3d nanotechnology and laser printing of nanoparticles and

Laser Zentrum Hannover, Germany

3D Nanotechnology and Laser Printing of Nanoparticles and Living Cells

Boris N. ChichkovLeibniz University Hannover 30.06.2014

[email protected]

Boris Chichkov, 3D Nanotechnology and Laser Printing of Nanoparticles and Living Cells

near IR fs-pulses

resin

Two-photon polymerization Laser printing Laser ablation

High resolution manufacturing with lasers


Micro- and nanostructuring for better implant adaptation

3

ImplantInteraction with the tissue

consisting of different cell types

„bad“ cells

Isolation and loss of functions Reconstruction and regeneration

„good“ cells

fibroblasts neuronal cells, osteoblasts, endothelialcells, chondrocytes, etc.


Material Functionalization

4

Biology Chemistry Physics

Growth FactorsAdhesion Peptides

In generalWettability

ElasticityTopography

Surface Charge

Selective cell control and antibacterial effect


Laser microstructuring of metals: Titanium “lotus-like” structures

Combination of micro- and nanostructures


Impact on Fibroblasts: Morphology

6Schlie S et al. (2010), Fadeeva E et al. (2010a), Fadeeva E et al. (2010b), Schlie S et al. (2012)

50 µm

Control

Pt

Ti

Si

Negative Response

Phalloidin (green)

10 µm


Primary neurons on laser-generated spike structures

Staining of neuronal markers and nucleus (blue)

Beta III Tubulin (green) MAP 2 (red) and synaptophysin (green)

Results:Spike structures do not have a negative effect on primary neurons


targettarget

laser

NanoNano--particleparticle

Liquid

• High purity and stability• Monoatomic materials• Alloy nanoparticles• Particle surface-functionalization• Polymer-embedded nanoparticle• Coatings with nanosized particles• Controlled drug-release• Stoichiometric nanoparticles• Novel methods better control

J. Phys. Chem. C, 2010Appl. Phys. A, 2010

Laser generation of nanoparticles


Laser printing of spherical gold and silicon nanoparticles

Opt. Express 18, 21198 (2010).

J. Opt. Soc. Am. B, 26, 130 (2009)

Opt. Express 17, 18820 (2009)


Jet and droplet formation

Ep = 70nJEp = 65nJ

Ep = 75nJ

Appl. Phys. A 79, 879 (2004)


Receiver substrate(glass)

Tightly focusedfs laser pulse

Thin Au film

Donor substrate(glass)

Fabrication of spherical nanoparticles by laser printing

11

„Laser-induced transfer of metallic nanodroplets for plasmonics and metamaterial application“JOSA B, Vol. 26, No. 12, B130, 2009


„Nano“-letters from Au

12

SEM-image of nanoparticles

The same image made by darkfield microscopy

20 µm

Boris Chichkov, 3D Nanotechnology and Laser Printing of Nanoparticles and Living Cells13

Receiver substrate (glass)

Bulk silicon


Laser induced transfer of silicon nanoparticles from bulk silicon

Evlyukhin et al. Nano Lett. 12, 3749 (2012)


Extinction of spherical Si nanoparticles in air

md

ed Extinction of a Si nanoparticle with R=65 nm

Magnetic response of Si spherical nanoparticles (Mie theory)

Electric dipole (ed)Magnetic dipole (md)

A. B. Evlyukhin et al. Phys. Rev. B 82,045404 (2010)

E k

1l

Ml

Elext Extinction cross section


Resonant response of silicon nanoparticles

Mie theory

ED

MD

Evlyukhin et al. Nano Lett. 12, 3749 (2012)


Controlled fabrication and precise deposition of silicon nanoparticles

Receiver substrate (glass)

Bulk silicon


Silicon nanoparticle

Silicon dioxide Silicon donor layer

U. Zywietz, C. Reinhardt, A.B. Evlyukhin, B.N. Chichkov, „Laser printing of silicon nanoparticleswith resonant optical electric and magnetic responses“, Nature Communications, 5, No. 3402, (2014).

200 nm

50 µm


Laser induced crystallization

Receiver substrate(glass)

Square-shapedfs laser pulse

Silicon nanoparticle (c-Si) Silicon nanoparticle (a-Si)

U. Zywietz, et al. Nature Commun. 5:3402 (2014).


Selective phase change of Si nanoparticles

U. Zywietz, C. Reinhardt, A.B. Evlyukhin, B.N. Chichkov, „Laser printing of silicon nanoparticleswith resonant optical electric and magnetic responses“, Nature Communications, 5, No. 3402, (2014).


Nonlinear interaction of fs-pulses

s0

s1

E0

E1

1P inducedFluorescence

s0

s1

E0

E1

2P inducedFluorescence

Linearexcitation

Non-linearexcitation

Fs-laser pulses allowenergy deposition into a Volume!


Two-photon polymerization


near IR fs-pulses

resin

Opt. Lett. 28, 301, (2003)Adv. Eng. Mat. 5, 551, (2003)

3D nanostructuring by two-photon polymerization

Nanotechnology with lasers

Ormocer

Deutsches Patent 101 52 878.7-43


Commercially available 2PP systemfrom LZH: [email protected]

Two-photon polymerization (5cm/s)


Regenerative medicine and tissue engineeringRegenerative medicine and tissue engineeringInterdisciplinary Research (Mathematics, Physics, Material Science, Engineering, Biology, Medicine)

Regenerative Medicine:

Replacement or repair of ill organs, which body cannot restore itself

Tissue Engineering:

Fabrication of living tissue from patient cellsTransplantation inside a damaged Organ


Organ transplantation demandOrgan transplantation demand


Organogenesis. 2010 Jul‐Sep; 6(3): 151–157

Kristine C Rustad, et.al. Organogenesis 2010 Jul‐Sep; 6(3): 151–157.

