nanobiomaterials for cell and tissue engineering
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Nathaniel Hwang, Ph.D.
Seoul National University,
School of Chemical and Biological Engineering
Nanobiomaterials for Cell and Tissue Engineering
Topics
Nanomaterials for Direct Conversion
Nanopatterned Substrates for Stem Cells
Injectable Hydrogels for Cartilage Tissue Engineering
Origami Tissue Engineering
Cell Surface Engineering for Stem Cell-based Therapy
Synthetic Inorganic Nanoparticles of in situ bone regeneration
Tissue Engineering
http://www.tissueeng.net/
Biological “living” replacements
http://bmsce.snu.ac.kr
Selected Publications (April. 2015)
1. Advanced Healthcare Materials 2015 10.1002/adhm.2014008352. Drug Delivery and Translational Research 2015, March 263. J. Controlled Release 2015 Feb 28;200:212-21.4. Biotechnology Journal 2014 10.1002/biot.2014000205. Journal of Biomedical Materials Research 2014 6. Acta Biomaterials 2014 Jul;10(7):3007-177. PNAS 2014 Jan 21;111(3):990-58. Biomaterials 2013;34(28):6607-6614. (2013 IF=8.312)9. Tissue Engineering 201410. Advanced Functional Materials 2012 Jul 24;22(14):2949-2955.11. Adv. Drug Deliv. Rev. 2013 Apr;65(4):536-58
Stem cell engineering via nonviraldelivery of reprogramming factors
Bioactive substrates for stem cell differentiation and epigenetic regulations
Cell surface engineering for stem cell based therapies
Injectable hydrogels for orthopaedics applications
Fabrication of customizable scaffolds for tissue engineering
Synthetic Biominerals for in situ Bone Formation
Yamanaka factor delivering nanoparticle
Bioactive substrates and photopolymerizing hydrogels
Controlling Stem Cells for Musculoskeletal Tissues
Regeneration
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Basic Strategy
Establishment of iPSCsEstablishment of
Differentiation ProtocolsApplication in TE and
Cell Therapy
Implantation of Carticel® :
Autologous Chondrocyte Transplantation
Periosteal flap
Defect
Biopsy
GMP Cell Processing
Carticel
J. Wenz, MD
Cell Number Issues
Cell Number Required to Engineer Cartilage: ~40 million cells/ml
3 ml = ~120 million cells
T150 = 150 cm2, Typically holds ~3 million cells
Loss of phenotype with expansion
Chondrocytes
Part I: Stem Cells
Induced pluripotent stem cells-the science and technology
(2012 Nobel Prize Physiology and Medicine)
Totipotent (zygote)
Pluripotent (ES, iPSCs)
Multipotent(adult stem cells)
Unipotent(differentiated)
Stem Cells and Reprogramming
Hochedlinger and Platch. Developmnent. 2009 (136); 509-23
24 candidate factors:
Ecat1, Dpp5(Esg1), Fbx015, Nanog, ERas,
Dnmt3l, Ecat8, Gdf3, Sox15, Dppa4, Dppa2,
Fthl17, Sall4, Oct4, Sox2, Rex1, Utf1, Tcl1,
Dppa3, Klf4, b-cat, cMyc, Stat3, Grb2
Transcription factors are delivered by retroviral vectors
and the colonies became visible by day 16
The generation of induced pluripotent stem cells –the Takahashi and Yamanaka paper, Cell, 2006
Gene Carrier/Gene Vector
Retrovirus Herpes
Simplex V
Adenovirus AAV Lipos
ome
DNA Polymer
Integration Yes Non Non Yes Non
Expression Stable Transient Transient Stable Transient
Transfection Efficient Efficient Efficient Low Low
Immune
Response
No Yes High No Yes Yes or
No
No
Generally, viral vector system show higher gene transfer efficiency than non-viral gene carrier system, but viral systems have potential risk of wild type virus regeneration, immunogenecity and cancer formation.
