Design and Preparation of Functional

Biomaterials and Polymer Scaffolds

for Tissue Engineering

Guoping Chen

1 Research Center for Functional Materials, National Institute for

Materials Science, Japan
2 Department of Materials Science and Engineering, Graduate

School of Pure and Applied Sciences, University of Tsukuba, Japan

Group Leader
Research Center for Functional Materials
National Institute for Materials Science, Japan

Professor
Department of Materials Science and Engineering, Graduate School of Pure
and Applied Science, University of Tsukuba, Japan

Guest Professor
Southeast University; Shanghai Institute of Ceramics; Sichuan University

Fellow, The Royal Society of Chemistry

Fellow, American Institute for Medical and Biological Engineering (AIMBE)
Associate editor, Journal of Materials Chemistry B
Editor, Science China Chemistry
Editorial board, Journal of Bioactive and Compatible Polymers: Biomedical
Application; Tissue Engineering; Journal of Tissue Engineering and
Regenerative Medicine
Advisory board, Biomaterials Science
National Institute for Materials Science (NIMS)
Tsukuba Science City

Tsukuba

Tissue Regeneration
Materials Lab.
Historical Highlights in Bionics and Related Medicine
 Tissue Engineering

Waste Muscle Scaffold Vein Muscle Bone Skin

Removal

Pump
Nutrients

Nerve Blood Vessel Bone Skin

Scaffold Scaffold Scaffold Artery Nerve Scaffold
Current Status of Tissue Engineering
Cartilage
Skin
Bone

Ligament
Tendon Myocardium
Tissue
Engineering

Blood Vessel Pancreas Liver

Three Factors for Tissue Engineering
Three Factors Tissue Regeneration Clinical
Bone Applications
Growth
Cells Factors

Cartilage

Skin

Liver
Scaffolds Patient

Good scaffolds are necessary for tissue regeneration

 Strategy to Mimic in vivo Microenvironment
Microenvironment
Collagen
Proteo-
glycan

Cell

Glycoprotein
Receptor
Soluble factors
Integrin

Cell

Temporary scaffolds with appropriate pore structures and surface properties

are required to support cell adhesion and to control cell functions.
 Research Topic 1
Manipulation of Cell Functions by
Micropatterned Surafces
Pattern differentiation Cell spreading area Cell-cell interaction
and geometry and protrusion

Cell density

500 m

J. Biomed. Mater. Res. A., 101, 3388-3395 (2013). Soft Matter, 9, 4160-4166 (2013).
Colloids and Surfaces B: Biointerfaces, 122, 316323 (2014). Journal of Materials Chemistry B, 4, 37-45 (2016).
 Stem Cells
Blastocyst

Fibroblasts
Skin
Bone marrow
Mesenchymal stem
cells
Inner cell mass

Defined factors

Mesenchymal stem cells

Hematopoietic stem cells

Embryonic stem cells Induced pluripotent stem Adult stem cells
cells Neural stem cells
etc.

Stem cells are promising cell sources for tissue engineering

 Multi-lineage Potential of Stem Cells
Self renewal Mesenchymal stem cell
Stem cell MSC proliferation

Tendogenesis/
Osteogenesis Chondrogenesis Myogenesis Marrow Stroma Adipogenesis
Ligamentogenesis
Progenitor

Transitory Transitory Myoblast Transitory Transitory

osteoblast chondrocyte stromal cell fibroblast

Mature cell
Osteoblast Chondroblast

Terminal
differentiation
Muscle
Bone osteocytes Cartilage chondrocytes myoblasts Stromal cells Tendon tenocytes Adipocytes

Manipulation of stem cell functions remains a great challenge

 Micropatterns Mimic in vivo Microenvironment
Soluble factors
Adjacent cells
Mechanical
force Uniform surfaces

Loss of biophysical restrict

Extra cellular matrix Cell

(ECM) Micropatterns
morphogenesis provide the
Mimiking in
vivo micro- biophysical
environment regulation

Micropatterning technology has been proved to be a useful tool

to decipher cell morphogenesis and to regulate cell functions.
 Photo-Reactive Polymer and UV Lithography
UV irradiation

