Fundamental of Tissue engineering

Introduction
“Tissue engineering is an emerging interdisciplinary
field that applies the principles of biology and
engineering to the development of viable substitutes
that restore, maintain or improve the function of
human tissues.”
Biology Engineering
Medicine
Tissue
Engineering

DEFINITIONS
 An interdisciplinary field that applies the principles of
engineering and life sciences toward the development
of biological substitutes that restore, maintain, or
improve tissue function or a whole organ – Langer
and Vacanti
 The use of a combination of cells, engineering and
materials methods, and suitable biochemical and
physico-chemical factors to improve or replace
biological functions.

• “The application of the principles and methods of
engineering and life sciences toward the fundamental
understanding of structure function relationships in
normal and pathological mammalian tissue and the
development of biological substitutes to restore,
maintain, or improve tissue function” – Y. C. Fung
• “The application of biological, chemical, and
engineering principles toward the repair, restoration,
or regeneration of living tissues using biomaterials,
cells, and factors alone or in combination.” – C. T.
Laurencin

A History of Tissue Engineering
 1000 BC – Sushrutha performed nose transplants
 16th Century – Tagliacozzi used a forearm flap for nose
reconstruction
 17th Century – Tooth transplantation
 18th Century – Skin & corneal transplantation
 19th Century – advent of sterile technique and anesthesia
precipitated and emergence of reconstructive surgery
 20th Century – proliferation of modern day transplantation
surgery and utilization synthetic materials for tissue repair

 Late 20th Century – shift in scientific focus toward cell based
reconstructive therapies and using biological components the
building blocks from tissue regeneration
 Post 1950 – Kidney, heart, lung, bone marrow
transplantation
 1985 – concept of Tissue Engineering was articulated in
detail
 1988 – First symposium on Tissue Engineering

Current Clinical Status
Grafting
method
Meaning Remark/ Drawbacks
Autograft To move tissue from one
site to another in the body
Patient is already suffering from disease,
thus grafting in the same body will be very
painfull and might lead to graft failure.
In old age patients, the grafted cells/ tissues
might not regenerate after harvest.
Allograft From another person (same
species)
This method suffers from issues like, Donor
scarcity and immune rejection.
The donor should be disease free.
Xenograft Tissue/organ taken from
other species
Problems like biocompatibility and immune
rejection are common in this method.
Moreover animal tissues are more prone to
contamination than human tissues, so after
grafting the chances of infection persist.
Patients are mainly treated surgically by grafting methods, by three ways- autograft,
allograft and xenograft.

REPAIR RESTOREREGENERATE REPLACE
Need of Tissue Engineering
o Donor tissues and organs are in short supply
o To minimize immune system response by using own cells or
novel ways to protect transplant
o ULTIMATE AIM of Tissue Engineering…

Overview of Tissue Engineering Process

CHALLENGES OF TISSUE
ENGINEERING…

Challenges
1. Microenvironment :
The proper
reconstitution of the
microenvironment for
the development of basic
tissue function and
properties
- Cell communicates
through local
microenvironment

Challenges
 Neighboring cells, ECM,
signaling molecules, cell
geometry, dynamics of
respiration, supply of
nutrients and removal of
metabolic products
 It mimics the dynamic,
chemical and geometric
variables

Challenges
2. Scale-up to generate
numerically enough,
properly functioning
microenvironments to be
clinically meaningful

Challenges
3. Microcirculation- It
connects the
microenvironments in
every tissue
 Metabolically active cells
are located within few
100m from a capillary
 Capillaries connect every
cells to a source and sink
4. Automation of system
operation at a clinically
meaningful scale

Important aspects of tissue engineering
Tissue engineered scaffold
Stem cell source
Bioreactor for making construct
Preservation

Lecture Outline
 What are Biomaterials?
 What is the need to know them?
 Scope of Biomaterials/ Scaffolds
 Types of Scaffolds used in Tissue Engineering
 Desirable Properties of Scaffolds for Tissue
Engineering
 Advances in Biomaterials/ Scaffolds Technology
 What are some of the challenges?

Suggested Books
 Biomaterials for Tissue Engineering Applications:
A Review of the Past and Future Trends - By Robert
L. Mauck
 Biomaterials: An Introduction - By Roderic S. Lakes
 Biomaterials and Tissue Engineering - By Donglu
Shi

Scaffold: Concept and functions
 Definition: It is a synthetic support material used to
replace part of a living system or to function in contact
with living tissue.
 Materials for Biomedical Application
 Scaffolds are 3D platforms for tissue engineering
 Used clinically or experimentally in implantable
electronic devices, drug delivery systems, hybrid
artificial organs, bone substitutes, ligament and
tendon replacements, etc.

Role of Engineered Scaffolds
 Allow cell attachment and migration
 Deliver and retain cells & biochemical factors
 Enable diffusion of vital cell nutrients and expressed
products
 Exert certain mechanical and biological influences to
modify the behavior of the cell phase

Scaffolds Applications:
Examples
 Orthopedic tissue construct /grafts
 Neural tissue regeneration
 Skeletal Muscle regereneration
 Joint replacements
 Bone plates
 Bone cement
 Hip Joint
 Artificial ligaments and tendons
 Dental implants for tooth fixation
 Blood vessel prostheses
 Heart valves
 Skin repair devices
 Cochlear replacements
 Contact lenses

Suitable Properties of Scaffolds
 Chemical Properties- Biologically active
- Sterilizable
 Physical Property - Mechanically supportive
 Biological Property - Biocompatible
- Biodegradable

Chemical Properties
 Suitable surface chemistry or bioactivity for cell
attachment.
- to facilitate binding the biomaterial with cell surface
receptor.
- in case of synthetic polymers, if they lack suitable
surface chemistry then their surface modification is
performed.
- Eg.- RGD peptide inclusion
 Sterilizable- without property loss
- to prevent contamination

Physical Property
 Mechanical Strength
- it should withstand shear stress generated by biological
fluid flow
Elastic Limit Break Point

Types of forces that can be applied to
Scaffolds
 Tensile- a force tending to tear it apart
 Compressive- A force that squeezes an object's
surfaces together and causes its mass to bulge.
 Shear- Shearing forces are unaligned forces pushing
one part of a body in one direction, and another part
the body in the opposite direction.
 Torsion- torsion is the twisting of an object
due to an applied torque.

 Stress- An applied force or system of forces that tends to
strain or deform a body.
 Strain- change in dimension of a body under load.
it is expressed as the ratio of total deflection or change in
dimension to the original unloaded dimension.
It may be ratio of lengths, areas or volumes (thus is is
dimensionless).
It gives the extent to which a body is distorted when it is
subjected to a deforming force, when it is under stress.
 Load- weight/ force applied
Terminology

 Elastic limit- stress that can be applied to an elastic body
without causing permanent deformation.
The stress point at which a material will no longer return to
irs original shape if it is subjected to higher stress.
Brittle materials tend to break at or shortly past their elastic
limit, while ductile materials deform at stress materials
beyond their elastic limit.
 Break point- A point of discontinuity, change or cessation.
 Yield point- The point in the stress-strain curve at which
the curve levels off and plastic deformation begins to occur.
Terminology

 Yield stress- The yield strength or yield point of material is
defined in engineering and materials science as the stress at
which a material begins to deform plastically.
Prior to the yield point the material will deform elastically and
will return to its original shape when the applied stress is
removed. Once the yield point is passed, some fraction of the
deformation will be permanent and non-reversible.
 Young's modulus- the slope of the elastic portion of stress-
strain curve, is a quantity often used to assess a material
stiffness.
Terminology

Mechanical properties
 Ultimate strength- Maximum value of load bearing
after which it may get permanently deformed.
 Tensile Strength- load bearing upto which a scaffold
could be elongated prior to breaking
- tested for fibrous materials
 Compressive Strength- degree of compression
- tested for porous scaffold
+ suitable Tailor Properties like pore size, % porosity

Biological Property
 Biocompatible
- The ability of a material to elicit an appropriate
biological response in a specific application by NOT
producing a toxic, injurious, or immunological
response in living tissue.
 Biodegradable
- rate of scaffold degradation = rate of tissue formation

Metals
Semiconductor
Materials
Ceramics
Polymers
SCAFFOLDS•Dental Implants
•Orthopedic screws/fixation
•Bone replacements
•Heart valves
•Dental Implants
•Implantable Microelectrodes
•Biosensors
•Ocular implants
•Drug Delivery Devices
•Skin/cartilage

Examples of Scaffold Materials
Type Material Applications
Polymer Silicone rubber
Dacron
Poly(methyl methacrylate)
Polyurethanes
Hydogels
Cellulose
Collagen (reprocessed)
silk fibroin, chitosan,
gelatin, alginate
- Catheters, tubing
- Vascular grafts
- Intraocular lenses, bone cement
- Catheters, pacemaker leads
- Opthalmological devices, Drug
Delivery
- Dialysis membranes
- Opthalmologic applications,
wound dressings
Metals Stainless steel
Titanium
Alumina
- Orthopedic devices, stents
- Orthopedic and dental devices
Ceramics Bioactive Glass (Silica)
Hydroxyapatite
Beta-TCP
Wollastonite
-Orthopedic devices
- Orthopedic and dental devices
-Orthopedic (Bone tissue)
-Orthopedic (Bone tissue)

