5_Scaffolds_(cont)

Prévia do material em texto

23/10/18	
  
1	
  
A1
1/
00
	
  
Cris%na	
  C.	
  Barrias	
  
ccbarrias@ineb.up.pt	
  
	
  
BIOMATERIALS,	
  
SCAFFOLDS	
  AND	
  
ADVANCED	
  
BIOFABRICATION	
  
TECHNOLOGIES	
  
MIB	
  -­‐	
  FEUP,	
  Porto,	
  October	
  17,	
  2018	
  
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Cells in their natural (3D) microenvironment: the ECM 
Progress in Polymer Science 2014;39:2010–2029 
adhesion 
domain 
protease-cleavable 
domain 
integrin cell protease 
cadherin matrix growth factor 
tethered growth factor 
cell membrane receptor 
23/10/18	
  
2	
  
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Extracellular	
  matrix	
  
-­‐  Extracellular	
  spaces	
  
-­‐  Connec?ve	
  ?ssues	
  
-­‐  Basement	
  membrane	
  
Fibrous	
  
proteins	
  
(collagen,	
  
elas?n	
  fibers)	
  
adhesive	
  
glycoproteins	
  
Gel-­‐like	
  
matrix	
  
GAGs	
  
(free	
  or	
  
linked	
  to	
  
proteins)	
  
Deconstructing the natural ECM 
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Hydrogels as ECM mimics 
3D matrices for cell entrapment 
High water 
content 
Permeable 
Compliant 
“true” 3D 
Versatile 
(e.g. chemical 
functionalization) 
Tunable 
viscoelastic 
properties 
Recapitulate the natural 
microenvironment of cells: 
the ECM 
23/10/18	
  
3	
  
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Crosslinking 
Swelling 
 
 
 Hydrogel networks 
Polymer	
  chain	
  
	
  
Crosslinking	
  point	
  
	
  
Water	
  
Physical (ionic) crosslinking Covalent crosslinking 
Polymer	
  chain	
  
	
  
Crosslinking	
  zone	
  
	
  
Water	
  
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Synthe?c	
  polymers	
  Proteins	
  ECM-­‐deriva?ves	
   Polysaccharides	
  
Collagen	
  
Fibrin	
  
Matrigel	
  TM	
  
	
  
	
  
Hyaluronic	
  acid	
  
Chitosan	
  
Alginate	
  
Pec?n	
  
	
  
	
  
Natural	
  polymers	
  
Hydrogel	
  based	
  3D	
  matrices	
  
Hydrogel-­‐forming	
  polymers	
  
“Cell-­‐instruc%ve”	
   “Bioinert”	
  
PEG	
  
PLEOF	
  
PHEMA	
  
…	
  
23/10/18	
  
4	
  
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Engineering	
  the	
  microenvironment	
  Biomaterial-­‐based	
  
designer	
  matrices	
  
	
  
Bio-­‐inert	
   Cell-­‐instruc%ve	
  
Matrix-­‐bound	
  factors	
  
Proteoly%c	
  
degrada%on	
  
Matrix	
  physical	
  
proper%es	
  
Soluble	
  
factors	
  
Blank	
  slate	
  	
  
3D	
   3D	
  
Cell-­‐matrix	
  
adhesion	
  
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Mimicking biological functionality with polymers 
Adapted from: Nature 540, 386–394 (2016) doi:10.1038/nature21005 
cell-matrix interactions 
cell-adhesion peptide 
matrix stiffness cell-cell interactions 
polymer growth factor Cell mimic 
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5	
  
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cell migrates 
to scaffold 
scaffold instructs 
and educates cell 
“educated” cell 
migrates from scaffold 
Adapted from: Nature 540, 386–394 (2016) doi:10.1038/nature21005 
Mimicking biological functionality with polymers 
cell-instructive 
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From simple cell entrapment to tissue 
morphogenesis (3D organoids) 
LIVE	
  DEAD	
   BM	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  ALP	
   OM	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  ALP	
  hMSC	
  (Day	
  0)	
  
