Post on 11-Aug-2021
IDROGELI
R.Barbucci Università di Siena
“hydrogels are three-dimensional,hydrophilic, polymeric networks capable of
imbibing large amounts of water or biologicalfluids”
HYDROGELS
AccordingAccording toto the the typetype ofofcrosscross--linkinglinking amongamong the the macromoleculesmacromolecules, ,
hydrogelshydrogels can can bebe divideddivided in in twotwo mainmaincategoriescategories::
hydrogelshydrogels
chemically based physically based
Chemical hydrogelnetwork
Hydrophilic OH groups
Physical hydrogelnetwork
Hydrophobic chains
Chemical cross-linking
Physical crosslinking
PVA gelation
Hydrogel
Is an hydrophilic polymericnetwork which may absorb
water in the amount from 10% up to 100 time its dry weight
polysaccharide
arm
junction point
Section ISection I IntroductionIntroductionHydrogels that react in response to a signal have recently received considerable research interestThese gels with additional functions are called environment-sensitive or smart hydrogels
Environment-sensitive hydrogelsRespond to changes in environmental conditions
• Swell • Shrink • DegradeHydrogels have been produced to respond to external environmental conditions such as:
Temperature pH Electrical fieldExternal stressLight
(and combinations of these)
Temperature sensitive hydrogelsNegative temperature-sensitive hydrogels have a Lower Critical Solution TemperatureLower Critical Solution Temperature
Chemically crosslinked Hydrogel (Permanent) (volume collapse)
(Do not dissolve)(Do not dissolve)
Above LCST: Deswells
Physically crosslinked Hydrogel (Non permanent)(Can Dissolve)
Above LCST: Hydrogel takes longer to dissolve/becomes insolubleBelow LCST: Hydrogel dissolves
Below LCST:Swells
(Soluble-insoluble transition)
Below LCST Above LCST
This is mainly due to the breaking of hydrogen bonds, causing the prevalence of polymer-polymer interactions and the growth of the polymeraggregates leading to phase separation
Cloud point measurement
The The mainmain polymerspolymers usedused forfor preparationpreparation of of hydrogelshydrogels
polymerspolymers
polysaccharides synthetic polymers
Gel components: 1) polysaccharides- hyaluronan- alginate- carboxymethylcellulose- etc.
2) arm specimen:- diamine- esterified diaminoacid- PEG- etc.
Changing polysaccharides, arms and cross-linkingdegree, many products useful for differentapplications can be obtained in a programmable and repeatable way (also concerning only the cross-linking degree)
The number of polysaccarides used for the preparation of hydrogels as delivery systemsor matrices for cells entrapping and drugdelivery is extramely large and it is verydifficult to list all them.
Several possibilities of chemical modificationand polymer mixing have given rise to a greatvariety of hydrogel matrices.
PolysaccharidesPolysaccharides thatthat havehave beenbeen investigatedinvestigatedforfor the the preparationpreparation of of hydrogelshydrogels in in recentrecent studiesstudies
are: are: starchstarchalginatealginatecarrageenancarrageenandextrandextrangellangellanguarguar gumgumpullulanpullulan
scleroglucanscleroglucanxanthanxanthanxyloglucanxyloglucanpectinspectinschitosanchitosancellulosecelluloseothersothers
hyaluronic acid
The most used polymers in the biomedical field
Hyaluronic acid
Carboxymethylcellulose
Alginic acid
Natural polysaccherides
O O
O
COONa
HOOH
CH2OHHO
NHCO CH 3
O
O
CH2OCH2COONa
OH
OHO
CH2OCH2COONa
O H
OH
OCOO Na
O
HOOH
O O
CO ONa
HO
OHGuar
OCH2H
O H
O HO OO
O H
O H
C H 2
O
O
O H
H O O H
C H 2O H
*
n
Chitosan
Hydrogel Synthesis
Characteristics:
-planned and reproduciblestoichiometry
-different diamine cross-linkingagents
-predictable cross-linking degree
-predictable properties
Technique to check C.D.:
-N.M.R.
