Designing natural bases scaffolds for

cartilage tissue engineering applications

Vitor M. Correlovitorcorrelo@dep.uminho.pt

3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the

European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas,

Guimarães, Portugal

ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

BRAGA

TAIPAS

GUIMARÃES

U. MINHO

U. Minho, Portugal - Braga & Guimarães

What are we doing?

Bone │ Cartilage │ Skin │ IVD │ Tendons │ Meniscus

Tissue engineering & Regenerative medicine

Natural polysaccharides:Starch; Chitosan; Carragenan; Alginates; Ulvan; Hyaluronic acid; Gellan gum; Chondroitin sulfate;

Natural, protein based:Silk fibroin; Marine collagen; Soy bean;

Natural mimics: Synthetic

peptides

Cell lines:SaOs-2, L292; etc...

Stem/progenitor cells:Bone Marrow; Adipose Derived;Amniotic Fluid/membrane; Umbilical

Cord; Blood and derivatives; hESC

Primary cells:Fibroblasts, Chondrocytes, Keratinocytes, Endothelial cells

Biomaterials Cells

│ CNS

Medicine

Materials

Science

Materials

Engineering

Life Sciences

NATURE

Biology

Biotechnology

Chemistry

3B’s

A TRULLY MULTIDISCIPLINARY GROUP

3600 m2 new state of the art full equipped facility specially

designed for TERM research

European Institute of Excellence on Tissue Engineering and Regenerative Medicine - Headquarters

Headquarters and the Home for 3B´s Research – Opened in July 2008,

AvePark, Taipas - Guimarães, Minho, Portugal

* CEO – Rui L. Reis

Our facilitiesThe Institute's laboratory facilities occupy 3600 m2 including:Chemistry labs Processing labs Characterisation labs

Cell culture labs

Bioreactor labs

Microscopy labs Histology labs

Animal testing facilities

Tissue Engineering (simplistic scheme)

Scaffolds Growth Factors

Cells

Medium

Natural based polymers at 3B’s

polysaccharides

neutr

al

poly

anio

nic

carboxylatedsulfated

polycationic

starchdextran

chitin

chitosan

carragenan(k, i, l)

chondroitin sulfate ulvan

alginate

hyaluronic acid

gellan gum

proteins

silk fibroin

collagen fibronectin

polyesters

poly(hydroxybutyrate)

J.F. Mano +, J.R.Soc.Interface ‘07

Bone Marrow (Rat, Rabbit, Goat, Human; mouse)

Adipose Tissue (Rat, Rabbit, Goat, Human)

Amniotic Fluid / Membrane ( Human)

Umbilical Cord (Human)

Blood (and derivatives)( Human)

Formal protocols with Hospitals and other Health Care Institutions:

Collaboration with other Universities

(Medical and Veterinary Sciences):

STEM/PROGENITOR CELLS SOURCES

Stem/progenitor cells sources:

Centro

Hospitalar do

Alto Ave

Material processing

What does distinguish us?

In house facilities for FULL biomaterial development

Cell isolation

Material characterisation

Cell expansion

Cell characterisation

Material extraction & purification

Cell seeding In vitro testing

In vivo testing

The 3B’s Research Group is developing innovative strategies aiming at

regenerating:

• Bone

• Cartilage• Osteochondral defects

• Skin

• Intravertebral disc (IVD)

• Neurological tissues

• (with ICVS – UM)

• Tendons

• Meniscushttp://www.ptei.org

THE MAIN TISSUES WE TRY TO ENGINEER

TYPES OF CARTILAGE

http://www.millerplace.k1

2.ny.us/webpages/lmiller

/photos/636532/Cartilag

e%20Types.bmp

ARTICULAR CARTILAGE

http://www.aclsolutions.com/anatomy.php

Avascular tissue that covers the

joint

Provides a low friction gliding

surface

Acts as a load-bearing and wear

control structure

Composed by chondrocytes and

a dense extracellular matrix (ECM)

ARTICULAR CARTILAGE STRUCTURE

Adapted from van Blitterswijk , Tissue Engineering, 2008

Matrix component tightly organized

Structure dependent on the collagen fibers alignment

ARTICULAR CARTILAGE CHARACTERISTICS

Lacks ihnate abilities to mount

a sufficient healing responseAdapted from Huey et al., Science, 2012

Avascular: lack of nutrient supply

No speciallized cells in cartilage

remodeling

Low metabolic activity

Inability of bone marrow MSCs or

resident chondroprogenitor cells to

generate hyaline cartilage

ARTICULAR CARTILAGE DAMAGE AND DISEASES

Rheumatoid arthritis

Osteoarthritis

Traumatic accident or injury

Wear and tear over time

http://myfitnessdepot.com/outdoors/runni

ng-outdoors/5-ways-to-avoid-knee-

injuries-while-running/

Treatments include medication and/ or

surgery:

Microfracture

Autologous Chondrocyte Implantation (ACI)

Matrix-Induced Chondrocyte Implantation (MACI)

CARTILAGE TISSUE ENGINEERING

CELLS

Stem cells from

different sources

Chondrocytes

SCAFFOLDS

Natural or synthetic

materials

Different processing

techniques

CULTURE CONDITIONS

Dynamic

Hypoxia; Co-cultures….