Derivation of iPSCs using non-viral delivery strategy
Safety issues related to the current strategy to make iPSCs: Yamanaka Factors (Oct3/4, Sox2, Nanog, Lin28)
Totipotent (zygote)
Pluripotent (ES, iPSCs)
Multipotent(adult stem cells)
Unipotent(differentiated)
Transdifferentiation
Hochedlinger and Platch. Developmnent. 2009 (136); 509-23
Cartilage vs. Muscle
Chondrocytes Muscle Fibres
Myogenic Conversion from Reprogrammed Chondrocytes
iPSCsPlastic statesChondrocytes
Two-three weeks process
Myogenic Induction (myogenic cells from chondrocytes?)
Cell morphology change during reprogramming
++
++
++
++
+++
Nucleofection
PBAE transfection
Plastic cells
TGF-b inhibitor
SB-431542
Human chondrocytes Myoblasts
Reprogramming
factor deliveryMyogenic
differentiation
Complex Formation of a Polymer and a Plasmid DNA
-- -
- +++++
DNA Ligand Polycation DNA Complex
+
Gene Delivery Pathways
1. Electro static interaction between carrier/DNA complex and anionic plasma membrane
2. Receptor mediate endocytosis, pinocytosis, or phagocytosis (depending on the size of the carrier/DNA complex
3. Endosomal release in the cytoplasm- leading to the release of the DNA
All gene therapy strategies depend on getting the gene or genetic materials into the targeted cells = TRANSDUCTION
Three barriers of gene delivery: Cell membrane, endosomal membrane, nuclear membrane
Combinatorial Polymer Library for DNA Delivery
Poly (b-amino ester)-based nanocarriers for iPSC generations
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Myogenic Commitment of Reprogrammed HumanChondrocytes
J.E. Hong et al., JCR 2015
Conclusion I: Stem Cells
Reprogramming of human chondrocytes via non-viral minicircle DNA delivery
Conversion of partially reprogrammed chondrocytes into myogenic cells
Feasibility in various cell-based therapeutic application
Yamanaka factor delivering nanoparticle
Bioactive substrates and photopolymerizing hydrogels
Controlling Stem Cells for Musculoskeletal Tissues
Regeneration
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70% 1 30 % A
100% 1
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85% 1 15% B
80% 1 20 % B
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OOOO
Basic Strategy
Establishment of iPSCsEstablishment of
Differentiation ProtocolsApplication in TE and
Cell Therapy
Substrate-dependent differentiation
Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency
The control of human mesenchymal cell differentiation using nanoscale symmetryand disorder
Dalby et al., Nature Materials 2007
McBurray et al., Nature Materials 2011
Signaling Between the Cyotoskeleton and Nucleus
Cells are inherently sensitive to local
mesoscale, microscale, and nanoscale
topographic andmolecular patterns in
the cellular microenvironment
Substrate/Nanotopography InduceEpigenetic Regulation of Stem Cells?
Molecular Mechanisms that Mediate Epigenetic Phenomena
Harp, J.M., et al., Asymmetries in the nucleosome core particle at 2.5 A resolution. Acta crystallographica. Section D, Biological crystallography,
2000. 56(Pt 12): p. 1513-34.
H2A
H2B
H3
H4
Histone ModificationDNA methylation
5’ ApTpGp meCp GpApTpG 3
3’ TpApCp Gp meCpTpApC 5’
Structure & Epigenetics ofEuchromatin versus Heterochromatin
