Biomedical Materials for

Tissue Engineering
-- An Immensely Exciting Field

(1)
12-05-2011

Content
 Introduction
 Background Concepts
– Cell
– Extracellular Matrix
– Cell-Matrix Interactions
 Principle of Tissue Engineering
-- Why TE, definition, types of TE, principle
 Biomaterial Scaffolds
– Design requirements
– Materials for scaffolding
– Fabrication methods
 Cell Seeding & culture
 Summary
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1
 Evolution of Biomaterials

TE

Biomaterials for TE is the

most advanced use of
biomedical materials

Knowing the Animal Tissue

• An aggregation of morphologically similar cells and
associated intercellular matter acting together to perform
one or more specific functions in the body.
• Animal tissues can be grouped into four basic types:
– Epithelial
– Muscle Dominantly cellular tissues
– Nervous
– Connective
• An animal tissue is composed of three basic components:
cells,
cells extracellular matrix (intercellular substances),
and various body fluids.

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 5

Cell
¾ The smallest individual unit of living matter, the presence
of which signals a difference between a living tissue and
a nonliving material.
¾ A structurally highly complex, ordered, and functionally
integrated assembly of organelles, cytoskeletal elements,
and enzymes.
¾ Essential attributes:
● Self-replication
● Protection from the environment
● Acquisition of nutrients
● Movement
● Communication
● Catabolism of extrinsic molecules
● Degradation and renewal of senescent intrinsic molecules
● Energy generation

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A typical animal cell. Within the cytoplasm, the major organelles and cellular
structures include: (1) nucleolus (2) nucleus (3) ribosome (4) vesicle (5) rough
endoplasmic reticulum (6) Golgi apparatus (7) cytoskeleton (8) smooth
endoplasmic reticulum (9) mitochondria (10) vacuole (11) cytosol (12) lysosome
(13) centriole. 7

Hydrophilic head

Hydrophobic core

Model of a prototypical cell membrane. Note the lipid bilayer with outer,
hydrophilic (exposed to an aqueous environment) and inner, hydrophobic
(maintains a barrier to solute movement) domains. Inserted through the
membrane or in either inner or outer planes are various proteins that
permit transport, cell–cell and signal–molecule interactions, and linkage of
the membrane to the intracellular cytoskeleton. 8

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 Extracellular Matrix
• ECM is a composite consisting of large
molecules synthesized by cells, exported to the
intercellular space and linked together into a
structurally supportive network.
• ECM holds cells together by providing physical
support and a matrix to which cells can adhere,
signal each other, and interact.
• Hugely varied:
– Hard tissues of bone and teeth
– Transparent matrix of the cornea
– Ropelike organization of tendons

Extracellular Matrix
• Principal functions are:
– Mechanical support for cell anchorage
– Determination of cell orientation
– Control of cell growth
– Maintenance of cell differentiation
– Scaffolding for orderly tissue renewal
– Establishment of tissue microenvironment
– Sequestration, storage, and presentation of
soluble regulatory molecules

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 Key Constituents of ECM
1. Structural fibrillar proteins
¾ Function: give ECM strength and flexibility
¾ Examples: collagen, elastin

2. Protein-polysaccharide complexes
¾ Function: provide matrix in which the other
proteins are organized
¾ Examples: proteoglycans

3. Adhesive glycoproteins
¾ Function: attach cells to the matrix
¾ Examples: fibronectin, laminins 11

Collagen synthesis

Assembled by cells, modified by cells as they

proliferate, differentiate, and migrate 12

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 With age, the amount
of elastin that the
body naturally
produces begins to
decrease. When
there is less elastin
present in the skin, it
causes drooping and
sagging.

Diagram of elastin showing the relaxed and stressed states. Tensile

forces cause the individual elastin molecules to unfold and align in the
direction of loading. The network is held together by the crosslinks
between the elastin molecules. 13

Proteoglycans & Glycoproteins

Proteoglycans
z Proteins are conjugated with polysaccharides
with serial repeat units
z Carbohydrate >> protein
z GAGs: Hyaluronate, Heparin, Chondroitin
sulfate, etc.

Glycoproteins
z Proteins are conjugated with some saccharides
lacking a serial repeat unit
z Protein >> carbohydrate
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7
 • Primary structure of
glycophorin A, a
glycoprotein that
spans the plasma
membrane ("Lipid
bilayer") of human red
blood cells

• Sugars are very

hydrophilic thanks to
their many -OH groups.
Their presence
makes glycoproteins far
more hydrophilic than
they would be otherwise
and are often essential
for the proper folding of
the protein into its
tertiary structure.