3D	
  
Day	
  14	
   Day	
  14	
  Day	
  14	
  
Osteogenic	
  differen%a%on	
  Viability	
  
3D	
  
Adapted from: EBS Journal 2012;279(18):3475-87 
Time	
   Intes%nal	
  organoids	
  	
  enterocyte	
  progenitors	
  
23/10/18	
  
6	
  
A
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/0
0 
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0 
From 3D-culture to organoids to organs-on-chips 
gut-­‐on-­‐a-­‐chip	
  
wyss.harvard.edu	
  
Development 2015;142:3113-3125 
Organoids	
  and	
  microfluidics	
  
A
11
/0
0 
A
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/0
0 
Multiple organs-on-chip 
nanoscience.ucf.edu/hickman/bodyonachip.phpv	
  
Trends	
  in	
  Cell	
  Biology	
  2011;21:12	
  
	
  Nature	
  2015;	
  519:	
  S16–S18	
  
23/10/18	
  
7	
  
A
11
/0
0 
A
11
/0
0 
3D cell culture in alginate hydrogels 
Cell-­‐laden	
  3D	
  
matrices	
  
(reversible)	
  ionic	
  crosslinking	
  
under	
  cytocompa?ble	
  condi?ons	
  
Ca2+	
  
Mimicking	
  features	
  of	
  in	
  vivo	
  environments,	
  while	
  taking	
  advantage	
  
of	
  the	
  same	
  tools	
  used	
  to	
  study	
  cells	
  in	
  tradi%onal	
  2D	
  cell	
  culture	
  
ALGINATE	
  
reversible	
  
%me	
  
CELLS	
  
Ca2+	
  
Gel precursor Crosslinking Hydrogel 
A
11
/0
0 
A
11
/0
0 
Unmodified	
  ALG	
  
3D cell culture in alginate hydrogels 
Chemical	
  modifica%on	
  with	
  pep%des	
  
pep?de	
  
pep?de	
  
pep?de	
  
F-­‐ACTIN	
  DAPI	
  
hMSC	
  
	
  	
  CryoSEM	
  
23/10/18	
  
8	
  
A
11
/0
0 
A
11
/0
0 
RGD	
  
Fibronec%n	
  
RGD-­‐gra`ed	
  hydrogel	
   Cell-­‐matrix	
  adhesion	
  Hydrogel	
  
Key ECM-like features: Cell-matrix adhesion 
Molecular	
  design	
  
Cell-­‐matrix	
  
adhesion	
  
cell 
matrix 
adhesion domain 
integrin 
A
11
/0
0 
A
11
/0
0 
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  RGD-­‐Alginate	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  Unmodified	
  alginate	
  
ALG modifications: cell-adhesive RGD ligands 
23/10/18	
  
9	
  
A
11
/0
0 
A
11
/0
0 
3D cell culture in alginate hydrogels 
F-­‐ACTIN	
  DAPI	
  
RGD-­‐alginate	
  
Modification with biochemical cues 
GGGGRGDSP	
  
Adhesive	
  pep%de	
   Cell-­‐MATRIX	
  ADHESION	
  
 Acta Biomaterialia 2011;7:1674-1682 & Soft Matter 2013;9:3283-3292 
A
11
/0
0 
A
11
/0
0 
Cells in hydrogels 
Overcoming the physical barrier 
Dror Seliktar. Science 2012:336:1124 
Unmodified ALG 
hMSC	
  
	
  	
  CryoSEM	
  
Cells need to overcome 
the physical barrier 
 
 
CELL-DIRECTED 
and/or 
USER-DIRECTED 
strategies 
23/10/18	
  
10	
  
A
11
/0
0 
A
11
/0
0 
Key ECM-like features: proteolytic degradation 
Molecular	
  design	
  
Proteoly?c	
  
degrada?on	
  
Cell	
  
instruc%ve	
  
Bioinert	
  
Fonseca	
  KB…BARRIAS	
  CC.	
  Progress	
  in	
  Polymer	
  Science	
  2014;39:2010–2029	
  	
  
cell 
protease 
protease-
cleavable domain 
matrix 
A
11
/0
0 
A
11
/0
0 
3D cell culture in alginate hydrogels 
F-­‐ACTIN	
  DAPI	
  
Adhesive	
  pep%de	
  
Modification with biochemical cues 
GGGGRGDSP	
  
Adhesive	
  pep%de	
  
MATRIX	
  ADHESION	
  
 Acta Biomaterialia 2011;7:1674-1682 & Soft Matter 2013;9:3283-3292 
GGYGPVGêLIGGK	
  
