Liu Nanobionics Lab
Micro and Nanotechnology Laboratory
UNIVERSITY OF ILLINOIS AT URBANA - CHAMPAIGN
Cell and Tissue Engineering
Nanotechnology
Yi Chen
Outline
• What is Tissue Engineering
• What is Nanotechnology
• Why we apply Nanotechnology to Tissue
Engineering
• How is Nanotechnology applied to Tissue
Engineering
– Different nanofabrication techniques
– Applications
Tissue Engineering
Expand number in culture
Remove cells from the
body.
Seed onto an appropriate
scaffold with suitable growth
factors and cytokines
Re-implant engineered
tissue repair damaged
site
Place into culture
Tissue Engineering
Hydrogels
Nanofibrous scaffolds
Self-assembling scaffolds
Solid freeform fabricated scaffolds
Differentiated cells
Adult stem cells
Embryonic stem cells
Cells
Scaffolds
Tissue
Engineering
Biorectors
Dynamic cell seeding
Improved mass transfer
Mechanical stimuli
Signals
Small molecules
Growth factors/polypeptides
Nucleic acids (DNA, siRNA, and
antisense oligonucleotides)
Nanotechnology and Tissue Engineering: The Scaffold, CRC Press; 1 edition (June 16, 2008)
Nanotechnology Overview
Nanotechnology is a branch of science
and engineering which deals with
structures and devices in nanometer
scale.
View & Characterize
 Optical Microscopy
 Electron Microscopy
 Atomic Force Microscopy
Create & Manipulate
Nanofabrication: (Top down & Bottom up)
 Lithography (Optical, E-beam, NIP, DPL)
 Etching (Wet Etching, Plasma Etching)
 Deposition (Evaporation, PECVD,
Electrochemical Deposition, Sputtering, etc.)
 Epitaxial growth (MOCVD, MBE, etc.)
Why we apply Nanotech in TE?
Cells on microfibrous scaffolds have a polarized relationship, with one side
of the cell attached to the scaffold, the other exposed to physiological
media. In comparison, it is likely that cells are more naturally constrained
by nanofibrous scaffolds.
Nanofibrous Scaffold
• Electrospinning
• Self-Assembly
Electrospinning
• This process involves the ejection of a charged polymer fluid onto an oppositely
charged surface.
• Multiple polymers can be combined
• Control over fiber diameter and scaffold architecture
Research on Parameters of Electrospinning Process
• Solution properties






Viscosity
Conductivity
Surface tension
Polymer molecular weight
Dipole moment
Dielectric constant
• Controlled variables





Flow rate
Electric field strength
Distance between tip and collector
Needle tip design
Collector composition and geometry
• Ambient parameters
 Temperature
 Humidity
 Air velocity
Tissue Engineering. May 2006, 12(5): 1197-1211.
Research on Materials
• Polyglycolic acid (PGA)
– Highly crystalline, hydrophilic,
byproduct is glycolic acid
• Polylactic acid (PLA)
– Hydrophobic, lower melting
temperature, byproduct is
lactic acid
• Polydioxanone (PDO)
– Highly crystalline
• Polycaprolactone (PCL)
– Semi-crystalline properties,
easily co-polymerized,
byproduct caproic acid
• Blends
–
–
–
–
PGA-PLA
PGA-PCL
PLA-PCL
PDO-PCL
•
•
•
•
•
Elastin
Gelatin collagen
Fibrillar collagen
Collagen blends
Fibrinogen
• Synthetic polymers
 PGA, PLA and PLGA most commonly
used
 PDO most similar to Elastin collagen
blend (limited by shape memory)
 PCL most elastic and mixed frequenlty
with other material s
 Provide nanoscale physical features
• Natural polymers
 Collagen Type I & III + PDO: best
possible match for blood vessels
Advanced Drug Delivery Reviews Volume 59, Issue 14, 10 December 2007, Pages 1413-1433
Self Assembly
Figure 1: Fabrication of various peptide
materials.
Figure 2: Self-assembling peptides form a
three-dimensional scaffold woven from
nanofibers ~ 10 nm in diameter.
(a) Representation of self-assembling peptide.