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aggregate

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8
 Heparan sulfate
proteoglycan in
matrix and
syndecan, cell
surface
proteoglycan. Its
core protein spans
the plasma
membrane and
can modulate the
activity of
fibroblast growth
factor (FGF).

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The fibronectin molecule consists of a dimmer held together

by S–S bonds. Note the various domains that bind to
extracellular matrix and the cell-binding domain containing
an arginine-glycine-aspartic acid (RGD) sequence.
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 The cross-
shaped laminin
molecule spans
basement
membranes* and
has extracellular
matrix (ECM)-
and cell-binding
domains.

*The basement membrane is a thin sheet of fibers that underlies the epithelium, which lines
the cavities and surfaces of organs including skin, or the endothelium, which lines the interior
surface of blood vessels. 19

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10
So, again…
¾ Collagen fibres strengthen and organize matrix
¾ Elastin fibres give resiliance
¾ GAG and proteoglycan molecules form highly
hydrated gel-like “ground substance” in which
the fibrous proteins are embedded
¾ Adhesive proteins help cells to attach to ECM
¾ Aqueous phase permits diffusion of nutrients

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Cell-Matrix Interactions

ECM
Regulating Secreting
cellular Assembling
behaviors Modifying

Cell
Integrins: Membrane-bound
molecules that can bind to ECM
molecules; Mediate cell–cell and
cell–matrix interaction
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and
communication.

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 (A) Initial contact of cell with
solid substrate.

(B) Formation of bonds

between cell surface
receptors and cell
adhesion ligands.

(C) Cytoskeletal
reorganization with
progressive spreading of
the cell on the substrate
for increased attachment
strength.

Progression of anchorage-dependent mammalian cell adhesion

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Why tissue engineering

• The loss or failure of an organ or tissue is a frequent,
devastating, and costly problem in health care, occurring
in millions of patients every year.
• Current therapeutic approaches:
– Transplantation (e.g., kidney transplant, heart transplant, liver
transplant): transplantation is substantially limited by a critical donor shortage
– Surgical reconstruction (e.g., heart bypass, skin grafting): cannot
replace all of the functions of the original tissue, long-term complications
– Artificial prosthesis (e.g., hip prosthesis, pacemaker, breast
implant): complicated by infection, limited durability of the material, lack of
mechanism of biological repair and remodeling
– Supplementation of metabolic products (e.g., insulin for
diabetes): cannot replace natural feedback mechanisms, frequently resulting
in dysregulation of hormone levels

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 Why tissue engineering
• Although these therapies have saved and
improved millions of lives, they remain imperfect
solutions.
• Tissue Engineering represents a new,
emerging interdisciplinary field applying a set of
tools at the interface of the biomedical and
engineering sciences that use living cells or
attract endogenous cells to aid tissue formation
or regeneration to restore, maintain, or improve
tissue function.
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Motivation of TE
• To create products that improve tissue
function or heal tissue defects.
– Replace diseased or damaged tissue
• Because……
– Donor tissues and organs are in short supply
– We want to minimize immune system
response by using our own cells or novel
ways to protect transplant.

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• An alternative to drug therapy, gene
therapy and whole organ transplantation
– Gene and drug therapy an option for treating
the underlying disease if the molecular basis
of the disease is understood
– Less suitable for replacing the entire function
of the cell
– “Grow” organs in the lab

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TE Definition
• “Interdisciplinary field that applies the
principle of engineering and life sciences
to the development of biological
substitutes that restore, maintain or
augment tissue function”
R. Langer and J. Vacanti “Tissue Engineering”. Science 260: 920-6, 1993.

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 Cell-based TE
• Uses mostly only cells and no materials,
aimed at certain diseases:
– Type I diabetes transplant of new pancreas
cells
– Adult stem cells for heart disease
– Neuronal transplants for Parkinson’s disease
– Bone marrow transplant for various blood
cancers
– Muscular dystrophy and polio

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Using adult
stem cells
• Inject into site of
damage
– Heart damage
– Muscular
dystrophy
• Texture,
stiffness and
chemicals in
surroundings
may influence
injected cells 30

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 What are Stem Cells ?

„ Cells with the capacity for unlimited or

prolonged self-renewal.