Polymer coating Washing

Organic substrate Photo-reactive polymer Photomask Polymer micropattern

Photo-reactive polymer Generated biradical

UV Intra-/inter crosslinked
N
N3 Graft polymer
N3 N

N3 N
N3 N
Organic substrate Organic substrate Organic substrate

13
 Synthesized Photo-Reactive Polymers
Positively Charged
Polyallylamine (PAAm) (CH2 CH)n
CH2-NH2
Negatively Charged
(CH2 CH)n
Poly(acrylic acid) (PAAc)
COOH
Neutral
Poly(ethylene glycol) (PEG) ( CH2 CH2 O)n

Poly(vinyl alcohol) (PVA) (CH2 CH)n

Bioactive Molecules OH

Gelatin Gelatin NH2

 Preparation of Photo-Reactive PVA and PVA-
Micropatterned PSt Surfaces
Synthesis of azidophenyl-derivatized poly(vinyl alcohol) (AzPhPVA)

x y
Dicyclohexyl-
Dicyclohexylcarbodiimide
O 4-(1-pyrrolidinyl)pyridine OH O O
n + N3
carbodiimide
OH HO
Poly(vinyl alcohol) 4-azidobenzoic acid 4-(1-pyrrolid-
Poly(vinyl alcohol) 4-azidobenzoic acid
(PVA)
inyl)pyridine
N3
Azidophenyl-derivatized poly(vinyl alcohol) (AzPhPVA)
Cells can not adhere on PVA Photo-reactive PVA (AzPhPVA)

Designed
Micropatterning of PVA hydrogel on PSt surface micropatterns
UV irradiation
non-transparent
transparent
Spreading area
Coating Drying Washing
Geometry
PSt surface Photo-reactive PVA Photomask PVA micropattern
Aspect ratio
 Culture of Stem cells on Micropatterns
F-actin assembly of human bone marrow-derived mesenchymal stem cells on micropatterns
after 24 hours culture.
20 m 40 m 60 m 80 m flat

50 m
50 m
circle triangle square pentagon hexagon

30 m
1 1.5
Aspect ratio

4 8
F-actin

nucleus
30 m

MSCs showed different actin filament nanostructures on the micropatterns.

 Osteo/Adipogenic Differentiation of MSCs
(Different areas but same shape)
40 mm 60 mm 80 mm 100
40 m Mean SD (n=3)
90 60 m p<0.5 p<0.01 p<0.001
80 m
80

Percentage of differentiation (%)

ALP

70

60


50

40

Oil Red O

30

20

10

0
Osteogenesis Adipogenesis
Scale bars: 50 mm Osteogenic or adipogenic culture for 7 days

Increased cell spreading facilitated the osteogenic differentiation but

suppressed the adipogenic differentiation of MSCs.
 Summary 1

PVA-micropatterned polystyrene surfaces

with various sizes, shapes and aspect ratios
were prepared by UV lithography.
Cell density, spreading, geometry, protrusion
and cell-cell interaction could be manipulated
by the micropatterns. Their effects on
osteogenic and adipogenic differentiation of
mesenchymal stem cells were disclosed.
 Preparation of Gold Nanoparticles with
Different Size and Morphology to Control
Stem Cell Functions
 Nanomaterials for Biomedical Applications
Nanomaterials
small size
vast surface area
high surface atomic insufficiency

Biomedical applications
application of nanomaterials and nanotechnology
in nanomedicine to dramatically change medical
science and technology

delivery imaging therapy separation

 Nanomaterials for Cancer Therapy

local
temperature
increase or free
radical
generation

photothermal therapy
photodynamic therapy
magnetic hyperthermia

iron oxide nanoparticles, carbon nanotubes,

gold nanoparticles, quantum dot, titanium
therapy dioxide nanoparticles,
polymeric nanoparticles
 Nanomaterials in Tissue Engineering
collagen fibronectin laminin proteoglycan

tissue engineering
integrin
iron oxide nanoparticles, carbon
As the similar nanoscale size and structure to proteins, nanotubes, gold nanoparticles,
nanomaterials can well mimic the extracellular matrices silicon nanoparticles,
(ECM). hydroxyapatite nanoparticles

Nanomaterials with excellent biomimetic features and physiochemical properties

show great potential in the fabrication of novel biomaterials and scaffolds for tissue
engineering.
 Control of Nanomaterial Properties
surface coating

size control

surface nanoparticles
modification

shape control

aggregation
composition
 Influence of Gold Nanoparticles with Different Size
and Morphology on Stem Cells Differentiation
Gold nanoparticle-cell
interactions