Basic idea of tissue engineeringBasic idea of tissue engineering


Materials Laser beam

Cover slip

Polymerized scaffold structure Liquid photosensetive polymer

• Ormocers®

• PEG-DA

• Organic-InorganicZr-hybrid materials

• PCL

• PLA

• Gelatin

• E-Shell®

Fabrication of scaffolds via two-photon polymerization (2PP)


200 μm 1 mm 150 μm

27

Scaffold-based approach / Examples

Fibrin scaffold


3D conductive polymer scaffolds

PEG-DA : poly(ethylene glycol) diacrylateEDOT : 3,4-ethylenedioxythiophenePEDOT : poly(3,4-ethylenedioxythiophene)

PEG-DA and EDOT blends are used for 2PP and sequential in-situ oxidative polymerization;

Real-3D, physically stable and biocompatible microstructures are produced; Interpenetrating polymer network of PEG-DA and PEDOT leads to conductivities

of up to 0.04 S/cm.

EDOT

PEDOT

Opt. Express, 21, 31029 (2013)


LIFT: Laser Induced Forward Transfer

Target

Transportingmaterial

Absorbingmaterial

Substrate

Laser pulse

Biological laser printing


MotivationApplications in Regenerative Medizin: - prototyping of complex biomedical products- biofabrication of tissue substitutes and living organs

Bioprinting Nozzle free laser printing


LIFTdynamics


EFFECT OF LASER PRINTING ON CELLS – SURVIVAL RATE

37

Nearly all cells survive the printing process

Live/Dead-staining withCalcein AM (green; vital cells) and Ethidium Homodimer 1 (red; dead cells)

Survival rate:

Fibroblasts (NIH3T3) 98% ± 1%Keratinocytes (HaCaT) 98% ± 1%human adipose-derived 99% ± 1%stem cells (ASC)cord blood derived 98% ± 3%endothelial colony forming cells (ECFC)

Koch et al., Laser Printing of Skin Cells and Human Stem Cells, Tissue Eng Part C: Methods 2010, 16(5): 847-854


Advantages of Laser assisted Bioprinting:

1. Printing of single to dozen of cells witha micrometer precision

2. No observable damage to the pheno-and genotype of the cells

3. Utilization of cross linkable hydrogels (e.g. Fibrin) enables 3D free form fabrication

Examined cells AssessmentshBMSCshASCsECFCsCardiomyocytesFibroblastsKeratinocytesChondrocytes

Survival ratesProliferationApoptoseComet assayRT-PCRImmunohisto-chemistry

Laser printing has no influence on the cell behaviourKoch et al. Tissue Eng Part C 16(5): 847-854 (2010)

Fundamental studies


CELL-CELL INTERACTIONS IN MULTICELLULAR ARRAYS

39

A printed predefined pattern of cell containing dropletsallows to study cell-cell or cell-environment interactions

EC

onl

y

AS

C o

nly

EC

+ A

SC

EC

+ A

SC

Gruene et al., Laser printing of three-dimensional multicellular arrays for studies of cell-cell and cell-environment interactions. Tissue Eng Part C Methods 17(10): 973-82 (2011)

250 µm

Printed droplets containing endothelial cells (EC, red) or adipose derived stem cells (ASC, green)

After 5 days cultured in VEGF-free media

o+

ASC (+), EC (o)600 µm


GENERATION OF 3D SKIN TISSUE

40

Each color layer consists of four printed sub-layer. The whole construct is about 2 mm high.

scale bars500 µm

Layered arrangement of red and green HaCaT (eGFP, mCherry), 18 h after printing

The cells have been embedded in collagen directly before printing.The layers do not intermix during or after printing.

Layers of fibroblasts (NIH3T3) and keratinocytes (HaCaT), in collagen I on Matriderm

Koch et al., Skin Tissue Generation by Laser Cell Printing. Biotechnol Bioeng. 109(7): 1855-1863 (2012)

Together with Prof. Vogt, MHH


Immunofluorescence staining of cytokeratin 14 (green) for keratinocytescell nuclei were stained with Hoechst 33342 (blue)

Generation of skin equivalents by laser printing

native skin Matriderm with cells

Matriderm without cells

19

Together with Prof. Vogt, MHH


MICROVASCULARIZATION

42

Accelerated vessel formation in printed structure (EC/MSC co-culture) in vitroImproved heart function after myocard infarction and implantation of printed cardiac patch

Vessel formationin the printed

structure after8 days

(HUVEC/hMSC-co-culture on

Matrigel-coatedcardiac patch)

Human cells, integrated in themurine vascularnetwork at theborder of thecardiac infarctzone, 8 weekspost-infarct

PEUU cardiac patchimmersed in Matrigel

Printed human endothelial cells (green) and human mesenchymal stem cells (hMSC, red) on a cardiac patch

Gaebel et al., Patterning human stem cells and endothelial cells with laser printing for cardiac regeneration, Biomaterials 32: 9218-9230 (2011)


Team/AcknowledgementsLZH NT DepartmentAnnette Barchanski,Andrea Deiwick,Andrey Evlyukhin,Elena Fadeeva,Ulf Hinze,Sven Ganske,Roman Kiyan,Jürgen Koch,Lothar Koch,Anastasia Koroleva,Olga Kufelt,Kestutis Kurselis,Carsten Reinhardt,Laszlo Sajti, Sabrina Schlie-Wolter,Andreas Schwenke,Urs Zywietz Thank you for your attention

3d nanotechnology and laser printing of nanoparticles and

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