Synthetic polymers
 More controllable from a compositional and materials
processing viewpoint.
 Scaffold architecture are widely recognized as
important parameters when designing a scaffold
 They may not be recognized by cells due to the
absence of biological signals.
Natural polymers
 Natural materials are readily recognized by cells.
 Interactions between cells and biological ECM are
catalysts to many critical functions in tissues

 Silk Fibroin
 Chitosan
 Starch
 Gelatin
 Alginate
 Cellulose
NATURAL POLYMERS SYNTHETIC POLYMERS
• Polyglycolic acid (PGA)
• Polylactic acid (PLA)
• Polycaprolactone (PCL)
• Polyvinyl alcohol (PVA)
• Polymethayl methacrylate
(PMMA)
Classification of Polymeric Biomaterials/
Scaffolds-

Advances in Scaffolds Technology
• Cell matrices for 3-D growth and tissue
reconstruction
• Biosensors, Biomimetic , and smart devices
• Controlled Drug Delivery/ Targeted delivery
• Biohybrid organs and Cell immunoisolation

What are some of the Challenges?
 To more closely replicate complex tissue architecture
and arrangement in vitro.
 To better understand extracellular and intracellular
modulators of cell function.
 To develop novel materials and processing techniques
that are compatible with biological interfaces.
 To find better strategies for immune acceptance.

Representative SEM micrograph of scaffolds prepared by different methods
Porous
 Solvent casting
 Salt leaching
 Phase Separation
 Gas Foaming
Fibrous
• Electrospining
Defined microstructure
• Rapid Prototyping
SCAFFOLD
Different ways of scaffold fabrication

Advanced Techniques
Rapid Prototyping
Micro fabrication:
1. Lithography
2. Solid freeform (SFF)
3. Cell Printing Technology
Scaffold Fabrication Techniques
Conventional Techniques
Salt Leaching
Phase separation
•Freeze – Drying
•Freeze Gelation
Solution Casting
Gas foaming
Electro spinning

SALT LEACHING
METHOD:
This method involves mixing water soluble porogen (e.g., NaCl) particles into a
biodegradable polymer solution. The mixture is then cast into the mold of the
desired shape.
After the solvent is removed by evaporation or lyophilization, the porogen
particles are leached out by water to obtain a porous structure.
Process Parameters:
 Size of the porogen
 Amount of porogen added
 Concentration of the polymer

Scaffold
Polymer Solution
Washing, Vaccum drying
Salt Leaching Process Overview:

ADVANTAGES:
 Simple operation
 Adequate control of pore size and porosity by
porogen/polymer ratio & Particle size of the added
porogen
 Highly interconnected pores
LIMITATIONS:
 Thermal degradation of polymer solution
 Possibility of toxicity due to residual solvent
 Non-uniform crystal shape of the porogen
 Precise control of pore size and porosity is difficult

Gas foaming
 Uses gas to form porous structure instead of organic
solvent systems
 CO2 is the most widely used gas for creating pores
 Incorporation of a particle leaching method on gas
forming help to get opened pores on the surface of a
scaffold

Principle
 Phase separation process works on separating different phases based on
following-
- Thermally induced phase separation - Changes in the solubility and
Gibbs free energy of a two-phase system with temperature.
- Non-solvent induced phase-separation – It is a ternary system
involving the polymer, a solvent and a non-solvent. Phase-separation is
induced by adding the non-solvent into the polymer and solvent
solution.
-Chemically induced phase-separation - Occurs in systems consisting of
a non-reactive polymer and a monomer that undergoes condensation
polymerization.
- After phase separation and appropriate treatment to eliminate the second
phase (if necessary), a porous network is obtained

Phase Separation
•It is a process of separation of polymer solutions into two phases,
one with low polymer concentration and other with high polymer
concentration. The concentrated phase solidifies shortly after phase
separation, and forms the scaffold.
•The phase separation process forms a porous structure within a
three-dimensional network
•These scaffolds have high surface area-to-volume ratio to enhance
cell adhesion, migration, proliferation, and differentiation function
•Freeze drying and freeze separation are the two methods which
work on the principle of Phase separation.

Control parameters
 Type of Solvent
 Type of polymer
 Polymer concentration
 Thermal treatment
 Order of procedures

Polymer is dissolved in its suitable solvent and stirred until a
homogenous solution is obtained
The polymer solution is poured into plastic/ teflon molds and frozen
o Gelation medium for the samples is
prepared and chilled
oThe frozen polymer solution is immersed
in the pre chilled gelation medium
oThe gelled samples are placed in a vaccum
drier until the complete evaporation of
solvent occurs
oThe prepared scaffolds are washed using
PBS and stored
Freeze Drying
Overview
 Works on the principle of Sublimation of
frozen solvent
 Frozen scaffolds placed under low
temperature (-110oC) vaccum conditions-
Lyophillization
 Frozen solvent removed: aids in pore
formation
 Helps to maintain biological activity of
scaffolds
Freeze Gelation

Freeze-Drying
A schematic diagram of Freeze drying process
Formation of pores after
sublimation of ice
Freezing Lyophilization
Ice crystals pores
Aqueous biopolymer suspension Reconstruction of ice crystal as
porogen in side the solid matrix
Requirement:
Removable phase (evenly distributed)
Solvent (should evaporate below -20˚C)
Important Parameters:
1. Temperature of freezing
2. Rate of cooling
3. Rate of lyophilization
4. Orientation inside the lyophilizer
5. Presence of Impurities

Advantages
 Can be used in water
based systems
 Helps to maintain
biological activity of
scaffolds
 Minimum damage to
scaffold property
Disadvantages
 High capital cost of
equipment
 High energy cost
 Long process time

Polymer Solution
Plastic Plate Storing at -20oC to -80oC
followed by vaccum drying
Porous Scaffold
Immersing in pre cooled
Gelation medium
A schematic diagram of Freeze gelation process
Freeze-Gelation

Advantages
• Simple procedure
• Process completes in lesser time
• Energy saving process (as no need
of furnace for heating)
• Lower residual solvent
Disadvantages
• Uneven pore distribution
• Brittle scaffolds

Advantages of Phase Separation:
 Desirable technique to produce scaffolds in a size range
similar to that of natural collagen
 High porosity ranges
 Prepared scaffolds possess ability to incorporate
biomolecules
Limitation:
Residual solvents may affect cell growth
No control over microgeometry of scaffold structure

 Method of fabrication of nanofibers using Electrostatic field.
• Used to form Nano fiber with diameter range 10-1000 nm.
 Polymer solution is forced through a capillary due to
intervention of high voltage resulting in formation of nanofibers
 Both random and oriented fibers can be fabricated depending
upon the collector
 Two methods are of use namely “Needle based” & “Free liquid
surface based”
Electrospinning

Schematic representation of Electrospinning Process
Principle: Continuous stretching of a viscoelastic jet derived from a polymer
solution or melt by the electrostatic forces. In electrospinning a solid fiber is
generated as the electrified jet is continuously elongated due to the
electrostatic repulsions between the surface charges and the evaporation of
the solvent.
Process of Electrospinning:
1. Charging of the polymer fluid.
2. Formation of the cone jet (Taylor cone)
3. Thinning of the jet in the presence of an electric field
4. Instability of the jet
5. Collection of the jet
Electrospinning

Parameters Affecting Electrospinning
Polymer solution parameters
Molecular Weight/Viscosity
Conductivity
Concentration
Surface tension
Dielectric Constant
Solvent Volatility
Process Parameter
Electrostatic Potential
Electric Field Strength
Shape of Electric Field
Distance between Tip and
Collector (Working distance)
Feed Rate
Diameter of Orifice
Environmental Parameters
Temperature
Humidity
Air Velocity Inside Chamber
Pressure

Polymer Solution Parameters
Molecular Weight and Solution Viscosity
 When a polymer of higher molecular weight is dissolved in a solvent, its viscosity
will be higher than solution of the same polymer but of a lower molecular weight.
One of the conditions necessary for ES to occur where fibers are formed is that the
solution must consists of polymer of sufficient molecular weight and the solution
must be of sufficient viscosity
 If the solution is more viscous, number of beads decreases and nanofibers of
uniform morphology is obtained.
Solution Conductivity
 Electrospinning involves stretching of the solution caused by repulsion of the
charges at its surface. Thus if the conductivity of the solution is increased, more
charges can be carried by the electrospinning jet.
 The conductivity of the solution can be increased by the addition of ions e.g. salt

Polymer solution Parameters (Cont…)
Concentration
increased polymer concentration in solution leads to increased fiber diameter
Surface Tension
 The initiation of electrospinning requires the charged solution to overcome its
surface tension
 This property depends upon type of polymer used and voltage applied
 A higher viscosity means that there is greater interaction between the solvent and
polymer molecules thus when the solution is stretched under the influence of the
charges, the solvent molecules will tend to spread over the entangled polymer
molecules thus reducing the tendency for the solvent molecules to come together
under the influence of surface tension.
Solvent Volatility
 solvent should be volatile enough to form dry fibers
 If solvent is less volatile the fibers will be wet and we will get film instead of
separated fibers, due to solvent deposition.