-pH-metry
O
COOTBA
O
CO O
N
NH3C
+
NH3C
+I-
CH3
ONH2(CH2)3NH2
(CH3CH2)3N+-I
+ -
O
CO
NH
(CH2)3
NH
O
CO
n
n
n
n
Cl
OO
HOOH
OHO
ONHCOCH3
O
OH
NaOOC
HYAL
OO
OHHO
OOH
OH3COCHN
O
OCOOH
OO
OHHO
OOH
OH3COCHN
O
OCOOH
A
OO
OHHO
OOH
OH3COCHN
O
HOCOO
OO
OHHO
OOH
OH3COCHN
O
OHCOO
A
R=DEO OO
R=PEGDGO
O OO
A:DEOCH2
H2C
OH
OH
A:PEG DG O O
OH OH
n
Other cross-linking pathways
HCl
,R
,R
NaOH
C1’
C1O
GlcA
O
O
GlcNAc
CH3
GlcARET.
176.
0190
175.
2000
170.
4983
54.1
1183
144.
4420
142.
5462
114.
1694
111.
9854
104.
2352
101.
6114
83.7
754
81.0
909
79.7
714
77.4
964
76.5
409
74.7
361
73.6
289
69.5
946
61.7
231
55.4
441
42.5
676
41.2
329
40.5
049
38.0
934
27.8
408
23.6
396
(ppm)
50 100 150
0
20
40
60
80
100
OO
NHO
O
OO
O
CH3O
OONH
NHO
ONa
+
Na+
Na+
Na+
12
35
64
1'2'
3'
4' 5'
1ret
2ret1ret
Hyal gel 50%
NMR spectroscopy
Water self-diffusion coefficients
Sample D (10-9m2 s-1)
Hyal 1.3DAP 2.43± 0.04
HyalS 1.3DAP 2.57± 0.03
Hyal 1.6DAE 2.82± 0.03
HyalS 1.6DAE 2.66± 0.06
The crosslinking agent affects hydrogel mechanicalproperties
1
10
100
1000
104
0.01 0.1 1 10
�Confronto
G' HYAL 1.3 DAP 50%G'' HYAL 1.3 DAP 50%G' HYAL 1.12 DAD 50% G'' HYAL 1.12 DAD 50% G' HYAL 800 PEG 50% G'' HYAL 800 PEG 50% G' HYAL 1.6 DAE 50% G'' HYAL 1.6 DAE 50%
G',
G''
[Pa]
Frequency [Hz]
0
10000
20000
30000
40000
50000
60000
5 50 100
Cross-linking Degree
Hyal
CMC
AA
Hydrogelmechanicalproperties
The crosslinking degree affects:
hydrogelWater Up-take
(H2O)
1
10
100
1000
10 4
10 5
10 6
0.01 0.1 1 10
Confronto G' AA G'' AA G' AA 50% G'' AA50%
G', G'' [Pa]
Frequency [Hz]
100%100%
The crosslinking degree affects hydrogel morphology
AA 5% AA 100%AA 50%
CMC 100%CMC 50%CMC 5%
Hyal 5% Hyal 100%Hyal 50%
The polysaccharide chemistry affects
1
10
100
1000
10 4
10 5
10 6
0.01 0.1 1 10
Confronto
G ' CM C 50%G '' CM C 50%G ' HA 50%G '' HA 50%G ' AAG '' AAG ' AA 50%G '' AA50%
G ',G ''[Pa]
Frequency [Hz]
02000400060008000
100001200014000
pH 2 pH 7,4 pH 9
Hyal 50%CMC 50%AA 50%
hydrogelWater Up-take
Hydrogelmechanicalproperties
Sample Residual EtO(μg/mL)
HA 50% 2,7x10-2
12000
17000
22000
27000
HA 50% HA 50%gamma rays
HA 50% EtO HA 50%steam (dry)
Materials 4h 24h
Polystirene 19.3±4.0 9.1 ±4.7
HA 50% 21.8 ±5.8 12.4 ±3.5
Water Up-take after sterilisation
Cytotoxicity of hydrogelssterilised by EtO on
endothelial cells
Properties of the gels- stoichiometry - planned cross-linking degree (from soft to hard gel)- sterilizability by EtO- thixotropy- coordination towards metal ions (Ag+, Cu2+)- chemical derivatisation (OSO3
-, -CONHCH3,etc.)- formation of predetermined microporosity- shape memory of microporous gels- amber effect
ThixotropyThixotropy
The property of certain gels to become liquid uponbeing shaken or agitated and to coagulate againwhen left in an undisturbed condition