Human mesenchymal stem cells Osteogenic

(hMSCs) differentiation

size and shape influence

star-70
sphere-40 sphere-70 sphere-110 rod-40 rod-70 rod-110 star-40 star-110
 Preparation of AuNPs and Cell Culture

40 nm 70 nm 110 nm 40 nm 70 nm 110 nm 40 nm 70 nm 110 nm

Au nanospheres (AuNSP) Au nanorods (AuNRO) Au nanostars (AuNST)

BSA coating

AuNSP-40 AuNSP-70 AuNSP-110 AuNRO-40 AuNRO-70 AuNRO-110 AuNST-40 AuNST-70 AuNST-110

1-3 days
Cell viability
Growth medium
21 days Cell Cellular Osteogenic
Osteogenic induction proliferation uptake differentiation
medium
 Morphology and Size of AuNPs
AuNSP-40: 36.08.0 nm

AuNSP-70: 68.310.4 nm

50 nm 200 nm 200 nm
AuNSP-110: 107.018.8 nm
AuNSP-40 AuNSP-70 AuNSP-110
AuNST-40: 36.87.8 nm

AuNST-70: 67.916.5 nm

AuNST-110: 112.623.8 nm
50 nm 100 nm 200 nm

AuNST-70 AuNST-110 AuNRO-40: 35.26.5 nm

AuNST-40
20.13.3 nm
AuNRO-70: 66.37.3 nm
13.71.8 nm
AuNRO-110: 117.313.6 nm
50 nm 100 nm 200 nm 27.06.1 nm
AuNRO-40 AuNRO-70 AuNRO-110
 Cell Viability Assay
WST-1 assay Live/dead staining
hMSCs were cultured with Control
various AuNPs for 3 days
(Au concentration = 0.5
mM)
AuNSP-40 AuNSP-70 AuNSP-110

AuNST-40 AuNST-70 AuNST-110

AuNRO-40 AuNRO-70 AuNRO-110

1 mm
Red: dead cells Green: live cells
AuNRO-40 at an Au concentration of 0.3 and 0.5 mM showed obvious cytotoxicity after 3
days of culture, while other particles did not.
 Cellular Uptake Assay
Optical micrographs

hMSCs were cultured with

various AuNPs for 21 days in
osteogenic induction medium
at the Au concentration of 0.5
mM.

Quantitative Au uptake (Inductively Coupled Plasma-Optical Emission Spectroscopy)

The cellular uptake of AuNPs

by hMSCs was performed in
a size and shape-dependent
manner:
AuNSPs>AuNROs>AuNSTs,
110 nm>70 nm>40 nm.
 Osteogenic Differentiation
Alkaline phosphatase (ALP) staining ALP activity assay

Alizarin Red S (ARS) staining Calcium deposition assay

 Gene Expression Profile
hMSCs were cultured with
ALP Runx2 various AuNPs for 3 weeks.

The AuNSP-40, AuNSP-70

and AuNRO-70 induced up-
regulation of ALP, Runx2,
IBSP and SPPI, while
AuNRO-40 induced down-
regulation of these genes.
IBSP SPPI The effect of AuNSP-110,
AuNST-40, AuNST-70,
AuNST-110 and AuNRO-
110 on the expression of
these gens was not obvious.

ALP: Alkaline phosphatase

Runx2: runt-related transcription factor 2
IBSP: bone sialoprotein 2
SPP1: secreted phosphoprotein
 Yes-Associated Protein (YAP) Activity
YAP immunofluorescence staining (3 days of culture)

YAP localized in cell nucleus

indicates the activation of YAP. YAP
activity was regulated by the
mechanical signals in cells, affecting
the osteogenic differentiation.

The AuNSP-40, AuNSP-70 and

AuNRO-70 increased the
percentages of activated YAP of
cells, while AuNRO-40 reduced
the YAP activity.
Speculated Mechanism

size and shape-dependent cellular

uptake.

AuNPs caused some mechanical

signals to regulate the YAP activity
in the cell nucleus, thus affecting
the gene expression.