Another important parameter that affects the electrospinning process is the various
external factors exerting on the electrospinning jet.
Voltage
 A crucial element in electrospinning is the application of a high voltage to the
solution. Initiation of fiber formation is dependent upon the voltage applied. The
high voltage will induce the necessary charges on the solution and together with
the external electric field, will initiate the electrospinning process
 The applied electric field also governs the diameter of Nano fibers formed during
this process.
 When the electrostatic force in the solution overcomes the surface tension of the
solution. Generally, both high negative or positive voltage of more than 6kV is
able to cause the solution drop at the tip of the needle to distort into the shape of a
Taylor Cone during jet initiation.
Diameter of Orifice
important in case of needle based electrospinning method
Process Parameters

Temperature- The temperature of the solution has both the effect of increasing
its evaporation rate and reducing the viscosity of the polymer solution.
 Eg.- When polyurethane is electrospun at a higher temperature, the fibers
produced have a more uniform diameter .This may be due to the lower
viscosity of the solution and greater solubility of the polymer in the solvent
which allows more even stretching of the solution.
 With a lower viscosity, the Columbic forces are able to exert a greater
stretching force on the solution thus resulting in fibers of smaller diameter.
Effect of Collector- There must be an electric field between the source
and the collector for electrospinning to initiate. Thus in most
electrospinning setup, the collector plate is made out of conductive
material such as aluminum foil which is electrically grounded so that
there is a stable potential difference between the source and the
collector.
Process Parameters (Cont…)

Feed rate:
The feedrate will determine the amount of solution
available for electrospinning. For a given voltage, there
is a corresponding feedrate if a stable Taylor cone is to
be maintained. When the feedrate is increased, there is a
corresponding increase in the fiber diameter or beads
size.
Distance between Tip and Collector (Working
distance)
- if fibers are formed at larger distance then they are found
to be thinner.
- For independent fibers to form, the electrospinning jet
must be allowed time for most of the solvents to be
evaporated. When the distance between the tip and the
collector is reduced, the jet will have a shorter distance
to travel before it reaches the collector plate.
Process Parameters (Cont…)

Environmental Parameters
• Temperature- inside the electro-spinning chamber, temperature controls the
solvent volatility, and hence the finer formation.
• Humidity- is important to form dry and separated fibers. At high humidity, it is
likely that water condenses on the surface of the fiber when electrospinning is
carried out under normal atmosphere. As a result, this may have an influence on
the fiber morphology especially polymer dissolved in volatile solvents.
• Air Velocity Inside Chamber- this plays the key role in fiber collection on the
collector. The air flow rate should be sufficient enough to allow complete fiber
deposition on collector. It also regulates the established voltage between the
electrodes.
• Pressure- the rate with which the polymer solution is forced to come out of
needle tip is an important parameter as it directly affects the feed rate.

Advanced Electrospinning Techniques
 Multi-jet electrospinning
 Porous tubes electrospinning
 Upward needleless electrospinning
-1. Two-layer-fluid electrospinning
-2. Free Liquid Surface Electrospinning
Purpose: Developing an electrospinning technique for
large-scale nanofiber production

Multi-jet electrospinning
Construction:
 A cylindrical electrode is used as an auxiliary electrode to cover the multi-
jet spinneret that stabilizes and optimize the electrospinning process.
 The presence of external electrode dramatically reduces the fiber
deposition area, thus improving the fiber production rate.
A multi-jet electrospinning with a cylindrical auxiliary electrode

Advantage:
 Increases electrospinning throughput by use of multi-jet spinnerets.
The fiber productivity can be simply increased by increasing the jet
number.
Limitations:
 Strong repulsion among the jets may lead to reduced fiber production
rate and poor fibre quality, which is the main obstacle to practical
application.
 Interferences in multi-jet electrospinning are unable to be eliminated
completely, which is a barrier to its industrialization.
 Successful operation of multi-jet electrospinning requires regular
cleaning system to avoid the blockage of the needle nozzles. Setting
the cleaning device for each needle is a limitation for mass production
of nanofibers.
 To reduce the jet repulsion, jets have to be set at an appropriate
distance, and a large space is required to accommodate the needles for
the mass nanofiber production.

Porous tubes electrospinning
Construction:
 Porous tubes are used as spinnerets to improve the fiber productivity.
 The electrospinning process is based on conveying solutions inside
the tube channels.
 The polymer solution under pressure is pushed through the tube wall
with many holes.
 A porous polyethylene tube with a vertical axis is used to electrospin
nanofibers . The production rate is reported to be 250 times greater
than that of single needle electrospinning.

Limitations:
 large variations in the fiber diameter.
 This setup can only produce 0.3 ~ 0.5 g/hr of nanofibers due to
the small number of holes (fiber generators) that can be
drilled per unit area. But the production rate can be easily
scaled up by increasing the tube length and the number of
holes
 The space between holes can’t be reduced much because of the
electric field repulsion between the jets.
 The strong jet interference in this setup results in nanofiber
belt instead of fiber web

Upward needleless electrospinning
1. Two-layer-fluid electrospinning
Construction and working:
 2 layers of fluid: lower layer – any ferromagnetic suspension and
upper layer- the polymer solution to be spun.
 During electrospinning, when a normal magnetic field is applied to the
system, steady vertical spikes are formed perturbing the interlayer
interface. As a result of applying a high voltage to the fluid at the same
time, thousands of jetting ejected upward.
Limitation:
 This upward electrospinning system requires a complicated setup
 the resultant nanofibers had large fiber diameter
 Wide diameter variation

A two-layer-fluid electrospinning setup

2. Free Liquid Surface Electrospinning
 Invented by Jirsak et al in 2005.
 It is a needleless electrospinning setup that uses a rotating
roller as the nanofiber generator.
 The roller is immersed into a polymer solution and slowly
rotates, the polymer solution is loaded onto the upper roller
surface.
 Upon applying a high voltage to the electrospinning system,
an enormous number of solution jets can be generated from
the roller surface upward.
 This setup has been commercialized by Elmarco Co with the
brand name “NanospiderTM”.

Setup of Free liquid surface electrospinning

Commercial Free Liquid Surface
Electrospinning Machine
Nanofiber formation from roller surface

Advantages
 Time efficient process
 Complete removal of solvent
 Diameter of the fibers can be formed to match the diameter of
ECM
Disadvantages
 Energy consuming process-
 High risk due to High voltage
 Preparation of 3D scaffolds with internal pore network
involves proper design of collector
Electrospinning (in general):

Aligned and Non-aligned Nano fibers
Electrospinning is a straight forward method for creating longitudinally oriented
nanoﬁbers.
However the process could be aided with some modifications to generate aligned
nanofibers.
Non-aligned nanofibers Aligned Nanofibers

Aligned Nano-fiber Preparation
1. By collecting electrospun fibers on rapidly rotating wheel
2. Using a collector consisting of two pieces of electrical
conductors separated by a gap.
3. Fabricating aligned yarn of nanofibers by rapidly oscillating a
grounded frame within the jet
4. Using a metal frame as a collector to generate parallel arrays of
nanofibres
5. Using magnetic field to produce aligned nanofibrous arrays
Schematic representation of electro
spinning set-up
(A) axle of rotation,
(B) polymer solution container,
(C) nozzle tip,
(D) encircling cylinder,
(E) Collector
(F) polymeric jet.

Rapid Prototyping Method
1. Imaging of the tissue defect via scanning techniques like, CT, X-
ray or MRI to obtain basic information about the defect.
2. CAD solid model- Complex shapes of the scaffold which would
fill the defect (as per noted in the scan) are designed using CAD
software
3. ‘.STL’ file is generated
4. Slicing the file into layers
5. Final build file
6. Fabrication of part- Scaffolds are fabricated using specific
technique
7. Post processing
STEPS in general-

Schematic Representation of Rapid Prototyping Method

Rapid Prototyping Techniques
Micro fabrication:
1. Solid freeform (SFF)
- Stereo lithography Analyzer (SLA)
- Stereo lithography Sintering or Selective laser Sintering (SLS)
- Fused Deposition Modeling (FDM)
- Three Dimensional Printing (3DP)
- Micro syringe Deposition (MD)
2. Lithography
- Photolithography and etching
- High aspect ratio Photolithography
- Micro molding
- Electroplating
3. Cell Printing Technology

Solid freeform (SFF)
Process:-
1. CAD is applied to generate a 3D replica of scaffold.
2. This image is organized into 2D slices typically of 100 microns thickness.
3. Scaffolds are produced by mechanically controlling the laser within the
horizontal plane forming pattern by polymerizing the monomers in specified
locations according to CAD.
Important points:-
• Lateral and vertical resolution depends upon various factors like-
• The lateral resolution is typically in the range of 50-250µm. Laser spot size is
the principal driver of the lateral resolution. Smaller is the laser spot, better is
the spatial resolution and thus manufacturing time is increased.
• Vertical resolution is determined by heat affected zone depth in photo-
polymerization process.

