18BTE414E-Biomaterials in

Tissue Engineering

UNIT-I – Part 1
 Biomaterial - Introduction
• In the broad sense of the word, natural or
synthetic materials used to support or completely
take over the function of the nonfunctional
tissues in the human body are called
biomaterials.
• The complete or partial replacement of damaged
or diseased organs and tissues have improved the
quality of life and prolonged the average life
expectancy, and this further increased the
interest in the biomaterials field.
• Even though the biomaterials discipline is a
new interdisciplinary field, its applications
date back to thousands of years BC. The glass
eyes, metal noses, and ivory teeth discovered
on the Egyptian mummies are good examples
Types of Biomaterials
Metals - Biomaterials
 Biomaterials Companies
• BioForma Research & Consulting, Inc., fibrinolytic systems, protein-material interactions

• Baxter International develops technologies related to the blood and circulatory system.

• Biocompatibles Ltd. develops commercial applications for technology in the field of biocompatibility.

• Carmeda makes a biologically active surface that interacts with and supports the bodys own control mechanisms

• Collagen Aesthetics Inc. bovine and human placental sourced collagens, recombinant collagens, and PEG-polymers

• Endura-Tec Systems Corp. bio-mechanical endurance testing of stents, grafts, and cardiovascular materials

• Howmedica develops and manufactures products in orthopaedics.

• MATECH Biomedical Technologies, development of biomaterials by chemical polymerization methods.

• Medtronic, Inc. is a medical technology company specializing in implantable and invasive therapies.

• Molecular Geodesics Inc., biomimetic materials for biomedical, industrial, and military applications

• Polymer Technology Group is involved in the synthesis, characterization, and manufacture of new polymer products.

• SurModics, offers Photo Link (R) surface modification technology that can be used to immobilize biomolecules

• W.L. Gore Medical Products Division, PTFE microstructures configured to exclude or accept tissue in growth.

• Zimmer, design, manufacture and distribution of orthopaedic implants and related equipment and supplies
 Properties of Biomaterials
1. Be biocompatible (nontoxic, non-carcinogenic,
non-allergenic, etc.)
2. Have physical properties (e.g., density, form, porosity,
surface roughness topography) comparable to those of the
tissue it replaces or is implanted in
3. Have appropriate mechanical properties (compressive,
tensile, shear, impact)
4. Have appropriate service lives (stable for life or degrade
within a matter of days or weeks depending on the goal)
5. Have chemical properties similar to that of tissues (e.g.,
hydrophilic or hydrophobic, have similar functional groups)
6. Be processable and sterilizable without difficulty
7. Have appropriate bioactivity (mostly inert, but could have
induction or conduction activities or carry bioactive agents if
needed)
8. Be economical and available
 Natural Biopolymer
• Cellulose
• collagen
• hyaluronic acid
• chondroitin sulfate
• chitosan
• microbial polyesters
• silk
• alginate
 Synthetic polymer
• Polymethyl methacrylate (PMMA)
• polyvinylchloride (PVC)
• Teflon
• Dacron
• Nylon
• poly(L-lactide) (PLLA)
• polyhydroxybutyrate-valerate (PHBV)
• polydimethylsiloxane (PDMS)
• Polyurethanes
• polyethylene (PE)
• polypropylene (PP)
• polytetrafluoroethylene (PTFE)
 Ceramics
• Aluminum oxide
• titanium dioxide
• hydroxyapatite
 Composites
• Composites are combinations of two or more
materials which form an integrated structure
combining the properties of its components to
produce a much improved product.
– Metal-ceramic
– polymer-ceramic
– metal-polymer
 Fibers
• A polymeric rod can easily be made by melting and extrusion at
relatively low temperatures.
• Metals on the other hand require a much higher temperature to be
processed into a fiber.
• Ceramics are the most difficult to process into fibers because of the
ionic nature not allowing the ceramic to flow at low temperatures.
They are produced by drawing from the melt, by spinning, or by
extrusion.
• Some important ceramic fiber types are alumina, magnesia, zirconia,
silicon carbide, and carbon fiber.
• As ceramics are crystalline and strong under compression, so are the
fibers. They withstand high temperatures and forces but are brittle
unless made into composites.
• Polymeric fibers are easily prepared by extrusion, drawing from melt
or solution. The temperatures needed are not excessive since most
polymers melt at around or below 200 °C.
• In the daily life of the humans, polymeric fibers find more use. Some
of the important ones are nylon, polyesters, aramides (e.g., Kevlar),
and polyurethane fibers (Spandex).
 Sheets
• Obtaining sheets of polymers, metals, and
ceramics is not as difficult as fiber forming
• The processing conditions are more easily
attainable because they do not require high
technology
• For polymers in addition to melt compression,
polymerization in sheet form and casting from
solutions are the methods available.
• Ceramic sheets are more difficult to obtain
because of the stringent conditions under which
the ceramics are processed.
 Foams
• Foams or sponges are very frequently used in the
biomaterials field
• The most common use is in tissue engineering where cells
of the targeted tissue or stem cells are seeded onto these
porous structures which preferably are biodegradable.
• Interconnectedness of the pores is required so that the cells
growing in the pores can move around, fully occupy the
structure, and modify the environment to suit their
biological needs.
• Some other applications where porosity is needed are the
interfaces where the integration of the biological system
with the implant is desired.
 Spherical Biomaterials

