Upload
others
View
16
Download
0
Embed Size (px)
Citation preview
Designing natural bases scaffolds for
cartilage tissue engineering applications
Vitor M. [email protected]
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
VERY STRONG PUBLICATION RECORD
QUALITY ASSURANCE
The 3B's Research Group performs all
its research and related activity under a
certified Quality Management System
according to ISO 9001:2008 Guidelines
A Unique – home built – IT platform for
managing online all the 3B´s Research
(see link)
Thank you!