Principle of SLA
SLA was developed in 1986 by 3D Systems
The process is based on the following principles:
 A laser is used to design precise regions of scaffolds through photo-polymerization
 Parts are built from a photo-curable liquid polymer that solidifies when sufficiently
exposed to a laser beam which scans across the surface of the resin
 The structure is made layer by layer, each layer being scanned by the optical scanning
system and controlled by an elevation mechanism which lowers at the completion of each
layer
This is a direct write method that requires substantial processing time to produce a
scaffold of sufficient scale & resolution for complex tissue engineering applications.

Process of SLA
1. A liquid state photosensitive polymer solidifies when exposed to a
lighting source
2. A platform that can be elevated is located just one layer of thickness
below the surface
3. According to the cross section of the part (starting with bottom layer).
The laser scans the polymer layer above the platform to solidify the
polymer
4. The Platform is lowered into the polymer bath to the layer thickness
5. Steps 3 and 4 are repeated until the top layer of the part is generated
6. Post-curing and part finishing can then be performed

Schematic of SLA for polymer solution

Applications of SLA
 Models for conceptualisation, packaging and presentation
 Prototypes for design, analysis, verification and functional testing
 Masters for prototype tooling and low volume production tooling
 Patterns for investment casting, sand casting and moulding
 Tools for fixture and tooling design and production tooling
Advantages of SLA
• High accuracy
• Surface quality is good
• Used for fabricating parts of varied sizes- from small pin to car dash board

Selective Laser Sintering (SLS)
Process:-
1. Roller spreads powder over a platform.
2. According to structural information obtained from CAD, the scanner performs
polymerization and sintering of powder (thus first layer from the bottom is
formed). This is done by CO2 laser that provides concentrated heating beam
which is traced over tightly compacted layer of fine heat fusible powder.
3. Platform moves one step down and above steps are repeated till the desired
scaffold structure is obtained.

Advantages of SLS:
• Rapid manufacturing
• Large and complex functional parts can be manufactured
in less time

Fused Deposition Modeling (FDM)
Also known as Biological Particle Manufacturing (BPM)
x-y-z plotter is utilized which has vertical stepping capability.
Process:-
According to the scaffold structure designed using CAD, molten scaffold
material such as polymer or ceramics are ejected from a nozzle on to a
surface
Continuous deposition of molten material on a solidified surface leads to
formation of a particular 3D scaffold structure.
• Porosity is generally not as high as it is with other technique.

Parts of a FDM Machine
 Raw material: The raw material mostly used in this process is generally
thermoplastic filaments or thermoplastic beads. ABS (Acrylonitrile Butadiene
Styrene) material, polyamide, polycarbonate, polyethylene, polypropylene, and
investment casting wax are the raw materials that are used in the technique.
Supporting materials are also used along with the main raw material.
 Extrusion nozzle: This is an important part of the apparatus from which metal gets
heated up and liquefied. The extrusion nozzle can be moved along the X-Y plane
only.
 Stepper motors: The stepper motor helps to move the nozzle according to the CAM
(Computer-Aided Manufacturing) program code which defines the path of the
motion of nozzle.
 Nozzle tip: This nozzle tip is the last point from which hot thermoplastic will get
deposited on the platform.
 Drive wheels: The drive wheels will provide the required feed for the filaments so
that it properly moves into the liquefier.
 Liquefier: This part of the set up liquefies the thermoplastic filament to molten state,
which is then deposited on the platform.
 Platform: This is the base on which fused deposition model is produced.

Advantages of FDM
• Economical technique for making medium sized parts
• Parts having greater stability can be manufactured
• Low end, economical machines.
• No post curing required
• Variety of materials can be used
• Easy material changeover
Disadvantages
• Can not be applied for polymer solution
• Not good for small features, details and thin walls.
• Surface finish
• Supports required on some materials / geometries.
• Support design / integration / removal is difficult.
• Weak Z-axis.
• Slow on large / dense parts.

Three Dimensional Printing (3DP)
3D printers use a variety of very different types of additive manufacturing
technologies, but they all share one core thing in common: they create a three
dimensional object by building it layer by successive layer, until the entire object is
complete.
Steps-
1. The file — a Computer Aided Design (CAD) file — is created with the use of a
3D modeling program, either from scratch or beginning with a 3D model
created by a 3D scanner. Either way, the program creates a file that is sent to the
3D printer.
2. Software slices the design into hundreds, or more likely thousands, of horizontal
layers.
3. 3DP utilizes a scanning system that directs a writer towards specific positions on
a 2D plane.
4. Then a jet of chemical binder is applied towards the powder bed which binds the
powder.
5. The platform then steps down in vertical direction to write next layer.
6. These layers will be printed one atop the other until the 3D object is formed.

Advantages of 3DP
• Very fast
• Cost effective
• Manufacturing of coloured parts is also possible

3D Printers CAD designed structure
Three Dimensional Printing (3DP)

Lithography Techniques
1. Photolithography and Etching
• Photolithography is used to pattern substrates for formation of topographic
features and spatial features like, formation of micro channels, adhesive or non-
adhesive regions.
• Comprises the application of thin layers of photoresist followed by plasma
etching – this produces topographic or spatial features on substrate.
• For nanoscale features- advanced lithographic processes are applied like-
Conformable Contact Lithography (CCL)
Deep reactive ion-etching (DRIE)

Lithography Techniques
2. High aspect ratio Photolithography
• A high energy beam is used to expose thick polymeric film to obtain
desired structure on the surface of Si wafers.
• Layers of thickness ranging from 25 µm to several hundred microns
are deposited and patterned to produce thicker layers of complex 3D
structure.

• Cells are dropped on previously printed successive layers. This allows printing of
complex 3D organs with computer-controlled system, by exact placing of different cell
types onto a polymer solution layer.
• The printer puts up solutions of cells or polymers into a specific place by the use of
specially designed software, and print two-dimensional (2D) tissue constructs.
• Addition for printing 3D constructs: Nontoxic, biodegradable, thermo-reversible gels can
be used which are fluid at 20°C and gel above 32°C, as a sort of “paper” on which tissue
structures can be printed, and the cells are the “ink.” Successive layers could be
generated just by dropping another layer of gel onto an already printed surface.
Cell Printing Technology

Advantages
 Best method to control
pore size
 Best method for
preparing complex
shaped scaffolds
 Energy & Time Efficient
Disadvantages
 Applicable to limited
polymers
 Sophisticated methods

Characterization Techniques
Physicochemical Morphology
Porosity
Thermal
Hydrophillicity
Viscosity
Binding Energy
Biodegradation
Swelling ratio
Microscopy
Porosimetry
DSC, TGA
Contact angle
Viscometer
XPS
Treatment in SBF
solution
Structural Crystallinity
Composition
XRD
FTIR,
Mechanical Stress-Strain relationship
Compressive strength (Porous)
Tensile strength (Fibrous scaffold)
Mechanical Tester
Biological Biocompatibility
Cellular organization
In vitro & in vivo
Fluorescence,
Confocal and FACS

Mechanical Characterization
 Ultimate strength- Maximum value of load bearing after which it
may get permanently deformed.
 Tensile Strength- also known as tension test, it is a test of load
bearing upto which a scaffold could be elongated prior to breaking
- tested for fibrous materials
 Compressive Strength- It gives degree of compression of a
material. A compression test determines behaviour of materials
under crushing loads. The specimen is compressed and
deformation at various loads is recorded.
- tested for porous scaffold

Types of forces that can be applied to scaffolds
 Tensile- a force tending to tear it apart
 Compressive- A force that squeezes an object's
surfaces together and causes its mass to bulge.
 Shear- Shearing forces are
unaligned forces pushing one part of a body in
one direction, and another part the body in the
opposite direction.
 Torsion- torsion is the twisting of an object
due to an applied torque.