(b) Electron micrograph of three-dimensional scaffold formed in vitro.
(c) Rat hippocampal neurons form active nerve connections; each green dot represents a single synapsis.
(d) Neural cells from a rat hippocampal tissue slide migrate on the three-dimensional peptide scaffold. Cells on the
polymer membrane (left) and on the peptide scaffold (right) are shown. Both glial cells (green) and neural
progenitors (red) migrate into the three-dimensional peptide scaffold.
(e) Brain damage repair in hamster. The peptide scaffold was injected into the optic nerve, which was first severed
with a knife. The cut was sealed by the migrating cells after 2 days. A great number of neurons form synapses.
(f) Chondrocytes from young and adult bovine encapsulated in the peptide scaffold. These cells not only produce a
large amount of glycosaminoglycans (purple) and type II collagen (yellow), characteristic materials found in
cartilage, but also a cartilage-like tissue in vitro53.
(g) Adult rat liver progenitor cells encapsulated in the peptide scaffold. The cells on the two-dimensional dish did
not produce cytochrome P450–type enzymes (left). However, cells in three-dimensional scaffolds showed
cytochrome P450 activity (right).
Nature Biotechnology 21, 1171 - 1178 (2003)
Self Assembly
Figure 3: Lipid, peptide and protein
scaffold nanowires.
Figure 4: Microlenses and fiber-optics
fabricated from protein scaffolds.
Nature Biotechnology 21, 1171 - 1178 (2003)
Self-Assembling Peptide Scaffolds for Regenerative Medicine
SAPNS heals the brain in young animals.
SAPNS allows axons to regenerate through the lesion site in brain.
PNAS March 28, 2006 vol. 103 no. 13 5054-5059
Phase separation
•
•
•
This process involves dissolving of a
polymer in a solvent at a high
temperature followed by a liquid–liquid
or solid–liquid phase separation induced
by lowering the solution temperature
Capable of wide range of geometry and
dimensions include pits, islands, fibers,
and irregular pore structures
Simpler than self-assembly
a) powder, b) scaffolds with continuous network, c) foam with closed pores
SEM of nanofibrous scaffold with interconnected spherical macropores
Advanced Drug Delivery Reviews Volume 59, Issue 14, 10 December 2007, Pages 1413-1433
Carbon Nanotube
Cell tracking and labeling
Sensing cellular behavior
Augmenting cellular behavior
Augmenting cellular behavior
Cytotoxicity
Murine myoblast stem cells incubated with
DNA-encapsulated nanotubes
neuron bridging an array of
carbon nanotubes thereby
creating neural networks.
Biomaterials Volume 28, Issue 2, January 2007, Pages 344-353
Block Coploymer
Synthetic scheme of block copolymers.
In vitro release profile of FITClabelled dextran (Mr 20,000) from
PEO–PLLA–PEO (Mr 5,000–2,040–
5,000) triblock copolymer.
Injectable drug-delivery system
Gel–sol transition curves.
Science 30 May 1997:
Vol. 276. no. 5317, pp. 1401 - 1404
Nature 388, 860-862 (28 August 1997)
Printing Technology
• Nanoimprinting Lithography
• Organ Printing
• Contact Printing
Nanoimprinting Lithography
Prof. Stephen Y. Chou
Thermal-sensitive Polymer
Optical-sensitive Polymer
Nanopattern-induced changes in morphology and
motility of smooth muscle cells
Alignment and elongation characterization
Wound healing assay for cell motility
SMC morphology
BrdU cell proliferation assay
Biomaterials Volume 26, Issue 26, September 2005, Pages 5405-5413
Organ printing: computer-aided jet-based 3D tissue engineering
Fig. 1. Fusion of embryonic myocardial ring.
Myocardium rings were cut from
Stage 15–16 HH chick ventricle, containing
only myocardium, endocardium and
some intervening matrix. Isolated rings beat
steadily for several days; (a) adjacent
apposed rings fused overnight and (b) beat as
one. (c). Schematic representation
of principle of organ printing technology:
placing of cell aggregates layer by layer
in solidifying thermo-reversible gel with
sequential cell aggregate fusion and
morphing into 3D tube.
Fig. 2. Cell printer and images
of printed cells and tissue
constructs.
