% 17.7 ± 8.7 (p<0.001) 11.1 ± 3.8

GG-HA 2% 10.9 ± 3.3 10.0 ± 4.7

Innervation in the condition with stem cells

Neovascularization tailored by hyaluronic acid

degradation products

Day 7 Day 14 8 Weeks post injury

Con

trol

GG-H

A

GG-H

A +

hAS

Cs

hASCs

A

GG-H

A +

con

d

0

10

20

30

40

50

604 3 4 4

IEN

F

(ne

rve

s/m

m)

Ctrl

GG-HA + hASCs 20μm

20μm

Control Heterotypic spongy-like

hydrogel 20μm 20μm

GG-HA 2 % 100μm GG-HA 1 %

14 days 21 days

Improved Re-epithelialization

Neoepidermis Maturation

3 days 7 days

Early Neoepidermis Keratinocytes Migration

da Silva LP, … Marques AP ACS Appl. Mater. Interfaces, 2016 Cerqueira MT, …,Marques AP, Tissue Eng Part A, 2014

Cerqueira

MT, …,

Marques AP,

ACS Appl

Mater

Interfaces,

2014 da Silva LP, … Marques AP, J Invest Dermatol, 2017

SKIN REGENERATION: PROBLEMATIC OF VASCULARIZATION

Common methods of TE skin Pre-vascularization using different cell sources

J. Boulard et al., Skin Tissue Models, 2018, Pages 177–200

M. T. Cerqueira, A. P. Marques, R. L. Reis, Using Stem Cells in Skin Regeneration: Possibilities and Reality. Stem Cells and Development, 21 (2012) 1201-1214, doi: 10.1089/scd.2011.0539

ALTERNATIVE TO ENDOTHELIAL CELLS: DIFFERENTIATION

OF STEM CELLS

VASCULARIZED DERMAL SUBSTITUTES

J. Boulard et al., Skin Tissue Models, 2018, Pages 177–200

0 10 20 30 40 500.0

0.2

0.4

0.6

0.8

1.0CtrlGGHA 1%GGHA 2%

Time (days)

Perf

usio

n R

atio o

f is

ch

em

ic

limb

/non

-isch

em

ic lim

b

(%)

GELLAN GUM-BASED STRUCTURES WITH ANGIOGENIC POTENTIAL

GG

-HA

1%

2 weeks 4 weeks

Ctr

l G

G-H

A 2

%

Low

High

GG-HA 1%

*

m m

a

m

*

GG-HA 2%

a

Control

* - material, a – adipose tissue; m – muscle

Amount of HA fragments existing in the solutions collected from degradation (HA-F) after 7 (HA-Fa,c)

and 21 (HA-Fb,d) days of hyaluronidase (50U) degradation

HA1 HA2 HA4 HA>20

GGHA 1% HA-Fa 55,2 ± 13,0 % 7,0 ± 1,5 % 0,2 ± 0,2 % 1,5 ± 0,2 %

HA-Fb 55,8 ± 0,3 % 6,2 ± 3,2 % 0,0 ± 0,0 % 0,0 ± 0,0 %

GGHA 2%

HA-Fc 47,2 ± 2,3 % 5,5 ± 4,8 % 0,5 ± 0,1 % 8,4 ± 0,2 %

HA-Fd 64,3 ± 4,0 % 7,5 ± 7,2 % 0,1 ± 0,1 % 1,5 ± 1,4 %

Vessel density (microvessels/mm2)

CD31 α-SMA α-SMA/CD31

Ctrl 8.5 ± 3.5 7.9 ± 3.5 1.1 ± 0.5

GGHA 1% 17.7 ± 8.7 (p<0.0001) 11.1 ± 3.8 0.8 ± 0.3

GGHA 2% 10.9 ± 3.3 10.0 ± 4.7 1.3 ± 0.9

L. P. da Silva… A.P. Marques, ACS Applied Materials & Interfaces 8 (2016) 33464

IN VIVO MODELS FOR SKIN WOUND HEALING

I. Pastar et al., Skin

Tissue Models,

2018, Pages

223–253

SIMILARITIES AND DIFFERENCES BETWEEN ANIMALS/HUMANS

G. Ignacio, Skin Tissue Engineering and Regenerative Medicine, 2016, Pages 387–400

Need of human skin models that are representative of human skin mechanisms in healing and in skin diseases/disorders

DIFFERENT IN VITRO SKIN MODELS

C. Pellevoisin et al., Skin Tissue Models, 2018, Pages 3–37

Overview of different types of reconstructed skin models, depending on the cells incorporated, as well as the matrix/organization employed

SKIN MODELS FOR DRUG PERMEATION, METABOLISM AND EFFICACY

H. Ruffner, Skin Tissue Engineering and Regenerative

Medicine, 2016, Pages 357–386

Drug Delivery and Metabolism

Efficacy Studies

EPIDERMAL MODELS FOR SAFETY CHEMICAL EVALUATION

C. Pellevoisin et al., Skin Tissue Models, 2018, Pages 3–37

SKIN MODELS TO STUDY SKIN DISEASES - Genodermatoses

E. Chacón-Solano et al., Skin Tissue Models, 2018, Pages 77–102

SKIN MODELS TO STUDY SKIN DISEASES - Genodermatoses

E. Chacón-Solano et al., Skin Tissue Models, 2018, Pages 77–102

SKIN MODELS TO STUDY SKIN DISEASES – Squamous Carcinoma

Normal 3D Organotypic Skin Models

3D Skin Cancer invasion Model

Characterization of the invasion process

Demonstration of tumor-cell-specific invasion

M. Berning et al., Skin Tissue Models, 2018, Pages 151–173

SKIN MODELS FOR WOUND-HEALING STUDIES

I. Pastar et al., Skin Tissue Models, 2018, Pages 223–253