AuNSP-40, AuNSP-70 and

AuNRO-70 treatments increased
while AuNRO-40 treatments
reduced ALP activity and calcium
deposition.
 Summary 2
The AuNPs with different size and shape were synthesized and they
were taken up by the cells in a size and shape-dependent manner.

Increased ALP activity and calcium deposition could be found in the

AuNSP-40, AuNSP-70 and AuNRO-70 treated cells, while the treatment
of AuNRO-40 reduced the ALP activity and calcium deposition.

The osteogenic marker genes were up-regulated in AuNSP-40, AuNSP-

70 and AuNRO-70 treated cells and down-regulated in AuNRO-40
treated cells.

This study will offer beneficial information for the design and
development of nanomaterials with defined size and shape for tissue
engineering and biomedical applications.
 Research Topic 3
Porous Scaffolds and Matrices for Tissue Engineering

Polymeric scaffolds with Biomimetic

controlled porous polymeric Regeneration of tissues and
structures matrices organs such as cartilage and skin

Tissue Eng. C, 16, 329-338 (2010). Biomaterials, 31, 2141-25 (2010).

Biomaterials, 31,5285-5835 (2010). Adv. Mater., 22, 3042 (2010).
Biomaterials, 32, 2489-2499 (2011). Biomaterials, 32, 9658 (2011).
Adv. Mater., 24, 4311 (2012). Biomaterials, 33, 2025-31 (2012).
Biomaterials, 34, 7632 (2013). Acta Biomaterialia, 10, 2005-2013 (2014). RSC
Advances, 5, 9440594410 (2015). Biomaterials, 73, 23-31 (2015).
Biomaterials, 52, 199-207 (2015). Acta Biomaterialia, 35, 185-193 (2016)
Some Typical Porous Scaffolds Developed by Our Group
Collagen Sponge Collagen Mesh PLGA-Collagen Hybrid Mesh

Autologous Scaffold Leakproof Scaffold PLLA-Collagen Hybrid Sponge

Preparation of Porous Scaffolds by Using Pre-
prepared Ice Particulates as Templates
Preparation of Funnel-like Scaffolds Using Ice Particulates
Spraying
Ice particulate template Collagen solution

Water droplets Ice particulates

Supporting plate Supporting plate Supporting plate

Bchner Funnel Funnel-like scaffold

Growth of ice crystals

Supporting plate

Biomaterials, 31,5825-35 (2010). Journal of Bioactive and Compatible Polymers, 25, 360-73 (2010)
Journal of Biomedical Materials Research: Part B, 93, 341-50 (2010).
Materials Letters, 107, 280-283 (2013). Acta Biomaterialia, 10, 2005-2013 (2014).
Biomaterials, 73, 23-31 (2015). RSC Advances, 5, 9440594410 (2015).
Tissue Engineering Part C Methods; 22(3), 189-98 (2016)
Micropatterned Ice Templates
Scaffolds with Micropatterned Pore Structures

Advanced Materials, 24(31), 4311-6 (2012)

 Scaffolds with Microgrooves
Ice template with stripe mciropattern
Ice line widthm 200 400 600 800

Collagen porous scaffolds with microgrooves

Top surface

Cross section
 Formation of Muscle Bundles in Microgrooved Scaffolds
(G200, 4 106 cells/ml seeded, 4-week culture)
F-actin F-actin
Nuclei Nuclei

Nuclei MHC MHC

Nuclei

Scale bar
= 200 m

Biomaterials, 73, 23-31 (2015).

 Control of Bulk Pore Structure Using Ice Particulates as a Porogen Material

Photos of ice particulates and collagen porous scaffolds

Ice particulates Ice-collagen scaffold Control

200 m 2 mm 2 mm

1. Ice particulates with a diameter range from 355 to 425 m were obtained.
2. Ice-collagen scaffold showed different appearance to that of control collagen scaffold.
 Collagen Porous Scaffolds with Ddifferent Pore Sizes
150-250 250-355 355-425 425-500 m

2 mm
150-250 250-355 355-425 425-500 m

35

500 m 500 m 500 m 500 m

100

200 m 200 m 200 m 200 m

 Cell Seeding Efficiency and Cell Distribution in Collagen
Scaffolds with Different Pore Sizes
150-250 250-355 355-425 425-500 m

