neural reapir tissue engineering - presentation

8/13/2019 neural reapir tissue engineering - presentation

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S

Heterogenic drug

release for neuralrepair

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech republic

Miroslav Vetrk, Martin Hrub, Martin Pdny, Ji Michlek


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Spinal Cord Injury

S Spinal cord injury is permanent injury

S Limitation in regeneration, Mesenchymal and Glial scarprevents spontaneous healing

S New system based on hydrogels, releasing neuromediators,which are promoting for growing and proliferation of neurons

S Goal: rebuild damaged neural connection


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State of art

Heterogenous system based on ion

exchanger DOWEX

Macroporous hydrogels matrix holding

exchanger in steady positions

Matrix pHPMA, pHEMA

Very stable in non ionic environment for

instance H2O

Gradient releasing of neuropromotors

by ionic straight change, PBS


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Model study

kinetik of releasing

Gelation 2D, p(HEMA)Dowex, swelling model

Model bonding neuropromotors methylene blue, radioaktive

labeled tyrosine amide

ReleasingAmax 668 nm methylen blue - graph

activity measurement [125I]tyrosine amide

Stability study


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Releasing


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Releasing

Methylen blue


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Maximum absorbance in

time


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Stability

Samples were measured during 46 days

Full spektrum detail abs. max formeth. blue


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125I-Tyrosin amide

Model kinetic study of releasing 125Ityrosin amide from pHPMA scaffolds, in

PBS, based on size of dowex elements


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CH2

CH3 O

O

OH

p(HEMA)poly N-(2-Hydroxypropyl)methacrylate

p(HEMA)poly(2-hydroxyethyl) methacrylate

p(MOETA+Cl-) - [poly(2-(methacryloyloxy)ethyl]trimethylammonium

3D scaffolds

Size distribution 0.03 mm - 0.05 mm, 10 Nm force


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Macroporous 3D hydrogels

SEM images of the macroporous hydrogels

Real size


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3D model

carbachol


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S

Acid cationic exchanger DOWEX 50, homogenicspread in the volume :

S Neuropromotors binging on DowexSerotonin,

Dopamine, Tryptamim, Carbacholyl chloride

S Washing H2O

S Biological studyproliferation (Human stem cells),

histochemistry

ExperimentsIn Vitro


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In Vitroproliferation

S Dopamine (DWX1) and serotonin (DWX2) decrease

proliferation of neuronsS Releasing tryptamin (DWX3) increase proliferation by 20-

30% in comparison with control cells culture. (DWX0scaffold same but without neuromediators.

S Releasing carbachol (DWX4) increase proliferation by25% in comparison with non-treated cells without scaffold(SPC-01)

S Proliferation curve of the control cells are other than cellswithout scaffold, what can be explained as adhesive cellsconnection on the surface of scaffold od DWX0


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Proliferation


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Histochemistry

S Histochemical analysis of the samples with human stem cells

S We observed growing cells colonies present in the scaffolds and

also seeding cells on the surface of the gels

S Dopamine (DWX1), serotonin (DWX2) are promoting only a

couple of cells, which survive in hydrogels. Serotonin promoted

cell growth only for 2-3 weeks

S Tryptamin (DWX3) a carbachol (DWX4) promote cells growth


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Histochemistry

For all pictures: blue color DAPIcells core marker, red color cells cytoskeleton


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Biodegradable scaffolds

S Biodegradable hydrogel (based on di-thionicotinic acid), is degradable

due to thiols groups presents in blood plasma, degradation is

irreversible

S Thiols present in human body (cysteine, albumine)

S Stable in non-reductive environment : PBS, H2O, DMSO


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Biodegradation

SS

SS

R

SH

SS

SH

RS

SR

polymer polymer

+

R-SH(cystein)

polymer

polymer

polymer polymer

+

2 R-SH(cystein)

polymer

+

+

2


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Tautomeric stabilisation of the

degradation products


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Hydrogel degradation

S Cysteine solution in the PBS

S Used cysteine concentration:

0.33 mmol.L-1 SH in blood plasma

4.2 mmol.L-1 SH in blood plasma + total albumine

12.6 mmol.L-1 model

42 mmol.L-1 model


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Degradation

-1,00

-0,80

-0,60

-0,40

-0,20

0,00

0 10 20 30 40 50 60

t, days

1

1. ccys= 0.33 mmol/L2. ccys= 4.2 mmol/L

3. ccys= 12.6 mmol/L

4. ccys= 42 mmol/L

Degradation of hydrogel in 4.2 mmol.L-1solution

(- SH in blood plasma + total albumin) during 50 days

234


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Conclusion

S Macroporous scaffold systems for neuropromoting effect were prepared,

characterised and tested.

S Gradient releasing has positive effect on neurons growth

S In vitroexperiments shown preferable neuropromotos - carbachol,

tryptamine

S Good kinetic degradation of the scaffold based on di-nicotinic acid up to 50

days, time needed for cells growth is very promising in spinal chord injuries

S Used hydrogels scaffold if has important role pHEMA, pHPMA, pMOETA+

Cl-


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Thank you for your attention

S Martin Hrub

S Martin Pdny

S Pavla Jendelov

for future consultations dont

hesitate to write

[email protected]

neural reapir tissue engineering - presentation

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