• Spherical biomaterials are called spheres or

capsules depending on whether they are full or
hollow in the center.
• Spherical biomaterials are especially important in
drug delivery applications where the micro- or
nanospheres or capsules are loaded with
bioactive agents and then introduced to the body.
• Micro- and nano-size, targeted drug delivery
vesicles are commonly used for cancer therapy.
 Tubular Biomaterials

• Biomaterials can also be processed in the form of

tubular structures.
• A wide variety of applications employ tubular
biomaterials as catheters, cannulas, tubes of
extracorporeal devices, nerve guides, drug eluting
systems with impermeable walls.
• On the other hand, stents and vascular grafts are
among the semipermeable tubular biomaterials
used in the treatment of a variety of impairments.
 Biomaterials with Engineered
Surfaces
• Engineered surfaces having a certain design and surface
topography or functionalized by tethering certain
molecules are important as biomaterials.
• The material surfaces having micro- or nano-designed
decorations are used to study cell-biomaterial
interactions and modified to improve implant-tissue
attachment properties.
• The ability to modify surfaces at such low dimensions is
a relatively recent capability learned from the
developments in the microelectronic industry.
• Biomaterials are also modified by immobilizing active
molecules onto these surfaces in order to create
antibacterial or antithrombogenic implants or surfaces
with enhanced cell adhesion capacity.
 Metals – General
properties
• Metals are generally hard, opaque, shiny, malleable, ductile, and
conductive materials.
• Metals can form alloys by mixing with other metallic elements at the
molecular level. The main purpose of forming alloys is to enhance some
properties of the metal such as make it less brittle, harder, and more
resistant to corrosion or have a more desirable color and luster.
• Due to their higher density and strength compared to polymers, metals
and metal alloys are extensively used as surgical and dental instruments,
biomedical devices, implants, joint replacements, and skull plates.
• Among the most common types of medical implants are pins, rods,
screws, and plates used to anchor fractures, and they are mostly made
from metals.
• As every medical device and implant, metals used in the clinics should
fulfill some requirements, such as they should have high biocompatibility,
high mechanical strength, high wear resistance, and high corrosion
resistance.
• Metals are preferable in the production of
load-bearing devices such as hip joints and femur
plates, due to their high modulus of elasticity and yield
strength.
• One of the most well-known alloys of metals is
stainless steel which contains iron mixed with
chromium, nickel, molybdenum, and carbon and also
commonly used in medical applications.
• Metals currently used in manufacturing implants
mostly contain Fe (iron), Cr (chromium), Co (cobalt), Ni
(nickel), Ti (titanium), Ta (tantalum), Mo
(molybdenum), V (vanadium), and W (tungsten).
• Metallic implants are used for two primary purposes:
one of them is used to replace a portion of the body
such as joints, long bones, and skull plates, and the
other is used as fixation devices to stabilize the broken
bones.
 Medical application of Metals
• Stainless steel - Internal fixation materials (bone
plates, screws, pins, nails, etc.) Total hip implants
• Cobalt alloys - Total joint implants Dentistry
• Titanium alloys - Total hip implants (stem and cup)
Dental implants Pacemakers
• Nickel-titanium alloys (nitinol) - Orthodontic dental
arch wires Vascular stents Catheter guide wires
Orthopedic staples and clips
• Magnesium alloys - Biodegradable implants
• Tantalum alloys - Wire sutures Radiographic markers
 Stainless steel
• Stainless steel is a very strong iron alloy, and its first
introduction to humans for fracture treatments has started
at the beginning of the 1900s.
• Stainless steel is most often used in implants that are
intended to repair fractures, such as orthopedic implants,
joint replacements, surgical and dental instruments, bone
plates, bone screws, pins, rods, and coronary stents
• Stainless steel is mostly iron and contains other metals such
as chromium (at least 10.5%, w/w), cobalt, molybdenum,
and carbon (less than 1.2%, w/w) which are added to make
it more resistant to corrosion.
• Stainless steel is preferred for plates and screws, but not for
weight-bearing implants and for extended periods.
 Cobalt-Chromium Alloys
• Cobalt-chromium (Co-Cr) alloys have two basic elements, up
to 65 w/w % Co and 35 w/w % Cr. Molybdenum (Mo) can be
also added to obtain finer grain sizes which result in higher
strength after casting or forging.
• These alloys have high strength, temperature endurance,
and wear resistance, and therefore, they are used in a variety
of joint replacement implants, as well as in some fracture
repair implants that require a long service life.
• These alloys are among the most widely used metals in knee
implants. Co-Cr alloys are especially useful where high
stiffness or a highly polished and extremely wear-resistant
material is required.
• Commonly used areas are dental and orthopedics, as
cemented total hip or in knee arthroplasty
 Titanium Alloys