MMP-­‐sensi%ve	
  pep%de	
  
MMP-­‐sensi%ve	
  pep%de	
  
Adhesive	
  pep%de	
  
F-­‐ACTIN	
  DAPI	
  
PROTEOLYTIC	
  
DEGRADATION	
  
Dual	
  
Crosslinking	
  
23/10/18	
  
11	
  
A
11
/0
0 
A
11
/0
0 
MMP-­‐sensi%ve	
  alginate	
  hydrogels	
  
Alginate	
  
F-­‐	
  ACTIN	
  DAPI	
  
0	
  
10	
  
20	
  
30	
  
40	
  
50	
  
60	
  
Blank	
   MMP-­‐2	
   MMP-­‐14	
  
RF
U
	
  (x
	
  1
00
0)
	
  
0	
  hour24	
  hours	
  
*
*
Enzyma%c	
  cleavage	
  of	
  PVGLIG-­‐ALG	
  
GGYGPVGêLIGGK	
  
MMP-­‐sensi%ve	
  pep%de	
  
(MMP2,	
  MMP14)	
  
Carbodiimide	
  chemistry	
  
A
11
/0
0 
A
11
/0
0 
DMA	
  (compression)	
  Bimodal	
  MW	
  composi%on	
  
50%	
  v/v	
  HMW-­‐RGD	
  
50%	
  v/v	
  LMW-­‐PVGLIG	
  
RGD	
  	
  PVGLIG	
   RGD	
  
2	
  wt.%	
  ALG	
  
Individually	
  tunable	
  mechanical	
  
proper?es	
  
MMP-­‐sensi%ve	
  alginate	
  hydrogels	
  
Ca2+	
  
23/10/18	
  
12	
  
A
11
/0
0 
A
11
/0
0 
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  MMP-­‐insensi%ve	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  MMP-­‐sensi%ve	
  	
  
	
  	
  	
  	
  RGD-­‐Alg	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  PVGLIG/RGD-­‐Alg	
  
F-­‐ACTIN	
  DAPI	
  
Gela%n	
  zymography	
  
Inactive 
Active 
 rMMP-2 R-Alg P/R-Alg 
Inactive Active 
Control	
  
RGD-­‐Alg	
  
PVLGLIG/RGD-­‐Alg	
  
%
	
  C
O
N
TR
O
L	
  
Cleavage	
  of	
  FRET-­‐PVGLIG	
  
 RGD PVGLIG/RGD 
 Alg Alg 
25	
  
	
  
20	
  
	
  
15	
  
	
  
10	
  
	
  
5	
  
	
  
0	
  
RF
U
	
  (x
10
00
	
  a
.u
.)	
  
MMP-­‐sensi%ve	
  alginate	
  hydrogels	
  
A
11
/0
0 
A
11
/0
0 
Cellular therapies for tissue regeneration 
CT with 
single-cells 
NEED FOR IMPROVED 
DELIVERY STRATEGIES 
Low cell survival 
Poor cell engraftment 
Lack of control over cell fate 
Low efficiency CT 
(Large cell dosages) 
 
Patient 
Cell harvest 
MSC, EPC 
Clinical application 
of 
cellular therapies 
Cell isolation 
Cell expansion 
Cell delivery 
Lesion site 
Hypoxia 
Inflammation 
Anoikis 
Oxidative stress 
23/10/18	
  
13	
  
A
11
/0
0 
A
11
/0
0 
	
  	
  In	
  Vitro 	
   	
  
	
   	
  	
  
P/R-­‐ALG	
   R-­‐ALG	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  MMP-­‐sensi?ve	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  MMP-­‐insensi?ve	
  
MMP-sensitive vs. MMP-insensitive hydrogels 
Outward cell migration 
P/R-­‐ALG	
   R-­‐ALG	
   P/R-­‐ALG	
   R-­‐ALG	
  
4	
  
	
  
3	
  
	
  
2	
  
	
  
1	
  
	
  
0	
  
800	
  
	
  
600	
  
	
  
400	
  
	
  
200	
  
	
  
0	
  
Ex
te
ns
io
n	
  
le
ng
th
	
  
N
r	
  m
ig
ra
yi
ng
	
  h
M
SC
	
  (p
er
	
  1
00
	
  u
m
)	
  
Biomacromolecules	
  2014;15:380-­‐90	
  
Inactive Active 
P/R-­‐ALG	
  
%
	
  C
O
N
TR
O
L	
  
300	
  
	
  