Scaffold should withstand shear stress generated by biological fluid flow
A typical stress strain curve-
Elastic Limit Break Point

 Stress- An applied force or system of forces that tends to strain or
deform a body.
 Strain- change in dimension of a body under load.
it is expressed as the ratio of total deflection or change in dimension to
the original unloaded dimension.
It may be ratio of lengths, areas or volumes (thus it is dimensionless).
It gives the extent to which a body is distorted when it is subjected to a
deforming force, when it is under stress.
 Load- weight/force applied
 Break point- A point of discontinuity, change or cessation.
Terminology

 Elastic limit- stress that can be applied to an elastic body without causing
permanent deformation.
The stress point at which a material will no longer return to its original
shape if it is subjected to higher stress.
Brittle materials tend to break at or shortly past their elastic limit, while
ductile materials deform at stress materials beyond their elastic limit.
 Yield point- The point in the stress-strain curve at which the curve levels
off and plastic deformation begins to occur.
 Yield stress- The stress at which a material begins to deform plastically.
Prior to the yield point the material will deform elastically and will return
to its original shape when the applied stress is removed. Once the yield
point is passed, some fraction of the deformation will be permanent and
non-reversible.
 Young's modulus- the slope of the elastic portion of stress-strain curve, is
a quantity often used to assess a material stiffness.
Terminology

Physicochemical Characterization

Morphology Analysis
1. SEM: Scanning Electron Microscopy
 Principle: Accelerated electrons in an SEM carry significant amounts of
kinetic energy, and this energy is dissipated as a variety of signals
produced by electron-sample interactions when the incident electrons
are decelerated in the solid sample. These signals include secondary
electrons (that produce SEM images), backscattered electrons (BSE),
diffracted backscattered electrons (EBSD).
 SEM is routinely used to generate-
- high-resolution images of shapes of objects (Precise measurement of
very small features and objects is also accomplished using SEM)
- to show spatial variations in chemical compositions.
- Pattern and diameter of pores and fibers can be determined

 Porous Scaffold 
 Fibrous Scaffold 
SEM Images- Examples

 Principle: The TEM operates on the same basic principles as the light
microscope but uses electrons instead of light. What can be seen with a
light microscope is limited by the wavelength of light. TEMs use
electron as “light source” and their much lower wavelength makes it
possible to get a resolution a thousand times better than with a light
microscope.
 Applications:
- The transmission electron microscope is used to characterize the
microstructure of materials with very high spatial resolution.
- Used to determine morphology, crystal structure and defects
- Crystal phases and composition can be determined
- Information about magnetic microstructure can be obtained
Morphology Analysis
2. TEM: Transmission Electron Microscope

 Principle: The AFM consists of a cantilever with a sharp tip (probe) at its
end that is used to scan the specimen surface. When the tip is brought into
proximity of a sample surface, forces between the tip and the sample lead
to a deflection of the cantilever.
 The cantilever is typically silicon or silicon nitride with a tip radius of
curvature in the order of nanometers.
 Deflection is measured using a laser spot reflected from the top surface of
the cantilever into an array of photodiodes.
 Forces that are measured in AFM include mechanical contact force, van
der Waals forces, capillary forces, chemical bonding, electrostatic forces,
magnetic forces etc.
Morphology Analysis
3. AFM: Atomic Force Microscopy

 Advantages:
- provides a three-dimensional surface profile.
- samples viewed by AFM do not require any special treatments (such as
metal/carbon coatings) that would irreversibly change or damage the
sample
- final image is free from charging artifacts.
- Most AFM modes can work perfectly well in ambient air or even a
liquid environment. This makes it possible to study biological
macromolecules and even living organisms.
Probe
Sample surface

POROSIMETRY
 The term “porosimetry” is often used to include the measurements
of pore size, volume, distribution, density and other porosity-
related characteristics of a material.
 Porosity is especially important in understanding the formation,
structure and potential use of many substances.
 The porosity of a material affects its physical properties
(adsorption, permeability, strength, density) and, subsequently, its
behaviour in its surrounding environment.
 Ideally, for a scaffold fabricated for tissue engineering applications,
its porosity should be ≥ 70%.

Hydrophilicity
Contact Angle Measurement
 The contact angle is the angle, conventionally measured through the
liquid, where a liquid/vapour interface meets a solid surface.
 It quantifies the wettability of a solid surface by a liquid. A given system
of solid, liquid, and vapour at a given temperature and pressure has a
unique equilibrium contact angle.
 The equilibrium contact angle reflects the relative strength of the liquid,
solid, and vapour molecular interaction.
 Significance:
 Determines amount of liquid a substance/scaffold can hold
 A scaffold implanted in the body, comes in contact with the body fluids
 Hydrophillicity of the scaffold is necessary for cell survival and cell
attachment

 The equilibrium swelling ratio (Es) can be measured by the conventional
gravimetric method.
 The dry weight (Wd) of scaffold is measured and then wet weight (Ws) by
immersing in simulated body fluid (SBF).
 The equilibrium swelling ratio of the scaffolds is defined as the ratio of
weight increase (Ws-Wd) with respect to the initial weight (Wd) of dry
samples.
 Es is calculated using the following equation:
Es= (Ws-Wd)/Wd
 Water uptake percentage (Wu) can be measured using the equation:
Wu= (Ws-Wd)/ Ws x 100
Hydrophilicity
Swelling Behaviour and Water Uptake capacity

Thermal Property
1. DSC: Differential Scanning Calorimetry
 Principle: DSC is a thermoanalytical technique in which the difference in
the amount of heat required to increase the temperature of a sample and
reference are measured as a function of temperature.
 Both the sample and reference are maintained at very nearly the same
temperature throughout the experiment.
 The basic principle underlying this technique is that, when the sample
undergoes a physical transformation such as phase transitions, more (or
less) heat will need to flow to it than the reference to maintain both at the
same temperature.
 This difference in temperature is measured by thermocouple.

Applications
 Glass Transitions
 Melting and Boiling Points
 Crystallization time and temperature
 Percent Crystallinity
 Heats of Fusion and Reactions
 Specific Heat
 Rate and Degree of Cure
 Reaction Kinetics
 Purity

Thermal Property
2. TGA: Thermal Gravimetric analysis
 Principle: TGA is a method of thermal analysis in which changes in
physical and chemical properties of materials are measured as a function of
increasing temperature (with constant heating rate), or as a function of time
(with constant temperature and/or constant mass loss).
 TGA is commonly used to determine selected characteristics of materials
that exhibit either mass loss or gain due to decomposition, oxidation, or
loss of volatiles (such as moisture). Means TGA measures weight changes
in a material (subjected to temperature variation in a controlled
atmosphere).
 TGA can provide information about physical phenomena, such as second-
order phase transitions, vaporization, sublimation, absorption, adsorption,
and desorption.
 TGA can also be used to know about chemical phenomena such as
chemisorptions, desolvation (especially dehydration), decomposition, and
solid-gas reactions (e.g. oxidation or reduction).

Applications of TGA-
1. materials characterization through analysis of characteristic
decomposition patterns
2. Studies of degradation mechanisms and reaction kinetics
3. Determination of organic content in a sample
4. Determination of inorganic (e.g. ash) content in a sample, which
may be useful for corroborating predicted material structures or
simply used as a chemical analysis.
5. It is an especially useful technique for the study of polymeric
materials, thermoplastics, thermosets, elastomers, composites, pla
stic films, fibers, coatings and paints

Viscosity
 Viscosity of a fluid is measured by instrument known as Viscometer.
 For liquids with viscosities which vary with flow conditions, an
instrument called a rheometer is used. Viscometers only measure under
one flow condition.
 In general, either the fluid remains stationary and an object moves
through it, or the object is stationary and the fluid moves past it.
 Principle: The drag caused by relative motion of the fluid and a surface is
a measure of the viscosity.
 The flow conditions must have a sufficiently small value of Reynolds
number for there to be laminar flow.

Biodegradation
 When a scaffold is incorporated inside the body, it comes in contact
with various biological fluids and enzymes and thus degrades with
time. And the space generated by scaffold degradation facilitates new
tissue formation.
 A scaffold should not only degrade, it should “Bio” degrade, i.e. its
degradation should be accompanied by its removal from the body,
without generating any toxic effect to the surrounding tissues.
 Degradation rate of scaffold= rate of tissue formation
 Degradation testing is done by giving enzymes like lysozyme and
incubating in a fluid medium (PBS or SBF) for particular time period
and measuring change in weight.

XRD: X-ray Diffraction
 Principle: Braggs Law of diffraction. A law stating that when a crystal
is pictured as a set of reflecting planes uniformly spaced at a
distance d and a beam of X-rays of wavelength λ strikes the crystal at
an angle θ, reinforcement of the reflected waves occurs when sin θ = n
λ/2d, where n is an integer known as the order of reflection.
nλ = 2d sin θ
 XRD is a material characterization technique that can be used for
analyzing the lattice structure of a material.

Applications
 Phase identification.
 Quantitative analysis.
 Crystal structure analysis.
 Microstructure of real materials.
20 30 40 50 60 70 80
0
100
200
300
400
Intensity(a.u.)
2 theta(degree)
*
(214)
(210)
(220)

FT-IR: Fourier Transform InfraRed
 Molecular bonds vibrate at various frequencies depending on the
elements and the type of bonds.
 Each bond vibrate at several specific frequencies.
 The data from the sample is collected in a wide spectral range and
converted into specific frequency which can be recorded as a
function of transmittance.