BIOACTIVE AGENTS

SCAFFOLD MATERIALS FOR CARTILAGE TE

SYNTHETICNATURAL

Starch

Chitosan

Silk

Polycaprolactone (PCL)

Poly lactic acid (PLA)

Poly(butylene) succinate

(PBS)

SCAFFOLDS PROCESSING

http://www.nist.gov/mml/bbd/biomaterials/images/fig-4_1.jpg

CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS

To

luid

ine

Blu

eA

lcia

nB

lue

CPBS

Co

lla

ge

n t

yp

e I

I

Alves da Silva et al., Acta Biomaterialia, 2009

• bovine articular chondrocytes

• histological analysis after 28

days

CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS

To

luid

ine

Blu

eA

lcia

nB

lue

20 xCPBS

Co

lla

ge

n t

yp

e I

I

Alves da Silva et al., Acta Biomaterialia, 2009

• bovine articular chondrocytes

• histological analysis after 28

days

CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS

To

luid

ine

Blu

eA

lcia

nB

lue

20 x

20 x

CPBS

Co

lla

ge

n t

yp

e I

I

Alves da Silva et al., Acta Biomaterialia, 2009

• bovine articular chondrocytes

• histological analysis after 28

days

CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS

To

luid

ine

Blu

eA

lcia

nB

lue

20 x

20 x

CPBS

Co

lla

ge

n t

yp

e I

I

20 x

Alves da Silva et al., Acta Biomaterialia, 2009

• bovine articular chondrocytes

• histological analysis after 28

days

CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS

CPBTA

20 µm

200 µm

20 µm

200 µm

To

luid

ine

Blu

eS

afr

an

inO

Alves da Silva et al., JTERM, 2011

AGGRECAN COLLAGEN II COLLAGEN I

SOX9 COLLAGEN X RUNX2

• human mesenchymal stem cells isolated

from bone marrow aspirates

• 28 days

CARTILAGE TE USING NANOFIBER MESHES

To

luid

ine

Blu

eA

lcia

nB

lue

Co

lla

ge

n t

yp

e I

I

PCL

Alves da Silva et al., Tissue Engineering, 2009

50 µm50 µm

100 µm

100 µm

• using bovine articular

chondrocytes

• 28 days

CARTILAGE TE USING NANOFIBER MESHES

To

luid

ine

Blu

eA

lcia

nB

lue

Co

lla

ge

n t

yp

e I

I

SPCL

Alves da Silva et al., Tissue Engineering, 2009

50 µm

100 µm

100 µm

100 µm

• using bovine articular

chondrocytes

• 28 days

CARTILAGE TE USING PCL NANOFIBER MESHES

CULTURED ON A FLOW PERFUSION BIOREACTOR

Alves da Silva et al., Biomacromolecules, 2010

100 µm100 µm

100 µm100 µm

50 µm 50 µm

50 µm50 µm

BIO

RE

AC

TO

RS

TA

TIC

TOLUIDINE BLUE SAFRANIN O

AGGRECAN COLLAGEN I COLLAGEN II

SOX9 COLLAGEN X RUNX2

hBMSCs

28 Days

PCL NFM MODIFICATION WITH CHONDROITIN

SULFATE20 µm

CARTILAGE-RELATED GENES

ECM PRODUCTION

Piai J, Alves da Silva et al., Submitted, 2014

MORPHOLOGICAL ANALYSIS

CS

-Im

mo

bil

ized

PC

L

NF

Ms

PC

L N

FM

s

14 Days 21 Days 28 Days

CARTILAGE ENGINEERING

Adapted from Kang et al., Biomaterials, 2012

CARTILAGE CO-CULTURES CONCEPT

The secreted molecules/ proteins provide a rich culture

medium able to influence chondrogenesis without the

addition of growth factors

CONDITIONED MEDIUM

Paracrine signalling of

chondrocytes will influence

the stem cells chondrogenic

differentiation

http://humanphysiology2011.wikispaces.com/file/view/paracrine-

signal.jpg/198153030/paracrine-signal.jpg

STEM CELLS CULTURES

Static culture

during 4 weeks

Cell source

Bone marrow

Articular

cartilage

Umbilical cord –

Wharton´s Jelly

Dynamic cell

seeding in a rotator

during 24 hours

CPBS fiber meshes

Indirect co-culture

using conditioned

medium from human

articular

chondrocytes

(hACs), without

growth factor

supplementation

hBMSCs hWJSCs

Alves da Silva et al., JTERM, 2013

GLYCOSAMINOGLYCAN PRODUCTION AND

CARTILAGE-RELATED GENES EXPRESSION

hWJSCs produced

significantly higher amounts

of GAGs when compared to

hBMSCs.Cultures using hWJSCs expressed higher

levels of cartilage related genes compared

to hBMSCs.