200	
  
	
  
100	
  
	
  
0	
  
Nr	
  cells/area	
   Extension	
  length	
   MMP2	
  secre?on	
  
Alginate	
  
Fibrin	
  
P/R-­‐ALG	
   R-­‐ALG	
  
HuNu+	
  DAPI	
  
	
  	
  In	
  Vivo 	
   	
  
	
   	
  	
  
P/R-­‐ALG	
   R-­‐ALG	
  
40	
  
	
  
30	
  
	
  
20	
  
	
  
10	
  
	
  
0	
  %
Hu
N
u+
	
  c
el
ls	
  
ou
ts
id
e	
  
/u
ni
t	
  a
re
a	
   %	
  cells	
  outside	
  
Alginate	
  
Alginate	
  
A
11
/0
0 
A
11
/0
0 
15	
  x	
  106	
  cells/mL	
  
SCID	
  MICE	
  
hMSC-­‐laden	
  hydrogels	
  
	
  	
  	
  	
  	
  	
  	
  	
  2	
  wt.%	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  4	
  wt.%	
  
Transplantation of hMSC-laden ALG hydrogels 
P/R-­‐Alg	
  
R-­‐Alg	
  
4	
  weeks	
  
	
  	
  	
  	
  	
  	
  	
  	
  2	
  wt.%	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  4	
  wt.%	
  
Alginate	
  concentra%on	
  (wt%)	
  
	
  	
  	
  	
  	
  1	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  2	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  3	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  4	
  
M
od
ul
us
	
  (k
Pa
)	
  
200	
  
150	
  
100	
  
50	
  
E	
  
E’	
  
DMA	
  (compression)	
  
NP	
  
***	
  
*	
  
23/10/18	
  
14	
  
A
11
/0
0 
A
11
/0
0 
	
  	
  	
  	
  PVGLIG/RGD-­‐Alg	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  RGD-­‐Alg	
  
2wt%	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
4wt%	
  
In vivo degradation of ALG hydrogels 
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  MMP-­‐sensi%ve	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  MMP-­‐insensi%ve	
  	
  
Alginate	
  (Safranin)	
  
Connec%ve	
  %ssue	
  (Light	
  Green)	
  
Biomacromolecules 2014;15:380-90 
Level	
  of	
  degrada%on	
  
A
11
/0
0 
A
11
/0
0 
	
  
§  Par%al	
  crosslinking	
  of	
  alginate	
  with	
  MMP-­‐sensi%ve	
  pep%des	
  renders	
  its	
  
hydrogels	
  par%ally	
  “degradable”	
  by	
  cell-­‐driven	
  proteoly%c	
  mechanisms	
  
§  More	
  physiological	
  3D	
  microenvironments.	
  
§  Improved	
  proper%es	
  as	
  ECM	
  mimics	
  
§  Mul%func%onal	
  hydrogels,	
  with	
  tuned	
  biochemical	
  and	
  physicochemical	
  
proper%es	
  can	
  be	
  formed	
  by	
  ra%onally	
  combining	
  different	
  pep%de-­‐
modified	
  alginates	
  
Summing up 
23/10/18	
  
15	
  
A
11
/0
0 
A
11
/0
0 
Guiding morphogenesis in bioengineered 
cell-instructive 3D microenvironments 
Cell-­‐matrix	
  interac%ons	
  
Matrix	
  physical	
  
proper%es	
  
3D	
  
Level	
  of	
  
adhesiveness	
  
Cell-­‐cell	
  interac%ons	
  
Single	
  cells	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
Micro%ssues	
  
Cell-­‐instruc%ve	
  
A
11
/0
0 
A
11
/0
0 
3D cell culture in alginate hydrogels 
Tuning the biomechanical properties 
Acta Biomaterialia 2014 ;10:3197–3208 
Bimodal	
  MW	
  
composi?on	
  
ALG	
  wt%	
  
RGD	
  density	
  
Ca2+	
  
G’	
  
	
  
G’’	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  STIFF	
  (	
  2wt.%	
  	
  alg)	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  SOFT	
  (1wt.%	
  alg)	
  
	
  
0	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  100	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  200	
   0	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  100	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  200	
  
RGD	
  (μM)	
  RGD	
  (μM)	
  
G’
,	
  G
’’	
  