• Titanium and its alloys are mostly preferred due to

good mechanical strength, relatively low density,
excellent corrosion resistance, and remarkable
biocompatibility.
• Titanium and titanium alloys have great corrosion
resistance, making them inert biomaterial .
• The most used titanium alloy in knee implants is
Ti-6Al-4V which is Grade 5 and has aluminum and
vanadium contents of 6 and 4%, respectively.
• The major limitation of titanium is its chemical
reactivity with other materials at elevated
temperatures.
• Titanium-based alloys have excellent properties for
use in porous forms for biological fixation of
prostheses
 Tantalum

• Tantalum is a pure metal which has a remarkable resistance to

corrosion, and excellent physical and biological characteristics. It is
a very hard, malleable, ductile, and one of the most unreactive
metals.
• Tantalum is used in artificial hips, knees, and other joints. Pins,
screws, staples, and other devices used to hold bones together are
also made of tantalum alloys.
• Tantalum based sheets, plates, rods, and wires are also used in the
production of prosthetic devices for humans such as skull plates,
meshes to repair the bone defects after cancer surgery, suture
clips, and stents for blood vessels.
• Because of its high density (16.6 g/cm3), it has been used in
radiography as a marking agent for diagnostic purposes.
• Tantalum can be fabricated in a highly porous form which has a
modulus of elasticity closer to that of the bone than stainless steel
or the cobalt-based alloys.
 Nickel-Titanium Alloy (Nitinol)