„ Cells that can produce at least one type of

highly differentiated descendant –
multipotent.

Commercial Interest in Stem Cells is High !!!

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Polio
• A virus attacks cells in
the spinal cord
– Signal no longer sent to
muscles in the leg
– Muscle wasting occurs
• Stem cell treatment
could
– encourage new spinal
neurons to grow
– help new muscle to grow

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 Scaffold-based TE
• Most strategies in tissue engineering have focused on
using biomaterials as scaffolds to direct specific cell
types to organize into three-dimensional (3D) structures
and perform differentiated function of the targeted
tissue.

• Synthetic bioresorbable polymers that are fully

degradable into the body’s natural metabolites by
simple hydrolysis under physiological conditions are the
most attractive scaffold materials.

• Using well designed scaffolds and optimized cell growth,

we can create tissues such as skin, cartilage, bone, etc.
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Principle of Tissue Engineering

)Appropriate cell source must be identified,
isolated and produced in sufficient numbers
)Appropriate biocompatible material that can be
used as a cell substrate or cell encapsulation
material isolated or synthesized, manufactured
into desired shape and dimensions
)Cells seeded together with growth factors onto
or into material, maintaining function,
morphology
)Engineered structure placed into appropriate in
vivo site

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 Types of Cells
• Tissue engineering starts at the level of the cell. It is
necessary to have a sufficient supply of cells that can be
ensured to be free of pathogens and
contamination. There are three sources of cells that can
be employed for tissue engineering, each with its own
advantages and drawbacks:
– Autologous: patient’s own cells; immune acceptable, does not
lend itself to off-the-shelf availability
– Allogeneic: cells from other human sources; lends itself to off-
the-shelf availability, but may require engineering immune
acceptance
– Xenogeneic: from different species; potentially unlimited supply,
but not only requires engineering immune acceptance, but must
be concerned with transmission of animal pathogens

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Screened for phenotype and

Seeded onto porous scaffolds increased in number through
together with growth factors to proliferation
enhance proliferation

A small number of
cells are removed
from the body

Seeded scaffolds are placed

in culture to further increase
cell number Implanted into the site of
damage to integrate with the
natural tissue
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 Human Cell Scaffold Nutrients,
Suspension Growth
Factors
electrical chemical
stimuli stimuli
Bioreactor
system

mechanical
stimuli
Implantation37
operation

Reactants:

Reactors: in vitro cell

culture; in vivo
(anatomical site)

Products: either scar or

regenerated tissue (or
intermediate cases)

TE can be viewed as processes of chemical synthesis

38

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 Tissue Engineering Triad
Scaffolds

Tissue
Engineered
Products
Biological

Cells Signals Physical

Mechanical
…. 39

One of the most striking wild things of

the late 20th Century:
Vacanti Mouse
-- mouse with the
human ear (ear
mouse)

z Created by Dr. Charles Vacanti & colleagues @

MIT in 1995, to demonstrate fabricating cartilage
structures for transplantation into human patients Dr. Charles A Vacanti
to treat victims of disease or accident
z Caused a buzz & public awareness with the
airing of a BBC broadcast on the potential of
tissue-engineered cartilage using images of the
ear mouse
Vacanti CA. History of Tissue Engineering and a Glimpse into its Future.
Tissue Eng. 2006 12(5): 1137-42. 40

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 Scaffold Design
z One essential criterion is biocompatibility,
i.e., the polymer scaffold should not invoke
an adverse inflammatory or immune
response once implanted.
z Porosity, pore size, and pore structure
are important factors to be considered with
respect to nutrient supply to transplanted
and regenerated cells.

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μCT representation of a highly porous Spongy bone from inside vertebrae

biphasic ceramic implant material obtained from a 21-year old male
(Camceram®, Cam Implants, Leiden, donor. Ritchie et al, Physics Today,
The Netherlands) with a HA: TCP ratio 2009.
of 60 : 40, porosity of 90%, and
macropores ranging from 300 to
500 μm. Bar represents 1 mm. 42

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 Scaffold Design
z Mechanical properties of the polymer scaffold
should be similar to the tissue or organ intended
for regeneration.
z Biodegradable or bioresorbable with a
controllable degradation and resorption rate to
match cell / tissue growth in vitro and/or in vivo.
z Be easily processed to form a variety of
shapes and sizes.
z “Ideal Scaffold” -- the scaffold is to be
engineered to mimic the natural ECM of a
specific tissue.