KETCHUPKETCHUP QUICKQUICK--SANDSAND
Property of these gels
ThixotropyThixotropy isis the the propertyproperty of some of some pseudoplasticpseudoplastic fuidsfuids toto varyvary theirtheir viscosityviscosity ififsubjectedsubjected toto shearshear stress.stress. RheogramRheogram
GG’’ ((ElasticElastic ModulusModulus) ) GG’’’’ ((ViscousViscous ModulusModulus) ) The The outwardoutward flow flow curvescurves do do notnot coincide coincide withwith the return the return onesones. . Area Area betweenbetween the the twotwo curvescurves intensityintensity of the of the thixotropicthixotropic phenomenonphenomenon whosewhose physicalphysicalmeaningmeaning isis the the energyenergy usedused in the network bond in the network bond dissociationdissociation per per unitunit of volume and time. of volume and time.
0 200 400 600 800 1000 1200 1400
500
1000
1500
2000
2500
3000
G';G
"(P
a)
Stress(Pa)
G'CMC G"CMC
0 200 400 600 800 1000 1200 1400200
400
600
800
1000
1200
140016001800
G';G
"(P
a)
Stress(Pa)
G'Hyal G"Hyal
0 200 400 600 800 1000 1200 1400
1000
1500
2000
2500
3000
3500
40004500
G';G
" (Pa
)
stress (Pa)
G' AA G" AA
1270Pa
505Pa505Pa
AA, Hyal and CMC AA, Hyal and CMC basedbasedHydrogelsHydrogels
are are thixotropicthixotropic
Hyal(b) Hyal(a) -- CMC(b) CMC(a) -- AA(b) AA(a)0
1000
2000
3000
4000
5000
6000
7000
8000
wat
er u
p-ta
ke
pH7,4
Morphological analysisWater up-take
Hyal, CMC and AA based 50% hydrogels under an appropriate mechanical stimulus, were able to change, temporarily, their physical state and, once removed the mechanical stimulus, they resumed the original consistence.
Before After
Biomedical application of hydrogels
Haemodialyse membranes
Tissue barriers
Temporal artificial skin
Coatings for cell cultures
Coatings for tubing and catheters
Bioartificial pancreas/liver
Cutaneous dressing
Artificial cornea
Contact lenses
delivery system
Soft tissue substitutes
Tissue engineering
Main biomedical applicationsofour polysaccharide hydrogels
•osteoarthritis therapy
•bioactive coatings (e.g. catheter, stent
•microporous hydrogels for the drugcontrolled release
•Electrochemotherapy
Replacement of nucleus pulposus
•cellular scaffold (artificial organs).
Polysaccharide hydrogels in osteoarthritis therapy
EFFECT OF HYAL 50%* HYDROGEL ON KNEE OSTEOARTHRITIS (in New Zealand adult-male rabbits)
Normal cartilage
Damaged cartilage treatedwith Hyal 50%. The arrow is pointing cellspiled on each other
Damaged cartilagewith Hyal 50%.
At 50 days cartilage lesion of the control group appeared covered by a thin and slightlyirregular layer of fibrous tissue.No samples showed evidence of proteoglycansproduction in the reparative tissue.
On the contrary Hyal 50% group showed a thick mixed hyaline and fibrocartilage layer.
