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