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Schematic of the mechanical contribution of an ECM scaffold over

time as it degrades and the mechanical contribution of the new host tissue
as it forms during ECM remodeling in the presence of appropriate
mechanical loading
44

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Factors Affecting Scaffold Degradation

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Scaffold Materials
• Today, four types of biomaterials have been
experimentally and/or clinically studied as
scaffold material for tissue engineering
applications:
– Synthetic organic materials: aliphatic
polyesters, polyethylene glycol;
– Synthetic inorganic materials:
hydroxyapatite, tri-calciumposphate, plaster of
Paris, glass ceramics;
– Organic materials of natural origin:
collagen, fibrin glue, hyaluronic acid;
– Inorganic material of natural origin:
coralline hydroxyapatite
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 Scaffold Materials
• Aliphatic polyesters such as polyglycolic acid (PGA),
polylactic acid ( PLLA), copolymers ( PLGA) and
polycaprolactone ( PCL) are most commonly used
materials in tissue engineering.
• They have been fabricated into thin films, fibers, porous
foams, and conduits and investigated as scaffolds for
regeneration of several tissues.
• The range of physical, chemical, mechanical, and
degradative properties that may be achieved using
synthetic bioresorbable polymers renders them
extremely versatile as scaffold materials. Their molecular
weight and chemical composition may be precisely
controlled during polymer synthesis.

47

Scaffold Materials
• Copolymers, polymer blends, and composites with other
materials may be manufactured to give rise to properties
that are advantageous over homopolymers for certain
applications.
• Moreover, many polymers can be functionalized by
converting end groups or addition of side chains with
various chemical groups to obtain polymers that can be
self-cross-linked or cross-linked with proteins and other
bioactive molecules.
• By choosing an appropriate processing technique,
scaffolds of specific architecture and structural
characteristics may be fabricated.

48

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 Applications of Scaffolds
• Tissue induction (in vivo)

The process by which ingrowth of surrounding tissue into a porous scaffold is effected.
49

Applications of Scaffolds
• Cell transplantation (in vitro)

--The most widely used TE strategy 50

--Possible to genetically modify cells before transplantation

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 Applications of Scaffolds
cells will not survive farther
• Prevascularization than a few hundred microns
from the nutrient supply

by injection

Suitable for large 3D transplants where nutrient 51

diffusion supply is critical for cell survival.

Applications of Scaffolds
• In situ polymerization
Injectable, polymerizable,
bioresorbable

Suitable for filling defects of any size, shape 52

with minimal surgical intervention.

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 Applications of Scaffolds
• Delivery of bioactive molecules
DNA, cytokines, GFs, hormones, angiogenic
factors, or immunosuppresant drugs

Microparticles or
Release directly from the nanoparticles loaded with
supporting matrix bioactive molecules are
53
impregnated into scaffolds
and serve as delivery vehicles

Scaffold Processing Techniques

pp741-744, Ratner et al 2004. 54

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 Solvent-Casting/
Particle-Leaching Method
• Biodegradable polymers (PLLA, PLGA, etc.) are used to
make scaffolding structure for supporting cellular
functions.

• Water-soluble particulates, such as salts and

carbohydrates, are used as the porogen materials. The
pore structure, porosity, and pore size can be easily
controlled by regulating the amount and size of salt.

• This method involves casting a mixture of polymer

solution (polymer/chloroform or polymer/methylene
chloride) and porogen in a mould, and then leaching out
the porogen with water to generate the pores and freeze-
drying the mixture.
55

From Khang G, Kim MS, Lee HB. A manual for biomateirals/scaffold fabrication 56
technology. World Scientific Publishing, 2007.

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(a) Salt sieving. (b) Preparing of polymer, solvent, salt. (c) Mixing of polymer, solvent, salt.

(d) Moulding. (e) Pressing. (f) Removal from mould.

(i) Removal of residual

(g) Dissolution of salt. (h) Freezing and freeze-drying. 57
solvent and storage.