Untreated damaged cartilage
Histological analysis
Macroscopic observation:
Hyal
lesionnative
ibuprofen
CH3 CH CH2
CH3
CH C O -CH3 O
lysineH3NCH2CH2CH2CH2CH C O-
NH3 O+
+
Anti-inflammatory drug:
Polysaccharidic hydrogels for the drugcontrolled release
Control: physiological solutionNo tissue regeneration
Treatment withHyal 50% loadedwith Ibu-Lys
Growth of new cartilagineous tissue close to the native cartilage
Effect of Hyal 50%-IbuLys* on knee osteoarthritis
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%)
*crosslinked Hyal by propandiamine(C.D. 50%) loaded with Ibuprophen-lysin salt.
After 50 days treatment
Treatment with Hyal 50%
Bone densitometry
30 days 50 daysHyal Hyal+Ibu Hyal Hyal+Ibu
F1 (whole femur) -12.8% -10.2% -24.8% -20.9%
T1 (whole tibia) -13.6% -14.8% -29.2% -20.7%
F2 (distal femoral epiphysis) -17.8% -17.4% -34.5% -27.7%
T2 (proximal femoral epiphysis) -17.9% -12.8% -31.9% -26.0%
For the pain the rabbit does not lay the leg inducing a decrease in the bonedensity.Mean percentage of the bone density reduction of treated site at 30 and 50 days in comparison with the initial one
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 reductionof the percentage of the defect was observed, at 50 days a significant difference was foundbetween Hyal-Ibu-lys group and Hyal group in terms of reduction of treated tibial site
O
O
O
RCOOX
RCOOXDMF, N2CMP-J
O
O
O
RCOOX
RCOO
NH3C
NH2CH3
O
O
O
RCOOX
RCONHCH3
polimers
AA
CMC : R=CH2OCH2
Realisation of new amidic polysaccharides
OCOONa
O
HOOH
O O
CONHCH3
HO
OH
O
O
CH2 OCH2COONa
OH
OHO
CH2OCH2CONHCH3
OH
OH
Amidic Carboxymethylcellulose (CMCA)
Amidic alginate (AAA)
0.1000 1.000 10.00frequency (Hz)
100.0
1000
10000
G' (Pa)
100.0
1000
10000
G'' (
Pa)
AA
AAA
The amidation affects the rheological properties and the water up-take
0.1000 1.000 10.00frequency (Hz)
100.0
1000
10000
G' (P
a)
100.0
1000
10000
G''
(Pa)
CMC
CMCA
0
2000
4000
6000
8000
10000
12000
CMC100% CMCA AA AAA Hyal
[(Ws-
Wd)
/Wd]
*100
Hydrogel: CMCA
After 50 days
Cartilageregenerationwas observed
Coating of Polyurethane cathetersCoating of Polyurethane cathetersPolyurethane based cathetersAdvantages
•optimal mechanical properties
•Low cost
•Disadvantages:
•High probability of microbial adhesion:solvable with proper coating(hydrogel-silver complexes)
•Thrombotic event: solvable with propercoating (antithrombotic materials)
water
Venous catheters
Hurinary catheters
0
5000
10000
15000
20000
25000
30000
35000
HA HA-Ag
swel
ling
degr
eeWater up-take
Hyal Hyal-Ag
The presence of silver ions provoked a drastic increase of the amount of absorbed water
EDAX analysis: a wide and homogeneous distribution of silver ions was presentThe presence of Ag ions induced a
disruption of the hydrogel laminae withformation of large holes
Physico-chemical characterisation:
Antimicrobial coatings: Hydrogel-Silver complexesEs: Hyal-Ag+
•The dried hydrogel was immersed for 24 hours in an AgNO3 solution
• The complex was washed until no more detectable ions were found in the the washing solutions
•The amount of metal ion up-taken by the hydrogel was determined by atomic absorption (0,046mgAg
+/mg gel dry)
Shift of the COO--
band from 1610cm--1 1
to 1590cm--11
Carboxylate groups involved in the coordination process
Sulphatation of polysaccharides
DMFN2SO3Py
NaOHpH 9EtOH (2:1) O/N 4°C
O
O
OH
COOTBAO
HO
O
OH
HO
COOTBA
O
O
OH
COOTBAO
PyO3SO
O
OSO3Py
HO
COOTBA
O
O
OH
COONaO
PyO3SO
O
OSO3Py
HO
COONa
Crosslinking of PolS
PolS
O
O
OH
CONH(CH2)3NH
O
PyO3SO
O
OSO3Py
HO
COOTBA
O
O
OH
COOTBAO
PyO3SO
O
OSO3Py
HO
CO
AAS
0100020003000400050006000700080009000
AA AAS
wat
er u
p-ta
kePhysico-chemical characterisation:
AA 50% AAS 50%
Evaluation of the antithrombotic activity:
PUPU
AASAASPU catheter coated with sulphated alginate. The coating induces an elongation of the thrombin time. The thrombin time test statesthe time necessary to thrombin to turn the fibrinogen to fibrin
TT (sec)
Control 35.3±1.2
AA polymer 68.6±3.0
AAS polymer >180
AAS hydrogel >180
Microporous hydrogelsfor the drug controlled
release
The porous structure in the hydrogel is obtained stratifying the 50% crosslinkedhydrogel onto a cell-colture strainer with a defined and controlled porosity (40, 70 and 100μm). The filter is placed on a beaker containing NaHCO3. Adding HCl, formation of CO2 bubbles is observed, their passage through the filter, firstly, and the matrix, secondly, induced the hydrogel to assume a porous morphology.
