Nanoscale Biomaterials for Cell and Tissue Interactions
for Potential Biotech Applications
Prof. Sungho Jin, UC, San Diego
• Cells are typically 1-100µm in size, but their interior and
exterior contains many nanometer size objects.
Cell nanostructures
Primary cell elements
• Nucleus (“Mayor of the City”) - This is often referred to as
the 'brain' of the cell. The function of the nucleus is to
control all other activities that are carried on within the cell.
• Cytoskeleton (“Steel Girders/Supports”) - makes up the
internal framework, like the steel girders that are the
framework for buildings in a city that gives each cell its
distinctive shape and high level of organization. It is
important for cell movement and cell division.
• Cell Membrane (“City Border”) - Like a city perimeter, cell
membranes surround the cell and have the ability to
regulate entrance and exit of substances, thereby
maintaining internal balance. These membranes also protect
the inner cell from outside forces.
The cytoskeleton:
Internal nanostructured scaffolding
• Strengthens cell & maintains the shape
– Allows the cell to adapt, reorganize and change shape
• Plays important roles in both intracellular (within-cell)
transport, cell signaling, gene expression (use of gene
information to produce proteins) and cellular division.
– Provides "tracks" with its protein filaments for transport
of organelles, molecules, vesicles, etc
Element
Diameter (nm)
Microfilament
F-actin
8-10
Intermediate
filaments
8-10
Microtubules
25
Cell membrane nanostructure
• Outer membrane of cell
that controls cellular
traffic.
• Contains proteins called
integrins that span
through the membrane
and allow passage of
materials (velcro-like
receptors that control
cell attachment, moving
and signaling).
• Proteins are surrounded
by a phospholipid
(hydrophilic head and
two hydrophobic tails)
bi-layer.
The importance of integrins:
Surface receptors
• Typically, integrin protein receptors
inform a cell of the molecules in its
environment and the cell evokes a
response.
– Integrins performs outside-in
signalling
– But they also operate an inside-out
mode
• They transduce information from
the external environment,
extracellular matrix (ECM), to the
cell as well as reveal the status of
the cell to the outside, allowing
rapid and flexible responses to
changes in the environment --Helps the cells to migrate.
Influence of external nanostructures: ECM
• The normal cell environment is comprised of a complex
network of extracellular matrix (ECM) with nano–micro
scale dimensions with intricate features.
• In many instances the capacity of a cell to proliferate,
differentiate, and to express specialized functions
intimately depends on the presence and maintenance
of an intact extracellular matrix.
ECM
Cell
interior
ECM features affect:
• adhesion complexes
• cytoskeletal
organization
• intracellular signaling
to adhere cells to
their substratum and
transmit signals from
the environment to
the cell exterior
Mechanisms for stimulating cell behavior
Motivation – Why nanostructured biomaterials?
• Cells have plenty of nanostructures essential
for survival.
• Fabricating nano-features on biomaterial
surfaces provides features that are on the
same scale as the bio-environmental features
that interact with cells.
• Looking at what happens on different nanotextures can help to uncover signaling
pathways that promote the desired cellular
response.
Basic definitions
• Nanomaterial – “Particles with lengths that range from 1 to
100 nanometers in two or three dimensions.” ASTM E 245606 : Terminology for nanotechnology. ASTM International
2006
• Biomaterial – “A non-living material intended to interface
with biological systems to evaluate, treat, augment or
replace any tissue, organ or function of the body.” Chester
1986
• Nanobiomaterial– “A biomaterial substrate composed of
nanometer-scale components.”
Example : the inorganic bone matrix is comprised of apatite
crystals with final dimensions 400 x 30 x 75 Å.
• Nanomedicine – “The monitoring, repair, construction and
control of human biological systems at the molecular level,
using engineered nanodevices and nanostructures.”
Why so much interest in the surface?
• For non-leaching materials, the body “reads”
materials through its surface.
• Surfaces are uniquely reactive.
• Surfaces are different from bulk.
