Properties of Hydrogel for biomedical applications and Hybrid ...

Properties of Hydrogel for
biomedical applications
and
Hybrid nanoparticle-polymer
hydrogels
Rolando.Barbucci

Hydrogel
Is an hydrophilic polymeric
network which may absorb
water in the amount from 10%
up to 100 time its dry weight
polysaccharide
arm
junction point

O
Hydrogel Synthesis
COOTBA
n
I - + N
H 3 C
Cl
O
CO O
+ N
H 3 C
+ NH 2 (CH 2 ) 3 NH 2 -
(CH 3 CH 2 ) 3 N +- I
N O
CH 3
n
Characteristics:
-planned and reproducible
stoichiometry
-different diamine cross-linking
agents
-predictable cross-linking degree
-predictable properties
O
CO
NH
(CH 2 ) 3
NH
CO
n
Technique to check C.D.:
-N.M.R.
-pH-metry
O
n

The polysaccharide chemistry affects
14000
12000
10000
8000
6000
4000
2000
0
pH 2 pH 7,4 pH 9
Hyal 50%
CMC 50%
AA 50%
10 5
hydrogel
Water Up-take
Confronto
G' CMC 50%
G'' CMC 50%
G' HA 50%
G'' HA 50%
G' AA
G'' AA
G' AA 50%
G'' AA50%
10 6 0.01 0.1 1 10
G',
G''
[P
a]
10 4
1000
Hydrogel
mechanical
properties
100
10
1
Frequency [Hz]

The crosslinking degree affects:
60000
50000
hydrogel
Water Up-take
(H 2 O)
40000
Hyal
30000
CMC
20000
AA
10000
0
5 50 100
10 5
Confronto
10 6 0.01 0.1 1 10
G' AA 100%
G'' AA 100%
G' AA 50%
G'' AA50%
Cross-linking Degree
G',
G''
[P
a]
10 4
1000
Hydrogel
mechanical
properties
100
10
1
Frequency [Hz]

Properties of the gels
-stoichiometry
- planned cross-linking degree (from soft to hard gel)
- sterilizability by EtO
- thixotropy
- coordination towards metal ions (Ag + , Cu 2+ )
- chemical derivatisation (OSO 3- , -CONHCH 3 ,etc.)
- formation of predetermined microporosity
- shape memory of microporous gels
- amber effect

What is thixotropy?
Thixotropy is the property of some
non-Newtonian pseudoplastic
materials that leads to a progressive
decrease in viscosity under a
mechanical stress followed by the
recovery of its consistence once the
stress is removed.

Thixotropy and hydrogels
INJECTABILIT
Y
MINI INVASIVE
SURGERY
(nucleous polposus
replacement, fillers for
damaged cartilage,
cosmetic applications)
CELL ENCAPSULATION
EASY MIXING WITH
DRUGS
IPN HYDROGELS

G';G" (Pa)
4500
4000
3500
3000
2500
2000
1500
G' AA
G" AA
1270Pa
AA, Hyal and CMC based
Hydrogels
are thixotropic
1000
0 200 400 600 800 1000 1200 1400
stress (Pa)
3000
2500
2000
505Pa
G'CMC
G"CMC
1800
1600
1400
1200
1000
505Pa
G'Hyal
G"Hyal
1500
800
G';G"(Pa)
1000
G';G"(Pa)
600
400
500
0 200 400 600 800 1000 1200 1400
Stress(Pa)
200
0 200 400 600 800 1000 1200 1400
Stress(Pa)

Main biomedical applications
of polysaccharidic hydrogels
•osteoarthritis therapy
•bioactive coatings (e.g. catheter, stent
•microporous hydrogels for the drug
controlled release
•cellular scaffold (artificial organs)

Polysaccharide hydrogels
in osteoarthritis therapy

EFFECT OF HYAL 50%* HYDROGEL ON KNEE OSTEOARTHRITIS
(in New Zealand adult-male rabbits)
Histological analysis
Macroscopic observation:
Hyal
Normal cartilage
Untreated damaged cartilage
native
lesion
Damaged cartilage treated
with Hyal 50%.
The arrow is pointing cells
piled on each other
Damaged cartilage
with Hyal 50%.
At 50 days cartilage lesion of the control group appeared covered by a thin and slightly
irregular layer of fibrous tissue.No samples showed evidence of proteoglycans
production in the reparative tissue.
On the contrary Hyal 50% group showed a thick mixed hyaline and fibrocartilage layer.