The Dynamic Nucleosome: An Epigenetic Signaling Module
Euch
rom
atin
Hete
roch
rom
atin
Bivalent Mark: H3K4me3 & H3K27me3
Bivalent Histone Modification in Stem Cell Differentiation
Bivalent Mark: H3K4me3 & H3K27me3
Fabrication of ECM Substrates with Nanotopography
PUA + acrylated-carboxylate mononer (10:1)
UV
EDC/NHS
300 nm 5 mm Flat
E. A. Kim, JBMR B
Immobilized vs. Adsorbed ECM Proteins
Immobilized Adsorbed
Post Seeding
Pre Seeding
E. A. Kim, JBMR B
Nano-Patterned/FN-immobilized Substrates
1 2 3 4
5 6 7 8
910
11
12
13
14
15
16
1w: 500p: 1000
2w: 450P: 900
3w: 400p: 800
4w: 350p: 700
5w: 800p: 1600
6w: 750p: 1500
7w: 700p: 1400
8w: 600P: 1200
9w: 1250p: 2500
10w: 1200p: 2400
11w: 1000p: 2000
12w: 900p: 1800
13w: 2000p: 4000
14w: 1800p: 3600
15w: 1600p: 3200
16w: 1500P: 3000
w (width), p (period) in nm16 different line patterned PUA on PS slide
J. Kim et al., In Review, Nature Methods
hMSC Staining for H3K3me3/H3K27me3
1 2 3 4
5 6 7 8
9 10 11 12
13 14 15 16
J. Kim et al., In Review, Nature Methods
Pattern-Specific Histone Modification and Nuclear Signatures
1 2 3 4
5 6 7 8
9 10 11 12
13 14 15 16
-25
-20
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-10
-5
0
5
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-10
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PC 1PC 2
PC
3
P4
P7
P10
P13
Greater levels of H3K27me3 expression as the line widths/spacing increases
Cells cultured on different patterns exhibited nuclear signatures that appear responsive to line/space width
J. Kim et al., In Review, Nature Methods
Topographical and ECM Effect on Myogenic CommitmentR
ela
tiv
e F
old
In
du
cti
on
0
0.2
0.4
0.6
0.8
1
1.2
0
0.5
1
1.5
2
2.5
3
0
0.5
1
1.5
2
2.5MHCd MHCa MYOG
300 nm 5 mm 300 nm 5 mm 300 nm 5 mm0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
0
0.5
1
1.5
2
2.5
3MHCd MHCa MYOG
300 nm 5 mm 300 nm 5 mm 300 nm 5 mm
Scale bar: 100 mm
FN-immobilized Substrates
300 nm 5 mm
Laminin-immobilized Substrates
300 nm 5 mm
E. A. Kim, G. Y, Jung et al., JBMR B
Application is Tissue Engineering?FN-Immobilized Nanofibers for MI
Myocardiac InfarctionModel
FibronectinIimmobilization
Aligned PCL Nanofiber pGMA Coated Nanofiber Fibronectin ImmobilizedNanofiber
A
pGMACoating by iCVD
Fibronectin ImmobilizedNanofiber Cardiac Patch
Fibronectin ImmobilizedNanofiber Mesh
BUCB Cells Seeding Transplantation
Cell adhesions and viability on pGMA-FN coated PCL Nanofibers
Increased cell adhesion and proliferation on pGMA-FN coated nanofibers
PCR arrays showed increased growth factorgenes (i.e., VEGF, IGF, FGF) on pGM-FN coated nanofibers
B. J. Kang, et al., Acta Biomat.
Evaluation of cardiac function after MI
Evaluation of cardiac function by echocardiography
B. J. Kang, et al., Acta Biomat.
Conclusion II: Substrate-dependent differentiation
Materials containing the topography with nanoscalefeatures can induce histone modification and modulate cell behavior
Cells cultured on different patterns exhibited nuclear signatures that appear responsive to line/space width Greater levels of H3K27me3 expression as the line
widths/spacing increases
Toward the myogenic commitment, immobilization of proteins to PUA nano-patterned substrates significantly enhanced the myogenic gene expressions.