• Surfaces readily contaminate
– Especially in fluid, more so in biofluids
• Surface structures are often mobile
• Much higher surface/volume ratio for micro/nano
Material factors that affect how the surface is
perceived...(by living cells)
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Surface chemistry
Surface energy/tension/wettability
Surface roughness
Crystallinity
Surface charge
Size of features (nano vs. micro)
Geometry of features
Redox potentials/ion release
Other-mechanical properties such as elasticity,
doping
Time sequence of response to a surface
• Protein adsorption onto surface (<1 sec)
• Development of protein monolayer or multilayers (110 sec)
• Adsorbed proteins denature (10-30 sec)
• Cellular attachment to proteins (30 sec –minutes)
• Cellular secretions to stimulate tissue response (soon
after)
Foreign material or
biomaterial
Cellular events at the surface
( A ) Initial contact of cell with solid
substrate of adsorbed proteins.
( B ) Formation of bonds between
cell surface receptors and cell
adhesion ligands (protein
functional groups).
( C ) Cytoskeletal reorganization
with progressive spreading of
the cell on the substrate for
increased attachment strength.
Why so much interest in nanostructured surfaces?
• Cells in our body are predisposed to interact with
nanostructured surfaces developing subtle biomimetism
(biomimicry).
• Protein adsorption characteristics are dependent on the
surface features of implanted biomaterials.
• Integrins are transmembrane cell receptors interacting with the
protein layer adsorbed to the scaffold. These interactions are
governed by molecular events at nanometer scale.
• Proliferation, migration, differentiation and extracellular matrix
production by cells during tissue repair are dependent on
protein adsorption on the surface of implanted biomaterials.
• Nanobiomaterials have an even greater increase in number of
atoms at their surface and possess a higher surface area to
volume ratio than conventional microscale biomaterials.
• Thus scientific developments of nanobiomaterials are multiple,
from wound healing, bone implants to bone (or cartilage)
tissue engineering scaffolds, to stem cell differentiation.
Discovery of Topography
• The ability of the substratum to influence cell orientation,
migration and cytoskeletal organization was first noted by
Harrison in 1911.
– Grew cells on a spider web.
– The cells followed the fibers of the web in a phenomenon
called physical guidance.
– Observed variations in the behaviour of cells due to the
solid support they grew on.
• Later, in 1964, it was first proposed that cells react to the
topography of their environment.
– Surface shape and physical features themselves.
• Since then, numerous studies have shown that many cell
types react strongly to topography, especially on the
nanoscale.
• The role of a substrate is more than merely providing
mechanical support…
– Act as intelligent surfaces.
– Providing chemical and topographical signals.
– Guides and controls cell behavior.
Topography shapes cell behavior
• Influencing cell behavior using substrate topography, particularly
in the sub-micron regime, is an attractive strategy for
regenerative medicine applications and advanced tissue
engineering.
• Many researchers are interested in investigating and
understanding which way a cell reacts to different kinds of solid
supports.
• Emerging literature presents many interesting findings on the
effects of nanotopography:
– Influences extracellular matrix organization, Enhances cell
adhesion, Alters cell morphology, Affects proliferation,
Initiates intracellular signaling, Provides contact/physical
guidance, Mediates stem cell differentiation
• Incorporating topographical consideration into the design of
scaffold environments is becoming increasingly important in light
of these studies.
• With advances in nanofabrication technologies the promise of
and the unknown information about topographical effects in
manipulating the cell-substrate interaction is now being
uncovered…
Inspiration
• New fabrication technologies and new
nanotechnologies have provided biomaterial
scientists with enormous possibilities when
designing customized tissue culture supports
and scaffolds with controlled nanoscale
topography.
• The main challenge:
– To effectively design these scaffold for specific
tissue engineering applications.
– Choose the appropriate combination of
biomaterial composition, size, structure, etc.
– Be able to tailor towards applications as
challenging and complex as…
• (a) wound healing
• (b) new bone growth and orthopedic implant
technologies
• (c) stem cell differentiation
• (d) reconnect nerves/spinal cord injuries/brain
damage
How can we create nanostructure
environments for signaling
differentiation?
Control and design of surfaces for desired
tissue interactions
• Nano lithographic periodic
surfaces/Nanomachined
surfaces—precise topographies
• Rough surfaces—promote
adhesion of cells through focal
contacts.
• Porous surfaces—promote
ingrowth of tissue for
mechanical interlocking, also
used for enhanced surface
effects.
• Fiber-meshed surfaces—can
generate gradients of pore
networks, imitates ECM fibers.
Nanopattern fabrication
• The definition of nano-structures on the substrates relies on the
clean room lithographic and Si processing methods.
• Lithography
– Usually, a computer-designed pattern is exposed by means of
light, electrons, ions or imprinted.
