Nanoscale Biomaterials for Cell and Tissue Interactions for Potential Biotech Applications

Prof. Sungho Jin, UC, San Diego

Cell nanostructures

• Cells are typically 1-100µm in size, but their interior and exterior contains many nanometer size objects.

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)

MicrofilamentF-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 nano-textures can help to uncover signaling pathways that promote the desired cellular response.

• Nanomaterial – “Particles with lengths that range from 1 to 100 nanometers in two or three dimensions.” ASTM E 2456-06 : 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.”

Basic definitions

• 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

Why so much interest in the surface?

• 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

Material factors that affect how the surface is perceived...(by living cells)

Time sequence of response to a surface• Protein adsorption onto surface (<1 sec)• Development of protein monolayer or multilayers (1-10

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.

• 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.

Discovery of Topography

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.

Controlled topography allows for controlled cell growth/alignment

• 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

Contact guidance by patterned biomaterials

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 multi-potential

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.

Stress fibers

Cell Signaling

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

Increasing elongation

Reduction of ECM

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• 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 exploration, manipulation, and application of living systems and biological phenomena.

• The nanostructured surface of a material can improve its interaction with cells and provide a desired response.

• Nanostructured tissue scaffolds and biomaterials are being applied for improved tissue design, reconstruction, and reparative medicine.

Basic Science

Living Cells

Nanosystems

Stem Cell Therapy/Directed Differentiation

Tissue Engineering

Nanofabrication

Questions