Immobilized nanofibers for efficient delivery of stem cells in to MI model
Injectable Hydrogels for Tissue Engineering
Hydrogel Integration into Defected Tissue
catehcol-methacrylated hyaluronic acidAldehyde-methacrylated hyaluronic acid
Thiolated HA + PEGDA
Catehcol-methacrylated chitosanAldehyde-methacrylated chitosanCatechol-methacrylated CSAldehyde-mathacrylated CS
Meniscus
Hydrogel
D.A. Wang et al., Nature Materials 2007
Bioactive hydrogels: providing physical signals
PEGDAPEGDA-HA
• Extracellular microenvironment plays a significant role in controlling cellular behavior
N.S. Hwang et al., Cell and Tissue Res 2011
Fabrication of ECM-based hydrogels for functional cartilage tissue engineering
Glycidyl
Methacrylate
Chondroitin Sulfate
Hyalruronic Acid
Methacrylated
Chondroitin Sulfate
Methacrylated
Hyalruronic Acid
PEG-RGD MeCS/HAPEGDA
RGD
Hydrogel
Construct
PEG CS HA
RG
DR
DG
Kim H et al., Tissue Engineering 2014
PEG-RGD PEG-RDG CS-RGD CS-RDG HA-RGD HA-RDG
DA
Y 1
DA
Y 3
DA
Y 7
Morphological analysis and biochemical analysis of chondrocytes in RGD/RDG-modified ECM hydrogels
Kim H et al., Tissue Engineering 2014
Cartilage Tissue Formation (3weeks in vitro)
H& E Staining Safranin-O Staining
Kim H et al., Tissue Engineering 2014
ECM-mediated Cell Behavior in Hydrogels
Kim H et al., Tissue Engineering 2014
Cartilage Specific Gene Expression Analysis
* * *
* *
Kim H et al., Tissue Engineering 2014
Alternative Biocompatible PI: Riboflavin-collagen gel
Riboflavin enables collagen crosslinking at visible light range
Collagen is a widely utilized biomaterials but portrays weak mechanical properties
Riboflavin(vitamin B2) as photoinitiator
Collagen gel 37℃(90min)
Collagen+0.006% riboflavinUV (10 min)
J.S. Heo et al., Drug Delivery and Trans. Med. 2015
Injectable Hydrogels for Cartilage Tissue Engineering
Bioactive photopolymerizing hydrogels for tissue engineering
CS-RGD microenvironment for enhanced SZP gene expression
Vitamin B for visible range photoactivation
Yamanaka factor delivering nanoparticle
Bioactive substrates and photopolymerizing hydrogels
Controlling Stem Cells for Musculoskeletal Tissues
Regeneration
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b
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90% 1 10% A
85% 1 15% A
80% 1 20 % A
75% 1 25% A
70% 1 30 % A
100% 1
90% 1 10% B
85% 1 15% B
80% 1 20 % B
75% 1 25% B
70% 1 30 % B
100% 1
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85% 1 15% C
80% 1 20 % C
75% 1 25% C
70% 1 30 % C
100% 1
90% 1 10% D
85% 1 15% D
80% 1 20 % D
75% 1 25% D
70% 1 30 % D
100% 1
90% 1 10% E
85% 1 15% E
80% 1 20 % E
75% 1 25% E
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b
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70% 1 30 % A
100% 1
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85% 1 15% B
80% 1 20 % B
75% 1 25% B
70% 1 30 % B
100% 1
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85% 1 15% C
80% 1 20 % C
75% 1 25% C
70% 1 30 % C
100% 1
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85% 1 15% D
80% 1 20 % D
75% 1 25% D
70% 1 30 % D
100% 1
90% 1 10% E
85% 1 15% E
80% 1 20 % E
75% 1 25% E
70% 1 30 % E
100% 1
90% 1 10% F
85% 1 15% F
80% 1 20 % F
75% 1 25% F
70% 1 30 % F
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a
b
100% 1
90% 1 10% A
85% 1 15% A
80% 1 20 % A
75% 1 25% A
70% 1 30 % A
100% 1
90% 1 10% B
85% 1 15% B
80% 1 20 % B
75% 1 25% B
70% 1 30 % B
100% 1
90% 1 10% C
85% 1 15% C
80% 1 20 % C
75% 1 25% C
70% 1 30 % C
100% 1
90% 1 10% D
85% 1 15% D
80% 1 20 % D
75% 1 25% D
70% 1 30 % D
100% 1
90% 1 10% E
85% 1 15% E
80% 1 20 % E
75% 1 25% E
70% 1 30 % E
100% 1
90% 1 10% F
85% 1 15% F
80% 1 20 % F
75% 1 25% F
70% 1 30 % F
OOOO
Basic Strategy: Biomimetic Materials and Stem Cell
Engineering Lab (BMSCE)
Establishment of iPSCsEstablishment of
Differentiation ProtocolsApplication in TE and
Cell Therapy
Customized Scaffolds for Tissue Engineering
Paper Origami
Suhwan Kim, B.S.