Porous formation technique
AA
Hyal
CMC
before afterAA
CMC
Hyal
CO2
Morphologic analysis
Pore diameter, density and thickness of the walls between the pores for polysaccharides based hydrogels (the dimensions were determined by Scion Image β 4.02)
Materials Native Hydrogel AA CMC Hyal Diameter
(μm) Density
(pores/mm2)Thickness
(μm) Diameter
(μm) Density
(pores/mm2)Thickness
(μm) Diameter
(μm) Density
(pores/mm2)Thickness
(μm) Cell strainer (φ 40μm) 13± 4 5,0 ± 1,5 2,2± 1,1 14± 4 2,0 ± 0,5 2,7± 0,9 15± 3 3,0 ± 0,5 1,8± 0,9
Cell strainer (φ 70μm) 30±2 1,5± 0,5 2,4± 0,9 30±4 1,50± 0,03 2,3± 1,4 35± 2 1,00 ± 0,01 2,7± 0,8
Cell strainer (φ 100μm) 40± 4 1,5± 0,3 2± 1 40± 9 1,00 ± 0,01 3,7± 2,1 40± 4 0,50 ± 0,02 0,70± 0,05
HyalpH 9
02000400060008000
100001200014000
Hyal Hyal 15um
AA AA 13 um CMC CMC 14um
wate
r up
-tak
e
pH 2pH 7,4pH 9
The presence of pores decreases
the water up-take at all the pHs
10.000.1000 1.000
frequency (Hz)
G',G
’’(Pa)
105
10
102
103
104
CMC 30
CMCCMC 40
CMC14
G’ G’’
10.000.1000 1.000
frequency (Hz)
102
103
104
105
AAAA40 AA30 AA13
G’ G’’
frequency (Hz)10.000.1000 1.000
.
104
10
102
103
Ha35
Ha Ha40
Ha15
G’ G’’
The presence of poresincreases the mechanical properties
AssessingAssessing of of porousporous matrixmatrix resistanceresistance of of alginicalginic acid acid gelsgels
pH 2
120 h pH 9
120h
pH 7.4
120 h
FCS 0,1% 120 h and
SDS 10% for 120 h
φ 30µm
φ 30µm
φ 15µm
φ 30µm φ 30µm
Incorporation of drugThe first involves placing a previously prepared hydrogel in a suitable drug solution until it swells to equilibrium, thus allowing the active agent to diffuse into the polymer
In the second approach, the hydrogel monomer(s) are mixed with drug, an initiator, with or without a crosslinker, and allowed to polymerise, thus trapping the drug within the matrix
…Drug controlled release system
Drug: Ibuprophene lysin
Porous hydrogelsa) Pore formation
and loading withthe drug
b) Loading of gel withthe drug and poreformation
00,10,20,30,40,50,60,70,80,9
1
0 1 2 3 4 5 6 7 8 9 10 11 12
time (days)
mg
Ibu-
Lys
tx/m
g Ib
u-Ly
s t0
Hyal 50%
Hyal 50% 35 um (a)
Hyal 50% 35 um(b)
The The drugdrug isis trappedtrapped hard hard bybythe the wallswalls of the of the porousporoushydrogelhydrogel (b)(b)
Hyal
CMC
Drug release from the hydrogels in relation to pore formation
00,10,20,30,40,50,60,70,80,9
1
0 50 100 150 200
time (h)
mg
Ibu-
lis tx
/mg
Ibu-
lis t0
CMC (a)CMC 30 um(a)CMC 30um (b)CMC 14um (b)CMC 40um (b)
Evaluation of drug release:
•The hydrogel, loaded with the drug is placed inside a polystyrene cell through which the washing solution (PBS pH 7.4)flows
•At regular time intervals an aliquot of the flowing solution is analysed (by UV) at 263 nm, evaluating the amount of released drug.