• This is a shape memory alloy of nickel and titanium, where

the two elements are present almost in equal atomic ratio.
• It has been used in the manufacture of endodontic
instruments in recent years.
• Nitinol alloys have greater strength and a lower modulus of
elasticity compared with stainless steel alloys.
• These properties are of interest in endodontology as they
allow construction of root canal instruments that utilize
these favorable characteristics to provide an advantage
when preparing curved canals.
• Nitinol is used to manufacture devices for dental,
orthopedic, and cardiovascular applications. The most
commonly used devices are catheter tubes, dental files,
guide wires, arch wires, gallstone retrieval baskets, filters,
needles, and other surgical instruments
 Magnesium-Based Biodegradable
Alloys
• Magnesium (Mg) is well known for its light weight
(density is 1.7 g/cm3) and biodegradability.
• Density, elastic modulus, yield strength, and
fracture toughness are close to that of the bone.
• In order to improve corrosion resistance, a
variety of elements such as Al, Zn, Mn, and rare
earth elements were alloyed with magnesium.
• Magnesium alloys showed improved corrosion
properties when compared to those of pure Mg.
 Advantage of Metals
• High strength
• Fatigue resistance
• Wear resistance
• Ease of fabrication
• Ease of sterilization
• Economical
 Disadvantage of Metals
• Corrosion
• High density
• High modulus
• Metal ion sensitivity
• Toxicity
 Techniques for modification of metal
implants
• TPS (titanium plasma spray): At very high speeds and very high
temperatures, titanium particles (40 μm and smaller) are sprayed
on the surface of the titanium implant producing a rough titanium
surface into which the bone tissue newly deposited by the cell can
penetrate and attach.
• SLA (sand-blasted, large grit, acid-etched): Sand particles are
sprayed on the implant surface to make macro grids and acid
etched to produce micro-scaled grids (2–4 μm). Usually
hydrochloric acid or sulfuric acid is used for the etching process.
With SLA technique, large, medium, or small grid rough surfaces are
obtained.
• Electropolished (oxidized): The surface of the implant is anodized.
Electrochemical anodic oxidation is performed on titanium surface.
With this technique, micro- pits are formed on the implant surface.
• TiO2 grit-blasted: In this method, TiO2 is blasted on implant
surface with several ways such as plasma spray.
• Machined surface: In this method, the surface roughness is created
by machining of the surface.
 Ceramics – General properties
• Ceramics are inorganic materials that are composed of
metallic and nonmetallic elements which are bonded to
each other with ionic or covalent bonds.
• Ceramics are produced from materials in powder form by
application of heat (sintering). They are hard, strong, and
brittle.
• There are numerous combinations of the metallic and
nonmetallic compounds, and the most commonly known
nonmetallic groups are oxides, hydrides, carbides,
phosphates, sulfides, and silicates. Aluminum oxides,
calcium phosphates, and titanium nitrides are in this class.
• Ceramics are now extensively used in dentistry, in the
production of orthopedic implants for the spine, and
particularly in total hip implants due to their resistance
against compression and wear
 Classification of Ceramics
• Clay: These are the naturally occurring ceramics that contain
minerals in the form of fine powders and have a certain plasticity
e.g., kaolinite.
• Glasses: This type of ceramics can be amorphous glasses
(silica-based ones) or crystalline glasses (e.g., the polycrystalline).
• Cements: These ceramics are used as binders to hold two or more
hard body components together (mostly calcium silicates).
• Refractories: These are the types of ceramics which are very
difficult to fuse, corrode, or draw. They can resist high
temperatures and cannot be melted for processing or shaping (e.g.,
alumina, silica, high purity oxides, graphite).
• Abrasives: These ceramics are very hard and are therefore used for
polishing purposes (e.g., diamond, silicon carbide, silica sand).
• Advanced ceramics: These ceramics are produced in different
forms for certain applications to carry the desired properties (e.g.,
nanotubes, fibers, particles) and are basically crystalline materials
with rigorously controlled composition.
 Manufacturing of Ceramics
• In the production of all types of ceramics, the main materials are in powder
form; in addition there are additives such as binders and stabilizers.
• After mixing and forming a homogeneous paste, a desired shape is given
by pressing, extruding, injection molding, or casting. Then the shape
obtained is fired (sintered) at high temperatures (ca. 1800–2000 °C), where
the particles melt and fuse and form a hard, dense material.
• Simple shapes using ceramics can also be produced by hot pressing where
forming and firing steps are combined. In this case, the mix of powders are
heated and pressed simultaneously so that the final form is achieved under
pressure.
• Another method is chemical vapor deposition (CVD) where the precursor
gases are deposited on the heated substrate under controlled temperature
and pressure. Some ceramics such as silicon carbide (SiC) or silicon nitride
(Si3N4) with superior physical properties can be produced in this fashion.
• Porous ceramics can be produced by applying reaction bonding technique
in which the powders are linked to each other by linkers via chemical
reactions. After giving the shape, the binders are burned, and their places
remain as pores.
 Advantages of Ceramics
• Biocompatibility
• Good degradation resistance in corrosive
environments
• High compression strength and moduli
• Superior hardness
• Wear resistance in comparison with metals.
• Depending on the nature of the tissue
attachment mechanism, ceramics are divided
into three subclasses:
• Bioinert ceramics - alumina, zirconia and
pyrolytic carbon
• Bioactive ceramics - hydroxyapatite (HA),
bioglasses or glass-ceramics
• bioresorbable ceramics - tricalcium
phosphate (TCP).
 Bioinert oxide-based ceramics
• Bioceramics have many superior properties such as high compressive
strength, wear and corrosion resistance, and ability to serve as a polish or
abrasive agent, and they can be prepared to be bioinert or bioactive. On
the other hand, they also have some disadvantages such as high modulus
of elasticity, low tensile strength, low fracture toughness, and difficulty of
fabrication.
• Bioinert bioceramics do not lead to chemical or biological reactions in the
biological media and therefore can maintain their physical and mechanical
properties in the host. They have reasonable fracture toughness and
are generally used as structural support implants such as bone plates,
bone screws, and femoral heads
• Alumina is one of the most commonly used bioinert, biocompatible and
highly stable bioceramic. It has low fracture toughness, high compressive
strength, high hardness, and high abrasion and wear resistance. Alumina
coatings yield smooth surfaces. Meanwhile, it may have a problem in
adhering to the tissue interface and therefore may lead to interfacial
loosening
• Its application areas include orthopedics such as the femoral head,
joint, knee prosthesis, bone screws and plates, porous coating for
femoral stems, dental crowns, bridges, and dental implants.
• Zirconia, like alumina, is one of the inert bioceramics. It is
obtained from mineral zircon, a gemstone of many colors. Zircon
is the most popular form of the zirconium mineral, and it turns
into zirconium when it is chlorinated twice and then precipitated
with sulfides or hydroxides, and finally it is calcined to its oxide.
• Zirconia has several advantages over other ceramic materials due
to the transformation toughening mechanisms operating in their
microstructure that can be detected in components made using
them.
• It is produced generally by hot press or by hot isostatic press
processes. Zirconia is generally used in orthopedic applications
such as femoral head, joint replacement, artificial knee, screws
and plates, as well as dental crowns and bridges
 Calcium Phosphate Ceramics (CPC)
• Calcium phosphate is naturally present in bone structure,
and therefore calcium phosphate salts were successfully
applied in replacing and augmenting bone tissue for many
years.
• Calcium phosphate can be in many different forms, and the
most widely used calcium phosphate-based bioceramics in
medical applications are hydroxyapatite (HAp) and
β-tricalcium phosphate (β-TCP).
• HAp ceramics react with the ions present in the body fluid
and form a surface apatite coat which induces protein
adsorption and cell attachment and lead to bone formation
and resorption of the biomaterial.
• Calcium phosphate ceramics can be prepared as powders,
tissue engineering scaffolds, self-setting bone cements, and
as coating materials for metals and heart valves to prevent
blood clotting.
 Bioactive Glasses (Glass ceramics)
• Bioactive glasses are generally silica-based having SiO44−
groups, and have a place in orthopedic and dental
prosthetic applications. They can be prepared as dense or
porous glasses.
• They have excellent mechanical properties. Studies carried
out both in vivo and in vitro have shown that many
bioactive glasses are nontoxic.
• Bioactive glasses form strong chemical bonds with tissue
and are therefore used in fixation of implants in the skeletal
system.
• Ions present in the body fluids are mineralized on the
surface of the bioglass implants and stimulate osteoblast
adhesion, differentiation, and bone growth.
• Depending on the combinations of silicon, calcium, and
sodium oxide levels, the properties of the bioglass varies
significantly and can be adjusted for a specific targeted
application.
 Polymers
• Polymerization can proceed according to two different mechanisms, chain
growth (or addition) and step growth (or condensation) polymerization.
• A distinction can be made between condensation and addition
mechanisms of polymerization; in condensation polymerization, two
functional groups bond with each other, generally by releasing a small
molecule such as H2O.
• While in addition polymerization, double bonds of monomers react
without releasing any molecule.
• The most important group of chain growth polymerizations is a
combination of vinyl monomers such as ethene (ethylene), propene,
styrene, and vinyl chloride which form polyethylene, polypropylene,.
• In any biomaterial application, the composition and structure of a
candidate biomaterial relevant to the properties under consideration
should be evaluated. The thermal properties of a biomaterial are very
important as the physical, electrical, mechanical, chemical, and biological
properties. Thermal properties provide information on stability of form,
effect of sterilization methods, processing and shaping, and storage
Unit 1