Origami-based Approach for Trachea Tissue Engineering
Bare paper
PSMa coated paper
PSMa-PLL/CaCl2coated paper
Hydrogel-cell-laden
Paper scaffold
3D paper tissue scaffold
<Front view>
<Upper view>
Key
Scaffold
implantation
Paper
scaffold
Pa
pe
r
orig
am
i
SH Kim et al., PNAS 2015
Initiated Chemical Vapor Deposition (iCVD) of PSMA
Sung Gap Im(KAIST)
SH Kim et al., PNAS 2015
Poly –l-Lysine Conjugation to PSMA coated Paper Substrate
600 400 200
Co
un
ts/s
(a
.u)
Binding energy (eV)
N1s
O1sC1s
PSMa
-PLL
PSMa450 400 350
Bare paper
-PLL
Bare paper
N1s
Binding energy (eV)
Co
un
ts/s
(a
.u)
SH Kim et al., PNAS 2015
Hydrogel Adhesion Control
Paper substrate
Hydrogel
adhesion
**
***
***
***
***
SH Kim et al., PNAS 2015
iCVD polymerization
PLL-CaCl2 dip
coating
Bare paper
PSMA (Poly(styrene-co-maleic
anhydride)) coated paper
O
** n
m
OO
PLL-CaCl2 coated paper
N H2
NH2
** n
m
N H O H
OO
Poly-l-
lysine
Hydrogel gelation
N H2
NH2
** n
m
N H O H
OO
Hydrogel coated
scaffold
Hydrogel
solution
Hydrogel-laden Paper-based scaffolds for TE
SH Kim et al., PNAS 2015
Hydrogel Thickness Control
A 1% ALG 1.5% ALG 3% ALG
10
min
30
min
517μm
960μm
231μm
497μm
C/C0
3% Alginate 1.5% Alginate 1% Alginate
BCalcium ions
Hydrogel
Time (min)
Calc
ium
co
nce
ntr
ati
on
(mg
/L)
5m
m
Paper substrate
SH Kim et al., PNAS 2015
Versatility of 3D Constructs Based on Paper Origami
SH Kim et al., PNAS 2015
Origami for 3D Scaffold Construction
SH Kim et al., PNAS 2015
In vitro Cartilage Tissue Engineering
SH Kim et al., PNAS 2015
Application of Tissue Origami in Animal Models
Paper origami
Hydrogel coating with
chondrocytes
Bioreactor
**
SH Kim et al., PNAS 2015
Application of Tissue Origami in Trachea Regeneration Models
** ****
**
Week 1 Week 2 Week 3 Week 4
**
**
w/
ch
on
dro
cyte
w/o
ch
on
dro
cyte
**
SH Kim et al., PNAS 2015
**
Week 2 Week 4
H&
ES
afr
an
in-
O
w/ chondrocyte
Week 2 Week 4
w/o chondrocyte
**
**
**
X 1
2.5
H&
ES
afr
an
in-
O
X 4
0 **
**
**
**
★★
w/ chondrocyte at
Week 4
w/o chondrocyte at
Week 4
*
*
Application of Tissue Origami in Trachea Regeneration Models
SH Kim et al., PNAS 2015
Conclusion III: Origami Tissue Engineering
This work describes an intriguing new strategy for formation of hydrogel-laden complex 3D structures starting with 2D paper sheets and suggests a new route for 3D tissue engineering scaffolds.
It combines concept extracted from origami and iCVD-based polymer coating.
This work also identifies a remarkable interesting problem in complex scaffolds fabrication: the CAD-based lock-and-key design of planar sheets that can be folded into 3D structures with spatial arrangements of tissue elements.
In principle, origami-based tissue engineering approach has successfully applied in trachea regeneration model.
Neural Degenerative Diseases
Huntington's Disease
Etiology• Autosomal dominant progressive chorea and dementia• Defective huntington protein (chromosome 4)• Degenerative of cholinergic and GABA-ergic cells in basal ganglia • Relative excess dopamine
Manifestation• Middle age onset• gradually worsening twitches• loss of muscle control• memory loss
Treatment• Dopamine antagonist• Genetic screening• Choroid plexus cell transplantation
Stem Cell Implantation
Implantation of cells have a nurturing role, mopping up toxins, secreting a range of chemicals that are essential for brain cell function.