a)a) PorePore formationformation and and loadingloading the the porousporous hydrogelhydrogel withwith the the drugdrug
b)b) LoadingLoading the the hydrogelhydrogel withwith the the drugdrug and and porepore formationformation
Methods:
ToF-SIMS analysis: drug distribution
External portion Internal portion
Hyal 50%
Hyal 35μm proc.B
Hyal 35μm proc.A
Total ions ibuprofen Total ions ibuprofen
Stimoli Elettrici e Rilascio di Stimoli Elettrici e Rilascio di FarmaciFarmaci
Il Caso della Il Caso della BleomicinaBleomicina (Farmaco (Farmaco antitumorale) nel gel di antitumorale) nel gel di GuaroGuaro sottoposto sottoposto ad ad elettroporazioneelettroporazione
O
O
O
O
O*
CH2OH
OH
OH
OH
OH
CH2
OOH
HO OH
CH2OH
n
GUAR
Guar-Bleomicina zona interna
100 μmTotal ion 100 μmCHNO
100 μmCHS 100 μmC2HS
Guar-Bleomicina zona esterna
100 μmTotal ion 100 μmCHNO
100 μmCHS 100 μmC2HS
Nuovi materiali
per la sostituzione
del nucleo polposo
Il nucleo polposo
Nucleo polposo (NP): materiale fibrocartilagineosoggetto alle leggi dei fluidi. Esso può essere considerato FLUIDO INCOMPRIMIBILE essendo confinato nella sua forma e posizione dall’ annulo fibroso che presenta natura elastica
Composizione del nucleo polposo:Acqua in un matrice di proteoglicani collagene e proteine
•H2O: 70-90% (90% alla nascita, 80% a 20 anni 70% a 60 anni)
•proteoglicani: varia da 65% (peso secco) al 30% con l’avanzare dell’età. Principali specie di PGs: condroitin 6-solfato, condroitin 4-solfato, cheratan solfato, dermatan solfato, versicano e acido ialuronico
•Collagene: 15-20%(peso secco). Tipi di collagene presente: II,VI, IX e XI
•Elastina e proteine non-collagenose: 20-45% (peso secco)
Gel polisaccaridici•70-90% H2O
•10-20% network di proteoglicani
Il nucleo polposo lombare umano non degenerato in condizioni di shear mostra un comportamento viscoelasticoMateriali analizzati:
•CMC (CMd: 0.95) 100%
•CMC (CMd: 0.77) 100%
•CMCA (CMd: 0.95) (Ad:40)
•CMCA (CMd: 0.77) (Ad:teorico 50)
•AA 50%
NP CMC (0.95)
CMCA (0.95)
CMC (0.77)
CMCA (0.77)
AA
⎢G* ⎜(kPa) 7-21 9.4-11.4 5-8 11.8-13.4 0. 9-1.5 19.5-26δ 23-31° 4.3-7.6° 15° 5.5° 7-11° 4.6-7.6°
Risultati:
O
O
O
RCOOX
RCOOXDMF, N2CMP-J
O
O
O
RCOOX
RCOO
NH3C
NH2CH3
O
O
O
RCOOX
RCONHCH3
polimers
AA
CMC : R=CH2OCH2
Realisation of new amidic polysaccharides
OCOONa
O
HOOH
O O
CONHCH3
HO
OH
O
O
CH2 OCH2COONa
OH
OHO
CH2OCH2CONHCH3
OH
OH
Amidic Carboxymethylcellulose (CMCA)
Amidic alginate (AAA)
Proprietà tissotropiche: CMCA
Scopo: possibilità di introdurre il materiale tramite iniezione all’interno dell’annulo fibroso
0 2000 4000 6000 8000 10000 12000osc. stress (Pa)
0
1000
2000
3000
4000