Part 2
 Evaluation of Biomaterials

• In-vitro Evaluation

• In-vivo Evaluation
 Material irritation, systemic toxicity,
hemocompatibility,
Biocompatibility carcinogenicity

oduct and Process

Validation

' periodic audit testing, release

testing
When lacking biocompatiblity ...

../ long-lasting chronic inflammation

../ cytotoxic chemicals

../ disruption of cells at interface

../ micron-sized materials

../ irritation

../ corrosion of meta Is

../ restenosis/thrombosis
Ian of biocompatibility tests

in vitro tests Clinical tests

Evaluation und er in vitro (literally "in glass") conditions

an provide rapid and inexpensive data on biological

Will the in vitro test measure parameters relevant to what

ill occur in the much more complex in vivo environment?

ln vitro tests minimize the use of animals in research, a

esirable goal
 hen appropriately used, in vitro testing provides useful
sights that can dictate whether a device need be further
valuated in expensive in vivo experimental models

he common approach is to start with simple in vitro tests

lf these experiments and investigations of a material's

fficiency deliver promising findings, then more
mprehensive studies on experimental animals (in vivo
valuation) will be performed

linical trials are the final step of this evaluation process

dissect

war:m!
- .
'-" I
 Biomaterial

Inhibiting areola
of different sizes
=Sensitive
in vitro tests
ss expensive way

irnulate biological reactions to materials when they are

aced on or into tissues of the body

• Experimentally controllable, repeatable,

• Fast, relatively inexpensive, relatively simple.
• Avoid the ethical and legal issues
• Transgenic cells carrying human genes can be used
• Small amount oftest material is required

• Questionable clinical relevance

• Chronic effects cannot be tested
• Pharmacokinetics cannot be evaluated
4
 e goal is to determine the biocompatibility or safety
biological environment

Carried out to determine that the device performs as

tended and presents no significant harm to the patient or

ADVANTAGES
• Higher level of significance
• Simulate real body conditions

• Expensive
• Time consuming
• Ethical issues
Factors considered during in vivo test

:Chemical composition of the materials

ature, degree, frequency, and

uration of exposure of the device and its constituents to

e intended tissues
To facilitate the selection of appropriate tests
biomaterials can be categorized by