Biocompatibility
 The properties of materials being biologically compatible by not eliciting
local or systemic responses from a living system or tissue.
 Biocompatibility is a series of tests that are used to determine the
potential toxicity resulting from contact of the components of medical
devices or combination products with the body.
 Biocompatibility testing:
 Invitro- SEM, Fluorescence microscopy Cell morphology, attachment
and spreading
- Alamar blue assay Cell proliferation
- MTT assay cell viability and metabolic activity analysis
 Invivo- preclinical tests in animal models- creating artificial wound and
accessing the response generated by the implant.
 Next step is clinical trial- performed by surgeons in hospitals

S.No TECHNIQUE APPLICATION
1. SEM Sample dia, Distribution, Orientation,
Fiber Morphology (Shape, Roughness etc.)
2. TEM Fiber Morphology (Shape, Roughness etc.)
No need to dry the sample as in SEM.
3. FESEM Fiber Morphology (Shape, Roughness etc.)
4. XPS (ESCA) Elemental composition of the surface (top 1–10 nm
usually)
5. FTIR Important functional groups and to determine the extent
of hydrogen bonding.
6. NMR To identify functional groups, number and type of
chemical entities in a molecule.
7. DSC Melting temperatures, Glass transition temperature.
8. AFM Surface profile by means of cantilever reading.
9. POROSIMETER Measures the pore sizes of Nano fibers.

 Stem cells are cells found in all multicellular organisms, that
can divide (through mitosis) and differentiate into diverse
specialized cell types and can self-renew to produce more stem
cells.
Importance:
• Serve as internal repair system, dividing continuously to replenish
other cells.
• Therapeutic applications: Treat diseases like cancer, Parkinson's
disease, spinal cord injuries, Amyotrophic lateral
sclerosis, multiple sclerosis, and muscle damage.
Unique properties:
•Stem cells are capable of dividing and renewing themselves for long
periods
•These are unspecialized cells and can give rise to specialized cells

stem cell
What is a stem cell?
stem cell
(Identical to parent cell)
SELF-RENEWAL
(copying)
specialized cell
e.g. muscle cell, nerve cell
DIFFERENTIATION
(specializing)
A cell that has the ability to continuously
divide and differentiate (develop) into
various other kind(s) of cells/tissues of the
body.

1 stem cell
Self renewal - maintains
the stem cell pool
4 specialized cells
Differentiation - replaces dead or damaged
cells throughout life
What is the need of self-renewal and
differentiation?
1 stem cell

Stem cell Potential for differentiation
Potency A measure of how many types of specialized cell a stem cell
can make
Totipotent can give rise to a complete individual/ All cells of the body
Eg. Cells from early (1-3 days) embryos are totipotent
Pluripotent Can make all types of specialized cells in the body
Eg. Embryonic stem cells are pluripotent
Multipotent Can make multiple types of specialized cells, but not all types
Eg. Tissue stem cells are multipotent
Unipotent stem cells can produce only one cell type, their own
but have the property of self-renewal, which distinguishes
them from non-stem cells
Eg. muscle stem cells

Important characteristics of Stem
cells Self renewal- They are unspecialized cells capable of renewing
themselves through cell division, sometimes after long periods
of inactivity.
 Regeneration- They can be induced to become tissue- or organ-
specific cells with special functions. In some organs, such as the
gut and bone marrow, stem cells regularly divide to repair and
replace worn out or damaged tissues. In other organs, however,
such as the pancreas and the heart, stem cells only divide under
special conditions.

Types of stem cell:
1) Embryonic stem cells come from a five to six-day-old
embryo. They have the ability to form virtually any type of
cell found in the human body.
2) Embryonic germ cells are derived from the part of a human
embryo or fetus that will ultimately produce eggs or sperm
(gametes).
3) Adult stem cells are undifferentiated cells found among
specialized or differentiated cells in a tissue or organ after
birth. They appear to have a more restricted ability to produce
different cell types and to self-renew.
4) Induced pluripotent (iPS) stem cells are adult cells of the
body which are reprogrammed to show pluripotency.

embryonic stem cells
blastocyst - a very early
stage of embryo
tissue stem cells
fetus, baby and throughout life
cells inside
= ‘inner cell mass’
outer layer of cells
= ‘trophectoderm’

Embryonic stem (ES) cells:
Where we find them
blastocyst
outer layer of cells
= ‘trophectoderm’
cells inside
= ‘inner cell mass’
embryonic stem cells taken from
the inner cell mass
culture in the lab
to grow more cells
fluid with nutrients
differentiated into all possible types of specialized cells

Embryonic stem cells (ESC)
Embryonic stem cells (ES cells) are pluripotent stem cells derived
from the inner cell mass (ICM) of the blastocyst, an early-
stage embryo

Detail about Blastocyst: ESC source-
• The blastocyst is a structure formed in the early development of mammals. It
possesses an inner cell mass (ICM) which subsequently forms the embryo. The
outer layer of the blastocyst consists of cells collectively called the trophoblast.
This layer surrounds the inner cell mass and a fluid-filled cavity known as the
blastocoel. The trophoblast gives rise to the placenta.
• In humans, blastocyst formation begins about 5 days after fertilization, when a
fluid-filled cavity opens up in the morula, a ball consisting of a few dozen cells.
• The blastocyst has a diameter of about 0.1-0.2 mm and comprises 200-300 cells
following rapid cleavage (cell division). After about 1 day, the blastocyst embeds
itself into the endometrium of the uterine wall where it will undergo later
developmental processes, including gastrulation.
• The inner cell mass of blastocysts is a source of embryonic stem cells.

Embryonic stem cells
Characteristics:
 Self-renewal in an undifferentiated state for long period
 Maintenance of “Stemness” or pluripotent markes
 Formation of teratoma when induced in SCID mice
 Maintenance of normal karyotype
 Clonality
 Stem cell marker expression (NANOG, Oct4)
Clinical Research:
 Myocardium diseases: regeneration of damaged heart muscle by
injecting hESC–derived cardiomyocytes directly into the site of the
infarct [Laflamme MA et al., 2007]
 Lung disease: alveolar type II epithelial cells derived from hESCs
(ATIICs) in a nude mouse model of acute lung injury (Spitalieri P.
et al. 2012)
 Nervous system: oligodendrocyte progenitor cells (GRNOPC1)
drived from hESCs can improve functional locomotor behaviour
after cell implantation in the damaged site, seven days after injury
in animal model

ESC Culture in laboratory
 Human embryonic stem cells (hESCs) are generated by transferring cells from
a preimplantation-stage embryo into a plastic laboratory culture dish that
contains culture medium.
 The inner surface of the culture dish is coated with mouse embryonic skin
cells specially treated so they will not divide. This coating layer of cells is
called a feeder layer.
 Cells divide and spread over the surface of the dish.
 the plated cells divide and multiply and crowd the dish, then they are removed
gently and plated into several fresh culture dishes. This process of re-plating or
subculturing the cells is referred to as passage.
 Once the cell line is established, the original cells yield millions of embryonic
stem cells. Embryonic stem cells that have proliferated in cell culture for a
prolonged period of time without differentiating, and are pluripotent are
referred to as an embryonic stem cell line.

 Importance of Feeder layer: The mouse cells in the bottom of the culture
dish provide the cells a sticky surface to which they can attach. Also, the
feeder cells release nutrients into the culture medium.
 Disadvantage of feeder layer: there is always a risk that viruses or other
macromolecules in the mouse cells may be transmitted to the human cells.

Differentiation of Embryonic stem cells

Differentiation of ESCs
When removed from the factors that maintain them as
stem cells, ES cells will differentiate and, under
appropriate conditions, generate progeny consisting of
derivatives of the three embryonic germ layers:
mesoderm, endoderm, and ectoderm
General approaches of differentiation:
1. ES cells are allowed to aggregate and form three-
dimensional colonies known as embryoid bodies (EBs)
2. ES cells are cultured directly on stromal cells, and
differentiation takes place in contact with these cells
3. involves differentiating ES cells in a monolayer on
extracellular matrix proteins

Tissue stem cells
or
Adult Stem Cells

Tissue stem cells:
Where we find them
muscles
skin
surface of the eye brain
breast
intestines (gut)
bone marrow
testicles

Adult Stem Cells (ASCs)
Characteristics:
 Have potential to self-renew for a long time
 they can give rise to mature cell types that have characteristic
morphologies and specialized functions along multiple lineages
Types of ASCs:
 Hematopoietic stem cells
 Mesenchymal stem cells
 Other stem cells: Neural stem cells, Endothelial stem cells,
Intestinal stem cells, Olfactory adult stem cells, Mammary stem
cells
Sources:
Bone marrow, umbilical cord and cord blood, adipose tissue,
deciduous teeth, brain, peripheral blood, amniotic fluid and
membrane, synovium, placenta