(k
Pa
)	
  
25	
  
	
  
20	
  
	
  
15	
  
	
  
10	
  
	
  
5	
  
	
  
0	
  
0.8	
  
	
  
0.6	
  
	
  
0.4	
  
	
  
0.2	
  
	
  
0	
  
High	
  MW	
  
Low	
  MW	
  
G’
,G
’’	
  
(k
Pa
)	
  
23/10/18	
  
16	
  
A
11
/0
0 
A
11
/0
0 
3D cell culture in alginate hydrogels 
Tuning biomechanical properties 
0h 
24h 
Stiff (17 kPa) 
FN F-ACTIN DAPI 
 
Endogenous	
  ECM	
  
Acta Biomaterialia 2014;10:3197–3208 
Soft (0.4 kPa) 
Hydrogel	
  contrac%on	
  
FN 
20 µm 
200	
  μM	
  RGD	
  
Microtissue 
MSC	
  migra?on	
  into	
  
0.4	
  kPa	
  alginate	
  
hydrogel	
  
Sprou?ng	
  bead	
  assay	
  
A
11
/0
0 
A
11
/0
0 
In	
  so`	
  hydrogels	
  cells	
  are	
  able	
  to	
  aggregate	
  even	
  in	
  the	
  
absence	
  cell	
  adhesion	
  ligands	
  
	
  No	
  RGD	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  200	
  μM	
  RGD	
  
10	
  μm	
   20	
  μm	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  4h	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  24h	
  
	
  	
  	
  	
  0.4	
  kPa	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  0.4	
  kPa	
  
Without	
  
RGD	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
FN F-ACTIN DAPI 
 
23/10/18	
  
17	
  
A
11
/0
0 
A
11
/0
0 
Viscoelastic properties vs. time 
é Local	
  polymer	
  concentra?on	
  
é Local	
  cell	
  density	
  (cellular	
  networks)	
  
é ECM	
  (FN)	
  deposi?on	
  
Adapted	
  from:	
  Kraning-­‐Rush	
  et	
  al.	
  CM	
  Phys	
  Biol	
  (2011)	
  
	
   	
   	
  
	
  	
  	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  	
  	
  
0	
  
0.5	
  
1	
  
1.5	
  
2	
  
2.5	
  
1-­‐0	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  1-­‐200	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  2-­‐0	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  2-­‐200	
  
G’
	
  (f
ol
d	
  
ch
an
ge
	
  re
la
%v
e	
  
to
	
  0
h)
	
  	
  
Oscillation rheometry 
Acta Biomaterialia 2014 ;10:3197–3208. 
!!!!!!!!!!!!!!!!No!RGD!!!!!!!!!!!!!!!!!!!!!!200!μM!RGD!!!!!!!!!!!!!!!!!!!!!!!No!RGD!!!!!!!!!!!!!!!!!!!!!!!200!μM!RGD!!
BM!
!
!
!
!
!
OM!
!!!!!!!!!!!!!!!!!!0.4!kPa!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!17!kPa!
!!!!!!!!!!!!!!!
Osteogenic 
differentiation 
A
11
/0
0 
A
11
/0
0 
Micro%ssue	
  matura%on	
  improves	
  angiogenic	
  proper%es	
  
VEGF
Da
y 0
Da
y 7
0.0
0.5
1.0
1.5
R
el
at
iv
e 
m
R
N
A
 e
xp
re
ss
io
n 
D0 D5 
* 
VEGF mRNA expression 
All data; Whiskers
Fr
es
hly
 P
re
pa
re
d
Pr
e-
inc
ub
ate
d
0
5
10
15
20
V
e
ss
e
l N
u
m
b
e
r
N
r n
ew
 v
es
se
ls
 * 
D0 D5 
Angiogenic potential (CAM assay) 
Time 
 
Cell aggregation 
ECM deposition 
23/10/18	
  
18	
  
A
11
/0
0 
A
11
/0
0 
Co-­‐culture	
  with	
  vascular	
  cells	
  
MSC	
  (CT-­‐blue)	
  
HUVEC	
  (Ac-­‐dil-­‐LDL)	
  
0h	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  24h	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  76h	
  