Fig. 3. (a) Printed bagel-like ring that
consists of several layers of
sequentially
(layer-by-layer) deposited collagen
type 1 gel. (b) Manually printed
living tube with
radial branches from the chick
27stage HH embryonic heart
cushion tissue placed
in 3D collagen type 1 gel.
Trends Biotechnol. 2003 Apr;21(4):157-61.
Contact Printing
J. Am. Chem. Soc., 2005, 127 (48), pp 16774–16775
Advanced Materials Volume 19 Issue 24, Pages 4338 - 4342
Summary
• Nanofibrous Scaffold
– Electrospinning
– Self-Assembly
• Nanoporous Scaffold
– Phase Separation
• Carbon Nanotube
• Block Copolymer
• Printing
– Nanoimprinting Lithography
– Organ Printing
– Contact Printing
Lab Chip, 2004, 4, 98 - 103
References
 Nanotechnology and Tissue Engineering: The Scaffold, CRC Press; 1 edition (June 16, 2008)
 Quynh P. Pham, Upma Sharma, Ph.D., Dr. Antonios G. Mikos, Electrospinning of Polymeric Nanofibers for Tissue
Engineering Applications: A Review, Tissue Engineering. May 2006, 12(5): 1197-1211.
 Catherine P. Barnes, Scott A. Sell, Eugene D. Boland, David G. Simpson, Gary L. Bowlin, Nanofiber technology: Designing
the next generation of tissue engineering scaffolds, Advanced Drug Delivery Reviews, Volume 59, Issue 14, Intersection of
Nanoscience and Modern Surface Analytical Methodology, 10 December 2007, Pages 1413-1433, ISSN 0169-409X, DOI:
10.1016/j.addr.2007.04.022.
 Shuguang Zhang, Fabrication of novel biomaterials through molecular self-assembly, Nature Biotechnology 21, 1171 - 1178
(2003)
 Rutledge G. Ellis-Behnke, Yu-Xiang Liang, Si-Wei You, David K. C. Tay, Shuguang Zhang, Kwok-Fai So, and Gerald E.
Schneider, Nano neuro knitting: Peptide nanofiber scaffold for brain repair and axon regeneration with functional return of
vision PNAS 2006 103 (13) 5054-5059
 Benjamin S. Harrison, Anthony Atala, Carbon nanotube applications for tissue engineering, Biomaterials, Volume 28, Issue
2, Cellular and Molecular Biology Techniques for Biomaterials Evaluation, January 2007, Pages 344-353, ISSN 0142-9612,
DOI: 10.1016/j.biomaterials.2006.07.044.
 Miri Park, Christopher Harrison, Paul M. Chaikin, Richard A. Register, Douglas H. Adamson, Block Copolymer Lithography:
Periodic Arrays of ~1011 Holes in 1 Square Centimeter, Science 30 May 1997: Vol. 276. no. 5317, pp. 1401 - 1404
 Byeongmoon Jeong, You Han Bae, Doo Sung Lee and Sung Wan Kim, Biodegradable block copolymers as injectable drugdelivery systems, Nature 388, 860-862 (28 August 1997)
 Evelyn K.F. Yim, Ron M. Reano, Stella W. Pang, Albert F. Yee, Christopher S. Chen, Kam W. Leong, Nanopattern-induced
changes in morphology and motility of smooth muscle cells, Biomaterials, Volume 26, Issue 26, September 2005, Pages
5405-5413, ISSN 0142-9612, DOI: 10.1016/j.biomaterials.2005.01.058.
 Mironov V, Boland T, Trusk T, Forgacs G, Markwald RR. Organ printing: computer-aided jet-based 3D tissue engineering.
Trends Biotechnol. 2003 Apr;21(4):157-61.
 Yu, A. A.; Stellacci, F., Contact Printing beyond Surface Roughness: Liquid Supramolecular Nano-Stamping, Advanced
Materials, 19, 4338-4342, 2007
 Yu A.A., Savas T., Cabrini S., diFabrizio E., Smith H.I., Stellacci F., High resolution printing of DNA features on poly(methyl
methacrylate) substrates using supramolecular nano-stamping, J. Am. Chem. Soc., 127, 16774-16775, 2005