Cell Surface Engineering
• Engraftment efficiency• Reduce rejection• Cell tracing
Cell Surface Modification for Stem Cell-based Therapy
Protection Imaging Tracking
TCEP
Recovery
Cell Surface Labeling
TCEP
Mal-Alexa
Fluor 488
A
2D 3D
Mal-Fluor PKH26 Merge
Ctrl
1mM
2mM
3mM
(-) Ctrl
(+) Ctrl
1mM TCEP
B
Cell Surface Engineering with CS
Mal-CS
Gold Thiol
PLL
0 500 1000 1500 2000 2500 3000-80
-60
-40
-20
0Mal-CSThiol
F (Hz)
Mass (mg/mL)
Time (sec)
F
(H
z)
Gold PLLMal-CS
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
M
as
s (m
g/c
m2)
-80
-60
-40
-20
0
F
(H
z)
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
M
as
s (m
g/c
m2)
Gold Thiol Mal-CS PLL Mal-CS
BA
TCEP + Mal-CS
PLL
Solution
(0.005%)
TEM image
Cell Surface Immobilization of Mal-CS
A B
1mM
5mM
Ctrl
10mM
TCEP
MCS Mal-Flour
** ** ** **
Ctrl 1mM of TCEP
Norm
aliz
ed b
y m
ode
Control1mM TCEP & 10mM Mal-CS
Molecular shielding effect: Reduced cell size and apoptosis related genes
▲Caspase3, p53 : apoptosis-initiating proteinsGreat protecting ability from inducing proteins
iNSC surface engineering
ControlAfter TCEP treatment
Scale bar : 100um
Yu et al., Cell Reports, 2015
• Inhibition of let-7 promotes direct reprogramming and self-renewal of hiNSCs
• HMGA2, a target of let-7, promotes the rapid and efficient generation of hiNSCs
• HMGA2 facilitates the direct reprogramming of human senescent cells and blood cells
Neuronal Cell Surface Engineering
Neuro-2A cell line
Scale bar : 10um
Tracking axonal growthInduced Neural
Stem Cell
InjectionCoating with Qdot
Collagen–MethacrylatedHA
Hydrogel
In vivo neuron cell tracking
TCEP
Mal-QD
Conclusion IV: Cell Surface Engineering for Stem Cell-based Therapy
Cell Engineering For Neurodegenerative Treatment
• Cell membrane disulfide bond reduction with TCEP for reactive thiol presentation
• Efficient macromolecular coating of cells for thiol-maleimidereaction
• Surface immobilization of Mal-CS was confirmed with FACS analysis and resulted in prevention of cellular clustering
• Universal methods for cell surface modification for immune tolerance and long term in vivo survival
• Efficient live cell monitoring platform for stem cell based therapies
Bone development
– Endochondroal ossificiation
– Transient cartilage tissue
– Primary ossification center
– Blood vessel invasion
Effects of Chondrocyte CM on MSCs
Rho et al., Cell and Tissue Res. 2015
Enhanced Osteogenic Response of CM expanded Cells
Rho et al., Cell and Tissue Res. 2015
Osteogenic priming of MSCS by Chondrocyte CM
Rho et al., Cell and Tissue Res. 2015
Biominerals for Bone Tissue Engineering
• 35% organic components
– Composed of cells, fibers, and organic substances
– Collagen – abundant
• 65% inorganic mineral salts
– Primarily calcium phosphate
– Resists compression
ACS Nano, 2014, 8 (1), pp 634–641
ACS Nano, 2014, 8 (1), pp 634–641
Characterization of WH Nanoparticles
Kim et al., Nature Materials (in review)
Biological Characteristics of WH Nanoparticles
Kim et al., Nature Materials (in review)
Enhanced Osteogenic Response of WH via SCL20a1 Pathway
Kim et al., Nature Materials (in review)
In Situ Bone Formation by WH Nanoparticle Incorporated Cryogels
Kim et al., Nature Materials (in review)
WH Nanoparticles for Bone Tissue Engineering
WH provides enhanced microenvironment for bone formation
Fabrication and characterization of synthetic WH nanoparticles for bone tissue engineering applications
Bone forming microenvironment via faster phosphate release along with negative charged surface for protein adsorption
WH may be utilized in dental applications
Acknowledgements
Undergraduate/Intern Students
Insun Kim
Graduate Students
Eunjee Lee Joon Lee
Hwan Kim Hyungu Yim Eunseo Lee
Suhwan Kim
Younghwan Choi
Jungha Park
Minui Han
Hyunbum KimYunsup LeeWook Sun
Seunghyun Kim
Jiyong Kim
Rachel Koh Young H. An
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