Alves da Silva et al., JTERM, 2013

CONCLUSIONS

CS-immobilized PCL NFMs represents a better substrate

for the maintenance of hACs phenotype, namely its

typical round shape and cellular agglomeration/

clustering, without affecting cells viability, proliferation

and ECM production.

Therefore, CS-functionalized electrospun nanofibers

represent a valuable substrate for culturing human

articular condrocytes, envisioning a cartilage tissue

engineering application.

Silk Fibroin-Based Scaffolds, Hydrogels

and Calcium-Phosphate Filled Materials

Aimed for Regenerative Medicine

Applications

Le-Ping YanDoctoral Program in Tissue Engineering, Regenerative Medicine and Stem Cells

Supervisors: Prof. Rui L. Reis, Dr. J. Miguel Oliveira and Dr. Ana L. Oliveira

13B's Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European

Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, S. Cláudio de Barco, 4806-909 Taipas,

Guimarães, Portugal. 2ICVS/3B’s, PT Government Associated Laboratory, Braga/Guimarães, Portugal.

2014

Bone fracture

http://www.springerimages.com/Imag

es/RSS/1-10.1007_s00590-010-

0732-3-5

Cartilage lesion

http://www.eorthopod.com/content/articul

ar-cartilage-problems-knee

Osteochondral defect (OCD)

http://www.hughston.com/hha/a_12_

4_1.htm

Background: Defects in skeletal tissues

Large defects in skeletal tissues are common problems in clinics

Current clinical strategies

http://thedenverclinic.com

Allograft

http://www.cartilagerestoration.org

Autograft ADVANTAGESDesirable clinical outcome

DISADVANTAGES

Autograft: limited supplies; donor site

morbidity;

Allograft: risk of disease transmission.

Silk fibroin

Easy access

Biocompatible

Biodegradable

Tunable properties

Advantages

http://wwwchem.uwimona.edu.jm

Main amino acid in silk fibroin

Silk fibroin

Biomaterials 2010,

31:4583-4591

Microspheres

Biomaterials 2005, 26:2775-2785

Porous scaffold

500 µm

Hydrogel

1 cm

Advanced Functional Materials

2005, 8:1241-1247.

Film

200 µm

Non-woven net

Biomaterials 2004, 25: 1069-1075

Main sequence of silk fibroin

http://en.wikipedia.org/wiki/Fibroin

http://phys.org/news/2012-

02-stretchable-spider-

silk.html

Spider silk

Antheraea cocoon

http://j-y-

m.deviantart.com/art/Antheraea-mylitta-

cocoon-91998659

Bombyx mori cocoon

http://www.shutterstock.com

Development of robust silk fibroin scaffolds and

used them as a platform for the following studies

Advantages:

Interconnected porous structure;

All aqueous procedures;

Environmentally friendly

Disadvantage:

Silk scaffolds cannot be prepared

with more than 10% silk solution

Kim UJ... Kaplan DL. Biomaterials, 2005, 26, 2775-2785

Aqueous derived salt-leaching approach

Silk scaffold: Preparation procedure

Cocoon Silk fibroin Concentrated silk solution

Scaffolds in wet status

DegummingDissolution and dialysis

Concentration by

PEG solution

Addition of

NaCl particles

Freeze-drying

Scaffolds prepared with 8, 10, 12 and 16% solutions were

named as silk-8, silk-10, silk-12 and silk-16, respectively.

Silk-12 Silk-16 3 mm

Silk-10Silk-8

Extraction

in water

Yan LP… Reis RL. Acta Biomater. 2012, 8(1):289-301.

16% silk solution

Addition of CaCl2 and

(NH4)2HPO4 solutions

Silk-NanoCaP suspension

Addition of NaCl particles

Salt-leaching/freeze-drying

Salt leached silk/CaP scaffolds

3 mm

3 mm

3 mm

3 mm

Silk/CaP-4

Silk/CaP-25Silk/CaP-16

Silk/CaP-8

Silk-NanoCaP scaffold: Preparation procedure

pH 8.5, aging for 24

hours

Groups

Theoretical

CaP content

(%)

Silk/CaP-4 4

Silk/CaP-8 8

Silk/CaP-16 16

Silk/CaP-25 25

Yan LP… Reis RL. Nanomedicine 2013, 8(3):359-378.