./ Nature of body contact of the medical device

and by

./Duration of contact of the medical device

TABLE 1 Medical Device Categorization by Tissue Contact
and Contact Duration

Surface devices Skin

Mucosa! membranes
Breached or compromised
surfaces

External communicating Blood path, indirect

devices Tissue/ bone/dentin
communicating
Circulating blood

Implant devices Tissue/bone

Blood
Contact duration Limited, ~24 hours
Prolonged, >24 hours and
<30 days
Permanent, >30 days
 o perspectives in in vivo testing

Utilization of in vivo tests to determine the general

ocompatibility of newly developed biomaterials for which
me knowledge of the tissue compatibility is necessary for
rther research and development

lt focuses on the biocompatibility of the final product,

at is, the medical device in the condition in which it is to
implanted

IO
rious in vivo Tests as per ISO 10993 standard and the
A guidance document are:

ln VIVO
tests
 10,993, Biological Evaluation of Medical Devices,
ernational Standards Organization
10.993-l. Evaluation and testing
10.993-2. Animal welfare requirements
I0.993-3. Tests for genoroxiciry, carcinogenicity.
reproductive toxicity
10,993-4. Selection of 1cs1., for interactions
lh blood
O 10.993-5. Tests for cytötox icity: /11 vitro met/unis
O 10,993-6. Tests for local cffCèL~after implantaLion
O I 0.993-7. Ethylene oxide sterilization residuals
I 0,993-9. Framework for the idemificaLion
quantification of potential
gradation productslSO10,993-10. Tests for irritation and sensítization
10,993-11. Testsfor systemictoxicity
10,993-12. Sample preparation and reference
1terlals
10,993-13. Identification and quantification of
esradation products from polymers
10,993-14. Identification and quantification of
adation products from ceramics
10,993-15. Identification and quantificationof
adation products from metals
11d alloys
10,993-16. Toxicokineticstudy desígnfor
adation products and leachables
Sensitization test
llergic response caused by the activation of complex
llular and humoral immunological mechanisms can occur
ter either single or multiple exposures

nimal used: Guinea pig

ethod: Buehler closed-patch test and the Magnusson­

ligman guinea pig maximization (ISO 10993-10)

f the test material is amenable to intradermal injection,

e maximization test is recommended. The closed-patch
st is the assay of choice for non-extractable, or when the
xtract or material may be topically applied

nduction phase followed by challange phase

e

16
Systemic toxicity
t estimate the potential harmful effects in vivo on target
ssues and organs away from the point of contact with
ther single or multiple exposure to medical devices,
iomaterials, and/or their extracts

Conducted by administering the extracts (polar and

onpolar in most cases) as a single dose to test animals,
nd the health status of the animals is verified
eriodically-typically 24, 48 and 72 hours after dosing

olvents, should be chosen to yield a maximum extraction

f leachable materials for use in the testing

Animals of choice for the conduct of these tests are mice,

ats, or rabbits
 Contd ...
lt may be acute, sub-acute or chronic

Acute toxicity is considered to be the adverse effects

that occur after administration of a single dose or
multiple doses of a test sample given within 24 hours

Subacute toxicity (repeat-dose toxicity) focuses on

adverse effects occurring after administration of a single
dose or multiple doses of a test sample per day du ring a
period of from 14 to 28 days

Chronic toxicity tests determine the effects of either

single or multiple exposures to medical devices,
materials, and/or their extracts during a period of at
least 10% of the lifespan of the test animal

18
No adverse physical symptoms after injection

Slight loss of motor function, slight difficulty

breathing, and symptoms of irritation in the
abdominal cavity

Difficulty breathing, loss of motor function,

dropping of eyelids, and diarrhea clearly
observed

Cyanosis, and trembling, or a sever case of

irritation in the abdominal cavity, diarrhea,
drooping of the eyelids, and difficulty of
breathing are observed

Mouse dies after injection

Genotoxicity

n vivo genotoxicity tests are carried out if indicated by the

emistry and/or composition of the biomaterial or if
vitro test results indicate potential genotoxicity

1nitially, at least th ree in vitro assays should be used and

o of these assays should utilize mammalian cells

[he initial in vitro assays should cover the three levels of

enotoxic effects:

Gene mutations, and

Chromosomal aberrations
21
 e most common test is the rodent micronucleus test:

he in vivo test normally uses mouse bone marrow or

ouse peripheral blood

Micronuclei, also known as Howell-Jolly bodies, are

nerally smooth, round remnants of nuclear chromatin

en in erythrocytes

An increase in the frequency of micronucleated

rythrocytes in treated animals is an indication of

duced chromosome damage

22
Micronucleated erythrocytes

23
Implantation test
mplantation tests assess the local pathological effects on
e structure and function of living tissue induced by a
mple of a material or final product

or short-term implantation evaluation out to 12 weeks

•Animals utilized in these studies are mice, rats, guinea

pigs, or rabbits

or longer-term testing in subcutaneous tissue, muscle, or

one
• Animals such as rabbits, dogs, sheep, goats, pigs, and
other animals with relatively long life expectancy are
suitable

24
 Contd...
Short-term effects are assessed by evaluating tissue

sponses to the implant at 1 and 4 weeks following the

•At least four rabbits per time period are recommended,

nd each rabbit is implanted with at least four test and

two negative control materials

•Evaluated responses include inflammatory reactions and

•If at least two of the four test sites exhibit a significant

response compared with the control sites, an adverse or

positive effect can be assumed

25
Eye irritation test (Draize test)

ocal tissue inflammation response to chemicals, without

systemic immunological component.