3/3/2017 Dr. Hariom Yadav
Adult stem cells

MULTIPOTENT
blood stem cell
found in
bone marrow
differentiation
only specialized types of blood cell:
red blood cells, white blood cells,
platelets

Types of Adult stem cells
and hierarchies

Principles of renewing tissues
Stem cell
committed progenitors:
- “transient amplifying cells”
- multipotent
- divide rapidly
- no self-renewal
stem cell:
- self renew
- divide rarely
- high potency
- rare
specialized cells:
- work
- no division

Haematopoietic stem cells (HSCs)
HSC
committed progenitors
neutrophil
NK cell
erythrocytes
dendritic cell
plateletsmegakaryocyte
macrophage
eosinophil
basophil
B cell
T cell
specialized cells
bone marrow

Mesenchymal stem cells (MSCs)
MSC
bone marrow
Bone (osteoblasts)
Cartilage (chondrocytes)
Fat (adipocytes)
specialized cells

Neural stem cells (NSCs)
NSC
brain
committed progenitors specialized cells
Neurons
Interneurons
Oligodendrocytes
Type 2 Astrocytes
Type 1 Astrocytes

Gut stem cells (GSCs)
GSC
Small intestine
Paneth cells
Columnar cells
Goblet cells
Endocrine cells
specialized cells

Induced pluripotent (iPS)
stem cells

Induced pluripotent stem cells (iPS cells)
cell from the body
‘genetic reprogramming’
= add certain genes to the cell
induced pluripotent stem (iPS) cell
behaves like an embryonic stem cell
Advantage: no need for embryos!
all possible types of
specialized cells
culture iPS cells in the lab
differentiation

Induced pluripotent stem cells (iPS cells)
cell from the body (skin)
genetic reprogramming
pluripotent stem cell
(iPS)
differentiation

Transdifferentiation
 Certain adult stem cell types can differentiate into cell
types seen in organs or tissues other than those expected
from the cells' predicted lineage. This reported
phenomenon is called transdifferentiation
 Example - brain stem cells that differentiate into blood cells
or blood-forming cells that differentiate into cardiac
muscle cells.

Stem cells at home:
The stem cell niche

Stem cell niches
Direct contact Soluble factors Intermediate cell
stem cell
niche
Niche
Microenvironment around stem cells that provides
support and signals regulating self-renewal and
differentiation

Stem Cells in Tissue
Engineering

Challenges in Stem cell
research
 It is uncertain that human embryonic stem cells in
vitro can give rise to all the different cell types of the
adult body.
 It is unknown if stem cells cultured in vitro (apart from
the embryo) will function as the cells do when they are
part of the developing embryo.
 Stem cell development or proliferation must be
controlled once placed into patients.
 Possibility of rejection of stem cell transplants as
foreign tissues is very high.
 Contamination by viruses, bacteria, fungi, and
Mycoplasma possible.

Cell seeding on 3D scaffold
It is process of dissemination of isolated cells within a
scaffold
 To maximize the utilization of donor cells
 To improve proliferation
 To minimize time in suspension culture for anchorage-
dependent and shear-sensitive cells
 To achieve high cell density and uniform cell distribution
Methods of cell seeding:
1. Static method
2. Dynamic method

Static seeding:
•Sterilization of
scaffold
•Trpsinization of
cells to be seeded
SEM image of cell
seeded scaffold
Disadvantages:
•Low seeding efficiency
•Non-uniform cell
distribution within scaffold
•Ineffective convection
Incubate at 370C in
humidified incubator
Incubate cell suspension
of concentration (7-
15x104) on scaffold
Incubate seeded scaffold
for 2 hr for attachment
and add media

1. Gravitational Seeding
2. Centrifugation Seeding
3. Low Pressure Seeding
4. Magnet-Assisted Seeding

a) Gravitational Seeding
- It is simply depositing cell suspension on top of the
scaffold and allowing the cells to settle by gravity, and
subsequently attach to the surface.
- Commonly known as Static Seeding.
- Advantage: Simple method
- Disadvantage: low efficiency and penetration

b) Centrifugation Seeding:
- In this method cells are seeded by applying a centrifugal force to the scaffold to
assist the penetration of cells.
- Advantages: better cell insertion and packing, as well as a more uniform
distribution .
- Disadvantages: difficulties in controlling scaffold orientation during seeding,
and effect of centrifugal forces on cell function.
c) Low Pressure Seeding:
- This method involves placing the desired cell population and the scaffolds into
sterile vacuum desiccators and applying vacuum to lower pressure in the chamber
in order to remove air from the scaffold and so enhance cell entry into the
scaffold.
- Advantage: ease of use and application to multiple types of porous scaffolds
- Disadvantages: cell function may be changed due to low pressure atmosphere and
their exists chances of genetic mutation.

d) Magnet-Assisted Seeding:
- To enhance the entry of cells into a porous scaffold, magnetic particles are
attached to the desired cell population and a magnetic force is applied across the
scaffold to physically pull the cells into the pores.
Process:
- The desired cell population is separated from a heterogeneous mixture by the use
of magnetic nanoparticles. In this process, the desired cells are characterized by
particular surface receptors which are then conjugated to magnetic particles such
as supermagnetic iron micro or nano beads.
- These nanoparticle-conjugated cells are then seeded onto the scaffold and a
magnet is placed below this system.
- The nanoparticle-conjugated cells get attracted (pulled) towards the magnet and
align themselves in the scaffold surface accordingly.

 Advantages: Increased efficiency of scaffold seeding and selectivity of the
desired cell type if antibodies are used to attach the magnetic particles to the
cells.
The ability to manipulate cells without direct physical contact and at a
distance, as well as the ability to localize the cells in one area
 Disadvantages: Possibility of nonspecific binding of magnetic particles to
undesired cells
Application of magnetic particles and subsequent force can change the levels of
gene expression in the target cells.
Top view of the scaffold showing
pattern arrangement of cells
Magnetic
nanoparticle
labeled cells
Porous Scaffold
Magnet
Incubation

Example of Magnet assisted seeding and its application in tissue engineering
Blood vessel development by seeding magnetic nanoparticle labeled smooth muscle cells
(SMCs) and Human umbilical vein endothelial cells (HUVECs)

References Dai, W et al. “Application of low-pressure cell seeding system in tissue
engineering.” BioScience Trends. 2009; 3(6): 216-219.
 Dar, A et al. “Optimization of cardiac cell seeding and distribution in 3D porous
alginate scaffolds.” Biotechnol Bioeng. 2002; 80(3): 305-12.
 Sasaki, T et al. “Magnetic nanoparticles for improving cell invasion in tissue
engineering.” J Biomed Mater Res A. 2008; 86(4): 969-78.

Dynamic seeding
Methods:
 Rotator or shaker
 Spinner flask
 Perfusion flow
 Rotational vacuum
Advantages:
 Higher seeding efficiency and uniform cell
distribution
 Convective transport for seeding

Dynamic seeding in various bioreactors:

Bioreactors for Tissue Engineering
 A tissue engineering bioreactor can be defined as a
device that uses mechanical means to influence
biological processes
 Enhance interaction between scaffold, cells and signalling
mechanism
 Forces: Fluid flow, compressive, shear, rotational forces and
magnetic forces, hydrostatic pressure
 It provides mechanical stimulus to produce ECM in shorter
time and homogeneous
 Important in cellular differentiation: to encourage stem
cells down a particular path
 To improve cellular spatial distribution

A systems view of tissue engineering processes

Types of bioreactors
 Spinner flask
 Rotating vessel
 Perfusion system
 Hollow fibre bioreactor

Spinner flask
Cells seeded on 3D scaffold are
suspended via wire in large volume of
culture medium.
Typically, spinner flasks are around
120 mL in volume (although much
larger flasks of up to 8 litres have
been used), Culture medium is
stirred using a magnetic bar at a
typical rotation of 50r/min.
Advantages:
•Reproducible and easy to use
•Improve cell viability, proliferation
and distribution throughout construct
Limitations:
•Nutrient diffusion
•Application of shear stress
Spinner flask

Rotating Vessel
 Rotating wall-vessel (RWV)
designed by Schwarz and colleagues
at NASA Johnson space centre.
 They based the bioreactor on two
basic design principles: (1) solid
body rotation (2) a silicone rubber
membrane for oxygenation.
 The solid body rotation is a vessel
that rotates horizontally and is
filled with culture medium.
 This method simulates some
aspects of microgravity by reducing
shear and turbulence associated
with stirred bioreactors.
Types:
Slow turning lateral vessel
(STLV)
High-aspect ratio vessel
(HARV)

Perfusion Bioreactor
 Perfusion bioreactors have been used to deliver cells to a
3D engineered construct via controlled flow, which
reverses back and forth within the construct
 In flow perfusion culture, the culture medium is forced
through the internal porous network of the scaffold. This
can mitigate internal diffusional limitations present in 3-D
scaffolds to enhance nutrient delivery to and waste removal
from the cultured cells.
 Flow applys mechanical stress to the cultured cells.
 The enhanced mass transfer, homogeneous cell
distribution and high seeding efficiency

Perfusion Bioreactor
a) Flow perfusion culture
b) The cassette with scaffold is sealed in place
by two neoprene O-rings above and below
the cassette
c) This three-part assembly (cassette and
two O-rings) is then held in place by a
Plexiglas screw top. Silicone tubing then
connects each of these flow chambers to the
pump and reservoir systems.