Yan LP… Reis RL. Nanomedicine 2013, 8(3):359-378.

Micro-CT image of pure CaP distribution Micro-CT image of CaP distribution in silk

3 mm

Silk/CaP-4 Silk/CaP-8

Silk/CaP-16 Silk/CaP-25

3 mm

Silk/CaP-4 Silk/CaP-8

Silk/CaP-16 Silk/CaP-25

Homogeneous distribution of CaP at macroscopic level

Silk-NanoCaP scaffold: 3D reconstruction (Micro-CT)

Grey: CaP phase White: CaP phase; Grey: Silk matrix

Development of robust and biomimetic silk based

scaffolds for OCD regeneration

Chondral layer: collagen,

glycosaminoglycan

Subchondral layer:spongy bone

Osteochondral tissue

http://www.aspetar.com/journal/viewartic

le.aspx?id=14#.U3seOPk7uSo

Bilayered scaffold: Scaffolds preparation and study designSilk-NanoCaP

suspension

NaCl particles

Dry for 2 days

Silk solution NaCl particles

Bilayered Silk/Silk-NanoCaP scaffold

Salt-leaching

overnight

Pores inside

the scaffolds

Subcutaneous implantation Implantation in knee OCDCulture with RBMSCs

Physicochemical

characterization

Yan LP… Reis RL. Submitted (2), 2014.

RBMSCs

rabbilt bone marrow

mesenchymal stromal cells

4 mm4 mm

0 2 4 6 80

20

40

60

80

100

Po

rosit

y d

istr

ibu

tio

n (

%)

Length (mm)

0 2 4 6 8

0

10

20

Ca

P d

istr

ibu

tio

n (

are

a %

)

Length (mm)

Integrated structure with distinct phase distribution

Bilayered scaffold: 3D reconstruction (Micro-CT)

Yan LP… Reis RL. Submitted (2), 2014.

Micro-CT image of the pure CaPMicro-CT image of the scaffold

Porosity distribution CaP distribution

Brown: Silk matrix

Blue: CaP phase

S16 SC16 Bilayered0

4

8

12

16

20

Co

mp

ress

ive m

od

ulu

s (

MP

a)

Dry status

S16 SC16 Bilayered0.0

0.1

0.2

0.3

0.4

0.5

Co

mp

res

siv

e m

od

ulu

s (

MP

a) Wet status

0.1 1 100.2

0.4

0.6

0.8

1.0

S16

SC16

Bilayered

E' (M

Pa

)Frequency (Hz)

Superior mechanical properties

Bilayered scaffold: Mechanical evaluation

n=6

n=5

n=6

Yan LP… Reis RL. Submitted (2), 2014.

Storage modulus (DMA)

S16: Silk-16

SC16: Silk/CaP-16

Silk-NanoCaP layer induced higher ALP activity of RBMSCs

0.0

0.1

0.2

0.3

Ab

so

rba

nc

e (

49

0 n

m)

Time (day)

1 3 7

Bilayered scaffold: RBMSCs’ viability and differentiation

n≥9

Yan LP… Reis RL. Submitted (2), 2014.

MTS assay

S16.B

asal

S16.O

steo

SC16

.Bas

al

SC16

.Ost

eo

Car

t.Bas

al

Bon

e.Bas

al

Car

t.Ost

eo

Bon

e.Ost

eo

Bila

yere

d.Bas

al

Bila

yere

d.Ost

eo

0.0

0.2

0.4

0.6

0.8

A

LP

ac

tivit

y (

µm

ol/h

ou

r/µ

g D

NA

) 1 Week

2 Week

Normalized ALP activity

n≥9

ALP: alkaline phosphatase RBMSCs: rabbilt bone marrow stromal cells

Tukey’s test

*

**

*

p<0.05 *

*

*

Tukey’s testp<0.05 *

*

RBMSCs were seeded onto the scaffolds

and underwent osteogenic differentiation for

1 and 2 weeks

1 mm

NB: New bone

S: Scaffold

Regeneration of osteochondral defect in rabbit knee

Bilayered scaffold: OCD regeneration in rabbit knee model

Masson’s

trichrome

staining

Yan LP… Reis RL. Submitted (2), 2014.

S

S

Defect control

Cross-section of

Silk/Nano-CaP

layer

Defect with

scaffold

Scaffolds were implanted into rabbit

critical size OCD (Ø 5 mm) for 4 weeks

200 µm

NB

S

S 200 µm

Regeneration of cartilage in osteochondral defect

S S

Safranin O staining

Collagen II

immunohistochemistry

staining

200 µm

200 µm

Bilayered scaffold: OCD regeneration in rabbit model

Yan LP… Reis RL. Submitted (2), 2014.

S: Scaffold

Control

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