Test animal: Rabbit

Volume of Extract: 0.1 ml

Extract is instilled in one eye of each animal, the other eye

eceives the control vehicle

The test and control eyes are assessedfor biological

sponses at 1, 24, 48, and 72 hours after instillation

The observation period need not exceed 21 days

26
 Grades for Ocular lesions

Cornea
Opacity: Degree of density (area most dense taken for reading~ No ulceration
or opacity O
Scattered or diffuse areas or opacity (other than slight dulling of nonna! luster),
detaíls of iris cleaity visible •1
Easity discernible t/llnsfucent area, delalls of Iris slightly obscured '2
Nacroos area, no details or Iris visible, size of pupil barely dlscemlble '3
Opaque cornea, Iris not discernible through the opacity '4
Iris
Nonnal O
Markedly deepened rugae, congestion, swelling moderate circumcorneal hy·
peremla, or Injection, any of these or combination of any thereof, Iris still re-
acting to light (sluggish reaction Is positive) '1
No reaction lo light. hemorrhage, gross destruction (any or all of these) ·2
ConJunctlvae
Redness (refera to palpebral and bulbar conjunctlvae, excluding cornea and
B~ vessels normal
0 O
Sorne blood vessels definitely hyperemíc (injected) 1
Diffuse, crimson color, individual vessels not easily discernible '2
Diffuse beefy red '3
Chemosls (refera to lids and/or nictìtating membranes)
No swelling O
Any sweßlng above normal (includes nictitating membranes) 1
Obvious swelling with partial aversion of lids '2
Swelling with lids about half closed ·3
Swelling with lids more lhan half-closed '4
'Starred figures indícala positive grades.
27
 Contd ...

SCORE OBSERVED EFFECT

o Non irritant

0-0.5 Minimal irritant

0.5·2 Mild irritant

2-5 Moderate irritant

5-8 Severe irritant

29
Skin irritation test

Measured by Trans-epithelia I Water Loss (TEWL)test

TEWL measurements are of great importance in

evaluating barrier functionality

The more perfect the skin protective coat, the higher

the water content and the lower the TEWL

30
 - - - - - srrntum stratum

o_õ_õ_
,--_-_-_-_-_-_..-
comeum
corneum

lowTEWL a) high TEWL

Figure l. Schematic illustration of the barrier function of
the stratum corneum. a) healthy skin, b) disturbed skin

31
Pyrogen testing

substances in devices that cause a febrile

endotoxin contamination is most commonly

ssociated with such an adverse effect; however, leachates

if materials can cause similar febrile res onses material-

SO 10993-11 recommends testin the

otential of extractable substances derived from material

Number of animals: Three rabbits required; comparison
of febrile response in test animals to baseline
temperature for evaluation of pyrogenicity potential

Test duration: Test measure ment intervals: every 30

minutes for 3 hours

Evaluation: Cutoff for positive febrile response: O.SºC

If any single animal of the three has a

temperature increase above the acceptable
range, the test can be continued with 5
additional animals 33
Carcinogenicity

Carcinogens induce tumors (benign or malignant),

crease their incidence or malignancy, or shorten the time

f tumor occurrence when they a re inhaled, injected,

ermally applied, or ingested

This test determine the tumorigenic potential of medical

evices, materials, and/or their extracts from either single

r multiple exposures

34
 Contd ...
Carcinogen i city tests should be conducted only if data

om other sources suggest a tendency for tumor induction

The conventional test for carcinogenicity is the long-term

odent carcinogenicity bioassay requiring 2 years

Carcinogenicity and chronic toxicity may be studied in a

Ingle experimental study

To facilitate and reduce the time period for carcinogenicity

esting of biomaterial, the FDA is exploring the use of

ransgenic mice carrying the human prototype e-Ha-ras

ene as a bioassay mode for rapid carcinogenicity testing.