– Carried out on a special light or electron-sensitive material or
imprinted into a special deformable polymer, which is then
used in subsequent pattern transfer processes as a mask, or,
alternatively, used for cell culturing as it is.
Contact guidance by patterned biomaterials
• Controlling cell adhesion, orientation and morphology through topographical
patterning is a phenomenon that is applicable to a wide variety of medical
applications such as implants and tissue engineering scaffolds.
– Wound healing
– Neuron alignment
• Effects on cells based on “ridge” features.
• Sharper angles orient cells better.
• Cells adhere better when aligned with surface
• texture.
• Possible explanations:
– Higher surface energy leads to preferential protein binding
– Alignment of adhesion plaques
– Abrupt edges lead to alignment or bridging
– Stochastic (non-deterministic ) spreading of cells
Controlled topography
allows for controlled cell
growth/alignment
Directed migration mechanism
• External guidance clues:
– Topography of the
extracellular matrix.
– Protein adhesion on the
features.
– Intracellular polarity
machinery and
adhesion receptors
direct the adhesion and
cytoskeletal remodeling
that is necessary for
lamellipodium
formation (the moving
front) to align.
Epithelial cells align
• Cell elongation and alignment on grooves and ridges of nanoscale
dimensions was compared with the morphology and orientation of cells
cultured on smooth substrates.
• It was found that ridges 70 nm wide induced human corneal epithelial
cells to elongate and align along the topographic features.
• Valuable for the development of implantable prosthetics.
Morphological changes in cells
• Aside from grooves inducing the elongation of cells, geometric features
(wells, pits, pillars) with dimensions in the nano range seem to effect the cell
morphology, organization of the cytoskeleton and internal framework.
• In general, significant increases in cell spreading and intricate shape changes
are measured in cells on nanotopographies compared to cells cultured on
planar controls.
• This behavior seems to be related to the fact that the cell membrane in
contact with the nanostructured surface will suffer tensile and relaxation
mechanical forces that will rearrange its components, such as integrin
complexes and thus signaling cell morphological changes.
Control
Nano
Mechanotransduction
• Cells are exquisitely sensitive to forces
of varying magnitudes, and they convert
mechanical stimuli into a chemical
response.
• Integrin–cytoskeleton linkage is
sensitive to force.
– Close-up of a focal adhesion showing
the balance of external and internal
forces (Fext and Fcell, respectively)
in driving stress at a mechanosensor.
– Depicted are actin stress fibers (red)
anchored into focal adhesions
(multicolored array of proteins) that
bind to the ECM (blue) through
integrins (brown).
• This balance of forces provides the
stress necessary for mechanical sensing.
• This triggers a cascade of reactions that
alter the balance of anabolic / catabolic
events within the cell.
• This is a major regulator in cell
homeostasis and development.
Mesenchymal stem cell differentiation
• Isolated from bone marrow.
• Self-renewing multipotential cells with the
capacity to differentiate into
many distinct cell types.
– Osteoblasts (Bone)
– Chondrocytes (Cartilage)
– Adipocytes (Fat)
• MSCs play an important role
in treatment for trauma,
disease, or aging.
Differentiation by topography only
“A key principle of bone tissue engineering is the
development of scaffold materials that can stimulate
stem cell differentiation in the
absence of chemical treatment …”
- Dalby
Polymer nanoscale order vs. disorder
• Defined e-beam lithography approach using polymethylmethacrylate
(PMMA) .
– 120-nm-diameter, 100-nm-deep nanopits
• Demonstrated the use of nanoscale disorder to stimulate mesenchymal
stem cells to produce bone mineral in vitro.
• Claimed that the focal adhesion complexes determined by the
nanostructure effected the differentiation pathways.
Increased Osteogenic Gene Expression
Osteocalcin (OCN)
Alkaline Phosphatase (ALP)
Cells sense the surface
• Cells are responding to the nanotopography even in a such a small
range of dimensions or disorder…
• Protein adhesion (fibronectin and albumin) are affected by the
surface features.
• Changes in adhesion formation:
– Impose morphological changes on cells.
– Impacts on cytoskeletal tension.
– Affects indirect mechanotransductive pathways.
• Surface nanotopography directly induces pronounced changes to
cell shape, and consequently gene expression, which can
potentially mediate differentiation of stem cells into various cell
types.