5000
6000
G' (
Pa)
0
1000
2000
3000
4000
5000
6000
G'' (P
a)
0
10,00
20,00
30,00
40,00
50,00
60,00
delta
(deg
rees
)
CMCACMCA
Punto di gelo
4017Pa (0,4Mpa)
Il materiale è iniettabile, la transizione gel-sol ècompletamente reversibile
Time sweep test: G’ e G” sono determinati in funzione del tempo a temperatura, frequenza e sforzo costanti: serve a seguire la variazione di struttura nel tempo
Parametri usati:
T=37°C
Frequenza=10Hz
Sforzo= (1MPa)
Sforzo=0,1MPa
Sforzo=0,01MPa
0 200,0 400,0 600,0 800,0 1000 1200 1400time (s)
10,00
100,0
1000
10000
G' (
Pa)
10,00
100,0
1000
10000
G'' (P
a)
CMCACMCACMCACMCA
Durante la notte il nucleo è sottoposto a stress bassi e si reidrata:
Cinetica di idratazione del gel di CMCA:
Parametri:
T=37°C
NaCl 0,9%
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
0h 1h 3h 6h 24h 48h 72h
25°C
37°C
Polysaccharidic hydrogels as cellular scaffolds
What is a cellular scaffold?
It can be defined as “a substrate able to favour celladhesion, 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.
Percentage of cells into gel using three different procedures of cell seeding: Amber effect (red), co-colture insert (green), normal seeding (blue).
99%
0%
64%
The “Amber Effect” appears as a more valid method to evaluatethe cell proliferation inside the hydrogel structure
amber effect co-colture insert normal seeding0
20
40
60
80
100Pe
rcen
tage
of c
ells
into
gel
Experimental methodology
polysaccharidic hydrogels as cellular scaffold
Cell type:
•Human diploid fibroblasts
•Rat hepatocytes
•Rat β-pancreatic cells
before after
AA 30μm AA 30μm
Amber effect
•Human diploid fibroblasts
Hyal_ 3mm Hyal40μm_ 3mm
Hyal40μm_ 1,5mmHyal_ 1,5mm
On both 1,5mm thicknative and poroushydrogels fibroblastsshowed a spread morphology
On 3mm thick nativehydrogels fibroblastsare mainly organisedin clusters
On 3mm thick poroushydrogels fibroblastsshowed a spread morphology
0.1000 1.000 10.00frequency (Hz)
100.0
1000
10000
G' (Pa)
100.0
1000
10000
G'' (
Pa)
AA
AAA
The amidation affects the rheological properties and the water up-take
0.1000 1.000 10.00frequency (Hz)
100.0
1000
10000
G' (P
a)
100.0
1000
10000
G''
(Pa)
CMC
CMCA
0
2000
4000
6000
8000
10000
12000
CMC100% CMCA AA AAA Hyal
[(Ws-
Wd)
/Wd]
*100
CMCA hydrogel:•Pancreatic cells
•Osteoblast-like cells MG63
•BAECCMCA Hyal
Pancreatic cells
Control CMC CMCA Hyal
After 14 days the Hyal hydrogel wascompletely destroyed,whereas the CMCA hydrogel was still present after 10 weeks
control CMC CMCA Hyal
WST1 (OD450650nm) 0.67±0.40 0.63±0.26 0.81±0.14 0.83±0.10LDH (IU/L) 12.4±1.90 18.0±3.90 15.8±5.20 21.4±1.90IL-6 (pg/mL) 0.7±0. 06 8.23±1.74 1.87±0.27 3.2±1.00TNF-α (pg/mL) 8.15±0.72 7.08±1.10 6.7±1.03 8.32±1.42