Effect of Hyal 50%-IbuLys on knee osteoarthritis
After 50 days treatment
C H 3 C H 3 O
C H C O -
C H 3 C H C H 2
N H 3 O
+
H 3 N C H 2 C H 2 C H 2 C H 2 C H C O -
Ibuprofen-lysine
+
Control:
physiological solution
No tissue
regeneration
Treatment with
Hyal 50% loaded
with Ibu-Lys
Treatment with Hyal
50%
Growth of new cartilagineous tissue close to the native
cartilage
Both Hyal 50% and Hyal 50% loaded with anti-inflammatory drug show the growth of new
cartilagineous tissue, which is absent in the control (physiological solution: NaCl 0,9%)

Bone densitometry
For the pain the rabbit does not lay the leginducinga decreasein the bone
density.
Mean percentage of the bone density reduction of treated site at 30 and 50 days in comparison with the
initial one
F1 (whole femur)
T1 (whole tibia)
F2 (distal femoral epiphysis)
T2 (proximal femoral epiphysis)
30 days 50 days
Hyal Hyal+Ibu Hyal Hyal+Ibu
-12.8% -10.2% -24.8% -20.9%
-13.6% -14.8% -29.2% -20.7%
-17.8% -17.4% -34.5% -27.7%
-17.9% -12.8% -31.9% -26.0%
At 30 days: no significant differences were found between Hyal-Ibu-lys treated group and
Hyal treated group in terms of reduction of treated site even if a clear trend in the reduction
of the percentage of the defect was observed, at 50 days a significant difference was found
between Hyal-Ibu-lys group and Hyal group in terms of reduction of treated tibial site

Polysaccharidic hydrogels
as cellular scaffolds

What is a cellular scaffold?
It can be defined as “a
substrateabletofavourcell
adhesion, proliferation and
differentiation”

Amber Effect
What is amber effect?
Seeding cells on hydrogel and leave the system pass
through a needle (thixotropic property) cells are
included into the matrix and are able to adhere and
proliferate. This means the possibility to inject cells
(e.g. chondrocytes) in situ.

polysaccharidic hydrogels as cellular scaffold
Cell type:
•Human diploid fibroblasts
•Rat hepatocytes
•Rat β-pancreatic cells
Amber effect
before
after
AA 30μm
AA 30μm

Effetto ambra: gel gAAE5 con fibroblasti
della linea C54 all’interno, 5gg

•Rat hepatocytes
O.D.(540nm)
1,1
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
24h
96h
168h
•Rat β-pancreatic cells
Hyal
Hyal
35um
CMC
CMC
40um
AA
AA40um
control Hyal AA CMC
softer native hydrogels performed better with cells which grow
insolution (rat hepatocytes and β-pancreatic cells) whereas
harder porous hydrogels were a better scaffold for cells which
need to adhere for proliferation (fibroblasts)

MeO 2 Nanoparticles as Crosslinkers for
the realization of Polysaccharide Hybrid
Hydrogels

Chemical Entrapment of NPs
Silanization of Me O 2 NPs
(3-Aminopropyl)trimethoxysilane (APT
CO
H Si NH 3 CO
2
H
O
3
OH
HO
APTMS
H O OH
TiO 2
HO
OH
TiO 2
H 3
C
EtOH /H 2 O pH = 5
CH 3 COOH
TiO 2
H 3 C
H 3C C
H3
TiO 2
O
O
O
Si
NH 2

Chemical Entrapment of NPs
Strategy for Hybrid Hydrogel synthesis
OR
O
HO
HO
CMC polymer
O
O
O
OH
O
+
HO
O
O
O
OH
OH
Silanized TiO 2
ONPs
H 3 C
C
O
n
EDC,
NHS
pH 4.75,
stirring
overnight
RO
O
NH
HO
O
TiO 2
HN
NH
O
O
OH
O
O
NH
O
HO
O
HO
O
O
O
OR
O
OH
O
OH
O
O
n
HO
O
NH
TiO 2
HN
O
O OH
O
O
n
NH
O
OR
TiO TiO 2
H 2 3C
H3
O
O
Si
NH 2
HO
O
OH
O
HO
O
OH
O
O
OR
O
n

Chemical Entrapment of NPs
Absorbance
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
FT-IR Analysis
1724
1640
CMC polymer
CMC-TiO 2
hydrogel
1598
1421
1323
1060
2000 1800 1600 1400 1200 1000 800
wavenumbers (cm -1 )
Evidence of amidic
bond formation
between polymer and
functionalized NPs.
The hydrogels has
been cross-linked
SEM/EDS
Si
Ti
The Si and Ti are
almost overlapped
and uniformly
distributed

Chemical Entrapment of NPs
Morphological Analysis
SEM
AFM
Surface
roughness
Ra 1.0
±0.1nm
CMC hydrogel
Surface
roughness
Ra 50-
100nm
CMC-TiO 2 NPs hydrogel

Without H
Magnetite NPs
In the hydrogel
With H

Conclusions
• Hydrogel formation occurs using functionalized TiO 2
or Fe NPs as crosslinking agent.
• No release of NPs occurs.
• The hydrogel with Fe NPs moves according to the H

Applications: Cell Alignment
- Neuronal guidance for nerve regeneration
- Mandatory for effective contraction of muscle (smooth, skeletal, cardiac)
- Tendon repair
- Endothelialization of vascular grafts

IMMUNOMAGNETIC LABELLING
- Paramagnetic microparticles
- Paramagnetic colloidal beads
- Molecular magnetic labelling

Magnetic Field
No Field
HUVECS- CD31 on PLA-Sheet
Field exposure: 5 min at 0,1 T
Picture captured after 4 hours culture

Alignment
Human monocytes CD-14
Field exposure: 5min at 0,05 T
Field exposure: none

HUVECS- CD31
Field exposure: 5 min at 0,1 T
Picture captured after 15 hours culture

Thank you for
your attention