Time	
  
hMSC	
  +	
  HUVEC	
  
MICROTISSUE	
  
FORMATION	
  
A
11
/0
0 
A
11
/0
0 
§  Cells in soft alginate hydrogels rapidly modified their biochemical/
mechanical environment: new niche 
§  Cells can be harnessed to tune artificial materials in situ, generating 
a new cell/matrix interface, that in turn affects cell behaviour. 
§  While they become less important over time, the original properties 
of the artificial matrix were key instructive cues 
Summing up 
23/10/18	
  
19	
  
A
11
/0
0 
A
11
/0
0 
Technology	
  based	
  on	
  highly	
   specific,	
  natural	
  binding	
  events	
   (e.g.	
  an%body/an%gen	
  
interac%on)	
  to	
  create	
  molecular	
  memory	
  on	
  a	
  material,	
  usually	
  polymeric.	
  
Neves,	
  M.	
  I.,	
  et	
  al.	
  (2017).	
  Tissue	
  Eng	
  Part	
  B	
  Rev	
  23(1):	
  27-­‐43.	
  
Molecular imprinting 
A
11
/0
0 
A
11
/0
0 
Impaired	
  diffusion	
  of	
  template	
  
molecules	
  
Molecular imprinting approaches 
Adapted from Neves, M. I., et al. (2017). Tissue Eng Part B Rev 23(1): 27-43.	
  
23/10/18	
  
20	
  
A
11
/0
0 
A
11
/0
0 
Molecular	
  imprin%ng	
  poten%al	
  applica%ons	
  in	
  TE	
  	
  
Neves, M. I., et al. (2017). Tissue Eng Part B Rev 23(1): 27-43. 
High-­‐selec%vity	
  
and	
  High-­‐loading	
  
scaffolds	
  
A
11
/0
0 
A
11
/0
0 
Tethering of ligand into 
pre-formed cell-laden 
hydrogels 
Moving forward: dynamic (4D) systems 
user-tunable platforms 
23/10/18	
  
21	
  
A
11
/0
0 
A
11
/0
0 
Activation of caged 
ligands in pre-formed 
cell-laden hydrogels 
Moving forward: dynamic (4D) systems 
user-tunable platforms 
A
11
/0
0 
A
11
/0
0 
Hybrid scaffolds 
§  When a single-component scaffold does not satisfy all the requirements of 
a particular TE application 
Examples: 
§  Insufficient stiffness and compressive strength (load-bearing 
applications) 
§  Lack of hierarchical structure (complex tissues) 
§  Lack of magnetic/electric properties 
§  … 
 
§  Multi-component (hybrid) scaffolds 
23/10/18	
  
22	
  
A
11
/0
0 
A
11
/0
0 
	
  
	
  
	
  
	
  
Hydrogels	
  
Combined/hybrid	
  scaffolds	
  
Hybrid scaffolds 
Examples Porous	
  (solid)	
   Fibrous	
  
	
  
	
  
	
  
	
  
Porous	
  (solid)	
   Par%cules	
  
micro	
  
nano	
  
micro	
  
nano	
  
Reinforcing	
  agents	
  
A
11
/0
0 
A
11
/0
0 
Hybrid scaffolds 
Examples 
Int J Nanomed 8:3641-3662 (2013) DOI: 10.2147/IJN.S43945 
23/10/18	
  
23	
  
A
11
/0
0 
A
11
/0
0 
Examples of hybrid scaffolds 
Advanced Materials 26(22) · 2014 DOI: 10.1002/adma.201400523 J Control Rel 173(1) · 2013 DOI: 10.1016/j.jconrel.2013.10.017 
A
11
/0
0 
A
11
/0
0 
Examples of hybrid scaffolds 
Advanced Materials 26(22) · 2014 DOI: 10.1002/adma.201400523 
23/10/18	
  
24	
  
A
11
/0
0 
A
11
/0
0 
Examples of hybrid scaffolds 
Enhancement	
  in	
  oligodendrocyte	
  
differen%a%on	
  on	
  PCL-­‐GO	
  
A
11
/0
0 
A
11
/0
0 
Examples of hybrid scaffolds 
J Tissue Eng Reg Med 8 (2014) DOI: 10.1002/term.1506 
Mechanical	
  
reinforcement	
  
for	
  bone	
  TE	
  
23/10/18	
  
25	
  
A
11
/0
0 
A
11
/0
0 
Multiphasic scaffold design 
A
11
/0
0 
A
11
/0
0 
 
Artificial ECM mimics 
How simple is complex enough?