Cytotoxicity e bioactivity on osteoblast-like (MG63) cells
BONE ALKALINE PHOSPHATASE
0
2
4
6
8
10
CTR CMC CMC-A HYAL
BALP
(U/l)
**
TYPE I COLLAGEN
0
1
2
3
4
5
6
7
CTR CMC CMC-A HYAL
**
AA AAA CMC CMCA Hyal control
WST1 (OD450625nm)
0.54±0.01 0.76±0.09
1255±117
4.4±0.5
307±56
0.85±0.09
92±25
2.72±0.34
0.73±0.02 0.70 ±0.04 1.16 ±0.03
Aggrecan (ng/mL) 898 ±80
0.57±0.01
924±61
6.3±0.5
203±12
7.43±0.62
378±109
1126±114 1294±82 731±83
IL-6 (pg/mL) 3.9 ±0.5 3.8±0.3 4.4±0.7 3.6±0.2
CollagenII(ng/mL)
233 ±29 338±30 313±17 133±11
2.61±0.43
MMP1 (pg/mL) 2.19 ±0.38 0.85±0.51 1.88±0.26 1.33±0.68
MMP13 (pg/mL) 164 ±25 93±15 121±16 68±12
Cathepsin B (ng/mL)
2.40 ±0.07 2.25±0.50 2.06±0.08 4.28±0.54
Cytotoxicity and bioactivity on chondrocytes
WST1: cell viability
Aggrecan, Collagen type II production: bioactivityMMP1, MMP13, Cathepsin B: differentiation
IL-6: inflammatory response
Synthesis of new scaffolds
02000400060008000
100001200014000
Hyal Hyal-Cu
wat
er u
p-ta
ke The presence of copper ions provoked a decrease of the amount of absorbed water
On the basis of the tendency of Cu2+ to form a “4+2” structure, the decrease of the water uptake can be due to both the charge neutralisation and involvement of hydrophilic groups in the complex formation.
EDAX analysis: in correspondence of the pellets a consistent presenceof copper ions waspresent as evidencedby an increase of colour tone.
Potential use (e.g. wound healing) of Copper complexes, as angiogenicfactors is shown by the following experiments
Fibrin net surrounding the completely swollen hydrogel (white mass)
New vessel
•Hyal- Cu2+ was implanted on the subscapolar panniculus adiposus of five months-old male Wistar rats and left for 21 days.
•Hyal-Cu2+ morcellised bone implanted on the subscapolar panniculusadiposus for 50 days.
New vessel
DNA (PDRN)DNA (PDRN)procedure a:procedure a:
NH2
O PO
OHOH
DNA
O PO
OHOH
NHC R
OC
NH
O
O PO
OHOH
DNA DNA
HOOC-R.COOH(acido bicarbossilicosolubile in acqua)
EDC(water soluble carbodiimide)
DNADNA gel(a)
DNA (PDRN)DNA (PDRN)NH2
O PO
OHOH
DNA
NH2
O P
O
C=N
O
OH
CH2 CH2 CH2 NCH3
CH3
NH
CH2CH3
NH2
O P
O
C=N
O
OH
CH2 CH2 CH2 NCH3
CH3
NH
NH2 (CH2)3NH2
NH2
O P
O
OH
N CH2H
CH2 NCH3
CH3
NH
CH2CH3
NH2
O P
O
OH
NH NH(CH2)3
C
EDC
DNA
DNA
DNA DNA
+
O
(derivato ureico)
procedure b:procedure b:
DNADNA gel(b)
Water up-take
•il prodotto presenta capacità di rigonfiare in soluzione acquosa
0
1000
2000
3000
4000
5000
0 24 48 72 96 120 144
time (h)
wat
er u
p-ta
keDNA(P)
DNA(N)
0
1000
2000
3000
4000
5000
DNA(N) DNA(P)
wat
er u
p-ta
ke