Hollow fibre bioreactor
 A hollow fibre bioreactor consists of a bundle of
hollow fibres encased in a cylindrical shell with ports
for flow of media around the fibres.
 It is a two compartment module with an intracapillary
and an extracapillary space

Criteria to define MSCs
1. Adherence to plastic
2. Specific surface antigen (Ag) expression
3. Multipotent differentiation potential

Adherence to plastic
 MSCs adhere to plastic tissue culture dish or flask
surface and attain flattened morphology.
Un adhered/ floating cells Adhered MSCs
Round morphology Flatened and elongated
structure

Specific surface antigen (Ag) expression
203
Analysis by Flow Cytometry (for MSCs)
Positive
expression
CD105 endoglin
Originally recognized by the MAb
SH2
CD73 ecto 5’ nucleotidase
Originally recognized by the MAb
SH3 and SH4
CD90 known as Thy-1
Negative
expression
CD45 pan-leukocyte marker
CD34 marks primitive hematopoietic
progenitors and endothelial cells
CD14 Prominently expressed on monocytes
and macrophages (the most likely
hematopoietic cells to be found in an
MSC culture)
CD19 markers of B cells that may also
adhere to MSC in culture and remain
vital through stromal interactions

Multipotent differentiation
potential
MSCs are able to differentiate into multiple lineages
including-
 Osteocytes
 Chondrocytes
 Adipocytes
When cultured in specific differentiation media

Cell Attachment & Morphology
SEM analysis
Observing the images of cells grown scaffold surface provides
information about cellular morphology, attachment and growth.
Process:
•Cell seeded scaffold are taken and washed with PBS (Phosphate
buffer saline).
•Then cells are fixed using freshly prepared 2% glutaraldehyde
solution.
•Next step is dehydration which is performed by washing with 35%,
50%, 70%, 90% and 100% ethanol gradient for 5 min each.
•Then the constructs are coated with gold or platinum by sputter
coating and observed under SEM (Scanning Electron Microscopy)

SEM images of cell seeded scaffold

Cell viability
MTT Assay or Cytotoxity Assay:
The MTT Cell Proliferation Assay measures the cell proliferation rate thus gives
indication of cellular viability when seeded on scaffolds.
MTT- 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide
Principle:
The yellow tetrazolium MTT is reduced by metabolically active cells, by the action
of dehydrogenase enzymes, to formazan. The resulting intracellular purple formazan
can be solubilized and quantified by spectrophotometry.
Process:
•Small disks of scaffolds seeded with cell suspension (~4 × 105 cells/ml approx).
•After incubation, MTT solution (5 mg/ml) is added to the culture well and
incubated for 4hr. Colour changes from pink to purple.
•Then purple colour precipitate of Formazan is solubilized by adding Dimethyl
sulfoxide (DMSO).
•The mitochondrial activity is measured by taking spectrophotometric reading at
595nm.

Cell Proliferation assay
Alamar blue assay
Principle:
Alamar Blue Reagent is a non-toxic, water-soluble resazurin
dye that yields a fluorescent signal and a colorimetric change
when incubated with metabolically active cells. Absorbance at
570nm and 600nm yields cell proliferation rate.
Process:

DNA Quantification
 Amount of DNA in a sample gives indication of cell growth on the
scaffold.
 This can be assessed Quantitatively and qualitatively.
 Quantitative- by Real time PCR analysis, which directly measures
amount of target gene in the sample.
 Qualitatively- using Hoechst dye (for labelling DNA) followed by
fluorometric analysis to yield count of cells showing fluorescence.
Cell Proliferation assay

Live dead cell count
Fluorescence
Cells are fixed on scaffold using 1.5% paraformaldehyde solution.
Then various dyes are used to stain different cellular components. These are
then examined under Fluorescence or confocal microscope to yield images
according to stains used.
Dye name Stains
Calcein-AM Live cells
EtBr (Ethidium Bromide) Dead cell nucleus
Phalloidin Cell cytoskeleton
DAPI Nucleus of live cells
Propidium Iodide (PI) Dead cell staining
Hoechst Nucleus of live cells
MitoRed Mitochondria

Flow Cytometry
 Principle:
Cells (or other particles) are illuminated as they flow
individually in front of a light source and then get detect and the
signal from those cells is correlated that result from the
illumination.
 Applications:
1. Cell counting: Each event of illumination is counted as one
cell and thus total events give number of cells in the
suspension.
2. Cell sorting: When cell suspension is ejected into air, it
will form droplets a droplet containing a cell is applied
either a negative or positive charge and sorted by passing
through an electric field.

Types of Interactions
 Scaffold influences cell viability, growth, function and motility.
 Types of cellular interaction under influence of scaffold
1. Adhesion
2. Migration
3. Aggregation

1. Adhesion
 Most tissue derived cells require attachment to a solid surface for viability
and growth.
 Cell adhesion to a surface is critical because it is followed by other
important phenomena like cell spreading, migration and differentiated cell
function.
 Phenomena
i. Cell attachment
ii. Cell spreading
iii. Focal adhesion
 Techniques used to determine cell adhesion are-
1. Sedimentation-detachment assay
2. Centrifugation assay
3. Fluid-flow chambers

i. Cell attachment- cells attach to the surface of
the scaffold and form monolayer on the
scaffold
ii. Cell spreading- surface attached cells divide
and proliferate to cover the surface of the
scaffold. The cells also penetrate inside the
interconnected pores of scaffold.
iii. Focal adhesion- Focal adhesions are large,
dynamic protein complexes through which the
cytoskeleton (protein present in the cell outside
the cytoplasm e.g intigrin, actin, myosin) of a
cell connects to the extracellular matrix
(scaffold).

Techniques to determine cell adhesion
 Sedimentation-detachment assay
i) sedimentation of cells onto a surface
ii) incubation of the sedimented cells in
culture medium for some period of time
iii) detachment of loosely adherent cells by
removal of the culture medium and
repeated washing
 The extent of adhesion is determined by
the number of cells that remain associated
with the surface or the number of cells
that were extracted with washes.

 Centrifugation assay
i) Seeding of cells onto a scaffold surface
ii) incubation of the cells in culture medium
for some period of time
iii) the plate is inverted and subjected to a
controlled detachment force by
centrifugation.
The extent of cell attachment is then
quantified

 Fluid-flow chambers
Fluid mechanical forces are utilized to produce cell detachment in a
well-controlled and quantifiable manner.
i) Cell suspension is injected into the chamber, and the cells are
permitted to settle onto the surface of scaffold and adhere.
ii) After incubation the fluid is forced between two parallel plates and
non adherent cells are removed with the flow of fluid, while adherent
cells remain on the surface, which can be quantified.

2. Migration
 Migration of individual cells within a tissue is critical for formation
of the architecture of organs.
 In tissue engineering, the ability of cells to move, in association with
scaffold surface or through other cells, will be an essential part of
new tissue formation or regeneration.
 Techniques used to determine cell migration are-
1. Under agarose test
2. Filter assaying
3. Direct visualization

Techniques to determine cell migration
 Under agarose test
i) a cell suspension is placed in a well of
semisolid agarose
ii) motile cells crawl on the solid substrate
underneath agarose.
 Filter Assay
i) cell suspension is placed on a filter with small
pores
ii) motile cells crawl through the pores of the filter
material to the other side, where they are
detected.
 Direct visualization assays
- the paths of movement of many individual cells are
directly observed for cells migrating on
surfaces and within solid gels

3. Aggregation
 Important in tissue development
 It correlates cell-cell interaction with cell differentiation, viability
and migration for subsequent tissue formation.
 Aggregate morphology allows re-establishment of cell-cell contact
in tissues, thus cell function and survival rate are enhanced in
aggregate culture.
 Formation of aggregates-
by incubating cells in suspension and adding serum proteins to
promote cell aggregation.
 Techniques used to determine aggregation are-
1. Direct visualization
2. Electronic particle counter
3. Aggregometers

Techniques to determine aggregation
1. Direct visualization –
Monitoring aggregate size to determine extent of aggregation
2. Electronic particle counter -
Invented by Moscona, determines kinetics of aggregation by
measuring aggregate size distribution over time.
This procedure utilizes computer image analysis to follow
disappearance of single cells with time.
3. Aggregometers -
Small angle light scattering through rotating sample cuvettes are
used to produce continuous record of aggregate growth.

From Chapter 4
Book- Tissue Engineering Principles for the design of
replacement organs and Tissues
W. Mark Saltzman

Fundamental of Tissue engineering

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