Freeze-drying
• Polymer is first dissolved in a solvent such as
glacial acetic acid or benzene to form a solution
of desired concentration.
• The solution is then frozen and the solvent is
removed by lyophilization under high vacuum.
Several polymers including PLGA and
PLGA/PPF have been prepared into porous
foams with this method.
• The foams have either leaflet or capillary
structures depending on the polymer and
solvent used in fabrication.
58

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 Filtrating Moulding Freezing

surface

cross-section

Lyophilizing Scaffold
SEM morphology
59

Solid Freeform Fabrication

• Mouldless manufacturing technique, commonly known
as solid free-form fabrication (SFF), rapid prototyping
(RP).
• Computer-aided design, manufacturing (CAD/CAM)
methodology
• SFF builds parts by selectively adding materials, layer by
layer, as specified by a computer program. Each layer
represents the shape of the cross-section of the model at
a specific level.
• SFF techniques offer unique ways to precisely control
matrix architecture (size, shape, interconnectivity,
branching, geometry and orientation) yielding biomimetic
structures varying in design and material composition,
thereby enhancing control over mechanical properties,
biological effects and degradation kinetics of the
scaffolds.
60

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Computed Tomography (CT)

Moris et al, J
Adv Manuf
Technol, 2005.
Trends in
Biotech. 2005

Hutmacher DW
Trends in
Biotech. 2004

CT scan data of the patients bone defect (a) are used to generate a computer-based 3D model (b). This model is
then imported into RP system software to be ‘sliced’ into thin horizontal layers, with the tool path specified for each
layer (c). The ‘sliced’ data are used to instruct the RP machine (d) to build a scaffold (e) layer by layer, based on
the actual shape of the computer model (c). RP technology produces excellent templates for the treatment of
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intricate bone defects (a and f). Custom-made scaffold and cell constructs (g, see arrows) exactly follow the
complex shaped 3D contour of the skull.

Osteomesh

http://www.osteopore.com.sg/

Osteopore International is a
Singapore based corporation
strategically poised to be a leading
medical device company in this region,
involved in designing, developing and
Osteoplug
marketing bioresorbable polymer
implants for neurosurgical, orthopedic,
and maxillofacial surgery use.
62

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 Nanofibrous Scaffolds

Molly M. Stevens, Science 2005,310:1135

Scaffolds with nanoscale architectures have larger surface areas to adsorb
proteins, presenting many more binding sites to cell membrane receptors. 63

Cell seeding and culture

in 3D scaffolds
• The major obstacles to the in vitro development
of 3D cell–polymer constructs for the
regeneration of large organs or defects have
been obtaining uniform cell seeding at high
densities and maintaining nutrient transport to
the cells inside the scaffolds.
• To achieve desired spatial and temporal
distribution of cells and molecular cues affecting
cellular function, cell culture conditions should
provide control over hydrodynamic and
biochemical factors in the cell environment.
64

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 B
A C

Common bioreactors used in tissue engineering. (A) static culture, (B) spinner flask,
65
(C) rotary vessel, (D) perfusion system.

Safranin-O-stained cross-sections of
engineered cartilage tissues after six
weeks of culture under different
hydrodynamic conditions
[glycosaminoglycan (GAG) is
stained red]. (a) Statically cultured
constructs contain central regions with
negligible GAG deposition. (b) Spinner-
flask cultured constructs stain more
intensely for GAG in their central regions
but are encapsulated by fibrous tissue at
their periphery. (c) Rotating-wall vessel
(RWV) cultured constructs stain intensely
and homogeneously for GAG throughout
their cross-sectional area. The improved
structural features following RWV culture
(observed histologically and confirmed
biochemically) were shown to
be related to increased equilibrium
modulus, dynamic stiffness and reduced
tissue permeability. Scale bars, 1 mm.
66

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 On-site bioreactor @ hospital

Surgeon takes a Reagents stored in

compartments under app.

Martin I et al. Trends in Biotech. 2004.

biopsy
conditions

Auto. isolate, expand, seed,

culture cells until suitably
developed graft is produced

biopsy

Data derived from clinical

records of the patient is
inputted to control & optimize
culture parameters

Culture parameters and tissue Timely implantation

development monitored & analyzed by the surgical team

Vision for a closed-system bioreactor for the

automated production of tissue-engineered grafts 67

Summary
‹ TE offers future promise in the treatment of loss of tissue
or organ function as well as for genetic disorders with
metabolic deficiencies.
‹ TE in principle is a balanced combination of biomaterials,
cells, bioactive molecules, and tissue culture systems.
‹ Biomaterials play important roles in scaffold-based TE,
future challenges are:
¾ To better understand extracellular and intracellular modulators of
cell function
¾ To more closely replicate complex tissue architecture and
arrangement in vitro by developing novel materials and
processing techniques that are compatible with biological
interfaces

68

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