500 m 500 m 500 m 500 m

Scaffold type Cell seeding efficiency

150-250 m 93.8 2.0%
250-355 m 93.2 1.6%
355-425 m 94.9 1.4%
425-500 m 94.1 1.2% Mean SD, n=6
Acta Biomaterialia, 10, 2005-2013 (2014).
Preparation of Hybrid Porous Scaffolds
Biodegradable Polymers
Biodegradable synthetic polymers
Poly(lactic acid) (PLA)
Poly(glycolic acid) (PGA)
Poly(DL-lactic-co-glycolic acid) (PLGA)
Processable Devoid of cell-recognition signal
High mechanical strength Hydrophobicity
Degradation time Large mesh interstices
Easy handling
Biodegradable natural polymers
Collagen
Specific cell interaction Weak mechanical strength
Hydrophilicity
 Hybridization Method
Mechanical Skeleton Hybrid Scaffolds
Synthetic polymer sponge Hybrid sponge

Microsponge or gel
of soft materials

Synthetic polymer mesh Hybrid mesh

 PLGA-Collagen Hybrid Mesh PLGA-Collagen Hybrid Sponge

Leakproof Hybrid Scaffold PLLA-Collagen Hybrid Sponge

Biomaterials, 26, 2559-66, 2005. Tissue Engineering C, 16, 329-38, 2010.

Biomaterials, 31, 2141-25, 2010. Biotechnology Progress, 26, 819-26, 2010.
 SEM Photomicrographs
Polylactin 910 (PLGA) knitted mesh PLGA-collagen hybrid mesh

Hybridization

PLGA
Collagen
microsponge
 Cartilage Tissue Engineering by Using
PLGA-Collagen Hybrid Mesh
Isolation of Chondrocytes

Digestion with
collagenase Cell seeding

Bovine articular
cartilage PLGA sponge Chondrocytes
Chondrocytes
suspension collagen microsponge
Phase-Contrast Photomicrograph of Chondrocytes
Immediately after cell seeding
Adhesion and Proliferation of Chondrocytes in
the Hybrid Mesh
Hybrid Mesh After cell seeding 1 day culture

1 week culture 2 weeks culture 4 weeks culture

 Size Adjustment

Single sheet

Rolled sheet

Laminated (5-sheet)
 Engineered Cartilage (Bovine)
Single Sheet Laminated (5-sheet) Rolled

12 weeks 12 weeks 12 weeks

Thickness: 200 m 1 mm 8 mm
 Ligament Tissue Enginieering
1 day
Rolling

4 days
Summary
Cartilage
Tissue Engineering
Bone
Tiss
ue
i n e erin
g
u e
Osteochondral Eng issues
Tiss ered T
ine
Eng
Tissue e erin
g
i n
Skin u e Eng sues
T i s s T i s
n e ered g
Eng
i
i n e erin
Eng
Tendon
Tis s u e
T i ssue
s
ered
ELigament ring
i n e
ng i n e e
Eng s
TMuscle
iss u e
T i ssue
e ered g
ng i n e erin
Blood
E
s u e Engvessel
i n
i ssue
s
T i s T
Nerve i n e ered issues
Eng ered T
Esophagus
E n g ine
i n e e ring
Eng
BladderTissues
Engineered
 ACKNOWLEDGEMENTS
NIMS University of Tokyo
Dr. Naoki Kawazoe Prof. Takashi Ushida
Dr. Yukiko Tsuda Dr. Kohei Tsuchiya
Dr. Hongxu Lu
Dr. Young-Gwang Ko University of Tsukuba
Dr. Hwan-Hee Oh Prof. Yutaka Miyanaga
Dr. Wei Song Prof. Naoyuki Ochiai
Dr. Qin Zhang Dr. Masataka Sakane
AIST Dr. Takashi Sato
Prof. Tetsuya Tateishi
Dr. Hajime Ohgushi NRICHD
Dr. Yoshio Shirasaki Dr. Akihiro Umezawa
Dr. Dechang Liu Dr. Kensuki Ochi
Dr. Mika Tadokoro
 ACKNOWLEDGEMENTS
Ikono Radyum
Nur Rofiqoh Eviana Putri

Funding agencies: World Premier International Research Center Initiative on

Materials Nanoarchitectonics and Grants-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Technology, Japan.
Thank you for your kind attention!