Cell Signaling
Stress fibers
Ceramic nanopores/nanotubes
• Electrochemical anodization
– Anodic Aluminum Oxide (AAO)
– TiO2 Nanotubes
• Controllable features
– Film thickness
– Pore size/diameter
– Wall thickness
– Interspaces
Nanoporous alumina (AAO)
• SEM of two-step anodization process for the
fabrication of nanoporous alumina surfaces.
• Observed difference in cell morphology.
– Cells on flat surfaces are spherical
whereas on nanoporous surfaces seem to
be spreading
• Different adhesion mechanism on
nano-features.
• Demonstrated:
– An increase in the alkaline phosphatase
activity, bone forming ability on
nanoporous surface.
– Enhanced matrix production of MSCs
when they are cultured on nanoporous
alumina substrates.
Mechanical interlocking – TiO2 nanotubes
• Osteoblasts (bone cells)
• Higher density of cells.
• 3-4X accelerated
growth.
• Up-regulated
mineralization.
• Intricate interaction.
TiO2 nanotubes for stem cell therapies
Reduction of ECM
Increasing elongation
Increasing cytoskeletal stress!
Mimicking bone properties
• Advantages of using nanoporous
AlO2, TiO2, and other ceramic
scaffolds.
– Bone (trabecular) is porous in nature.
– Nanoscale ECM properties have
similar ceramic material properties
• Granular minerals and collagen fibers
• TiO2 crystal structure has same crystal
structure as hydroxyapatite.
• Similar chemistry
– To help orthopedic implant “osseo”integration into existing bone.
ECM Collagen fibers
• It is the main component of connective tissue, and is the
most abundant protein in mammals.
• Abundant in cornea, cartilage, bone, blood vessels, the gut,
and intervertebral disc.
• “Tropocollagen” is approximately 300 nm long and 1.5 nm in
diameter.
Hydrothermal TiO2 nanotubes: Enhanced
mineralization
• Hydrothermal reaction in autoclave chamber of Ti
foils in 10M NaOH solution.
• 8nm diamter, 150nm tall tangled nanotubes.
• Could be advantageous for imitating the natural ECM
environment of collagen type I fibers having fibrous
and tangled nanoscale geometry.
• This is an important aspect to bone scaffold designs in
order to present host cells with an innate ECM
interface where it triggers repopulation and
resynthesizing a new matrix.
Nanofiber designs
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Electrospinning technique:
– Nanofibrous scaffolds are promising candidate scaffolds for cell-based tissue
engineering.
– More closely mimics the niche-like unit (ECM fibrils) for facilitating proper
cell behavior.
• Electrospinning generates loosely connected 3D porous mats with high
porosity and high surface area which can mimic ECM.
• Can be randomly or aligning on the surface based on ground collection
plate
Made mostly of organic polymers.
• Can be bioresorbable/biodegradable
• Natural materials or synthetic
Nanofiber advantage
• Scaffold architecture affects cell binding and spreading.
• The cells binding to scaffolds with microscale architectures flatten and
spread as if cultured on flat surfaces.
• The scaffolds with nanoscale architectures have bigger surface area for
absorbing proteins and present more binding sites to cell membrane
receptors.
• The adsorbed proteins further can change the conformations, exposing
additional binding sites, expected to provide an edge over microscale
architectures for tissue generation applications.
General remarks
• Toxicity must be avoided but inertness is not a high priority
in nano/biomaterials.
– Bioactivity: positive interaction and effects on the human
body.
• Metallics, alloys and ceramic biomaterials are useful for hard
tissue (bone) but not suitable to replace soft tissues because
of markedly different mechanical properties.
– Must match the material properties to the body material
• Consider modulus, shear, wear particles, fatigue,
strength
• Applicable for 3-D geometries
• Conventional polymers are used for many of today’s pliable
disposable or biodegradable medical devices.
• New functional biomaterials are anticipated…
Take Home Message
• The eukaryotic cell is unimaginably
complex and remarkable.
• Controlling interactions at the
level of natural building blocks,
from proteins to cells on the
nanoscale, facilitates the novel
Nanofabrication
exploration, manipulation, and
application of living systems and
biological phenomena.
Living Cells
Basic
Tissue
• The nanostructured surface of a
Science
Engineering
material can improve its
interaction with cells and provide
Nanosystems
a desired response.
• Nanostructured tissue scaffolds
Stem Cell Therapy/
and biomaterials are being applied
Directed Differentiation
for improved tissue design,
reconstruction, and reparative
medicine.
Questions