35
 Contei...
The gene, is capable of transforming normal cells into a
oplastic cell following its mutation, confers an
usually high susceptibility to tumor formation in rasH2
nsgenic mice

Various advantages of e-Ha-ras animals are:

./Mutagen detection within 6 months

./ Able to detect various non mutagenic carcinogens

./More rapid onset and higher incidence of tumors

Hemocompatibility
BIOMATERIAL
{l.
ur.: ========ii BLOOD-, ----u
CELLULAR EFFECTS HUMORAL EFFECTS
ex: thrombosis, ex: activation of the
embolism, lysis, and coagulation, kinin,
inflammation complement, and
fibrinolytic systems

, Device contact w~h circulating blood • il

IMPLANTS INDIRECT
Drainage e atheters, Sten ls. Cardiac val ve Blood bag
Butterfly needles

-.
~
 Mf'thods I Con1111(•uts
Light micro<copy(adhered Light microscopy can be replaced by
pLllclct,.leukocytes. scanning electron microscopy ,f the
a2gruate.. crythroC}1es. nature of the material presents technical
fibnn. CtC ) problems for light microscopy.
Coagulation Partial rhromboplastin
urne [ncnacrivated)

Platelet count

Leukocyte count and Hemolysis rs regarded as an especially

differential; hemolysis signifìcantscreening test to perform in
(plasma hemoglobin) this cat~ory because of its measurement
of red blood cell membrane fragility m
contact with materials and devices. The
method used should be one of the
normative standard test methods for
hemolysis.
Immunology C3a. Csa, TCC, Bb, iC3b, A panel including the last four tests
C¡d, SC5b-9 encompasses the various complement
aci,,·ation pathways.

38
 atelets adhesion and aggregation

•Thrombosis is the formation of a blood clot inside

a blood vessel, obstructing the flow of blood through
the circulatory system
Test article (e.g., tubing or catheter) is implanted in the

guiar veins of two (2) dogs.

The test article is implanted in the jugular vein

The test article are removed and examined for the

resence of thrombi, and the vein is examined for patency

Unit -2 Tissue engineering

Tissues
 TISSUES
Human body is made up of four basic tissues:
epithelial tissue, connective tissue, muscular
tissue and nervous tissue.
Four types of tissue

OK SF
COE: SIL
eo)
EA
Mea £3

Connective tissue
PSE 1 =
Epithelial tissue

it N
ae.
Muscle tissue Nervous tissue
 v V

i
4 +
— i. Central
Cells
nervous
A.Simple epithelium a. i. Fibroblasts Skeletal
i. Squamous muscles
ii. Fibrocytes ; system
li. Cuboidal li. Peripheral
b. Adipose /fat cells Smooth
iti, Columnar c. Plasma cells muscles nervous
iv. Pseudostratified d. Mast cells Cardiac system
|

tii, Autonomic
|

e. Macrophages muscles
nervous
f. Leucocytes
system
g. Pigment cells
-Siratified
stratified h. Mesenchymal
epithelium cells
i. Transitional
Fibres
li. Stratified
i. Collagen fibres
squamous non
ii. Elastic fibres
cratinined }
iii. Reticular fibres
iii, Stratified
wg:

squamous
keratinized
 EPITHELIAL TISSUE

Epithelial tissue lines the external & internal

surfaces of our body. It can be ectodermal,
mesodermal and endodermal in origin.
Function:
i. Protection
li. Secretion
iii. Absorption
iv. Forms glands & ducts
Epithelium can be simple or stratified.
 A. Simple Epithelium
An epithelium is called as simple when all the cells rest on
the basement membrane. It can be :
i.Squamous epithelium- is one in which cells are
flattened with nuclei raising a bulge in the cell. It allows
rapid exchange of substances through it. e.g. epithelial
lining of alveloi .
“yy FS
Or,
Air sacs
of lungs

Nucleus of Nuclei of
squamous squamous
epithelial cell epithelial
cells

Basement
membrane
“AS
Photomicrograph: Simple
(a) Diagram: Simple squamous squamous epithelium forming part
of the alveolar (air sac) walls (100x).
ii. Cuboidal epithelium- is one in which cells are
like a square where height of the cell is equal to
the width of the cell. The nucleus is central and
round. Its main function is absorption and
secretion e.g. epithelial lining of follicles of
thyroid
gland. Simple Cuboidal Epithelium

Nucleus Cytoplasm

Connective tissue Basement membrane

iii. Columnar epithelium- is the one wherein the
height of the cells is greater than the width of the
cells. The nuclei is basal and vertically elongated.
Functions of a columnar cell are protection,
secretion and absorption. e.g. epithelial lining of
fallopian tubes.

fp
Cilia
ul set

ae6 6 | |

@ |
6 |
|

eo eo |
Single layer of column of
rectangular cells

Crane
Basement membrane
iv. Pseudostratified epithelium- Pseudo= false,
Stratified= multilayered. As the name suggest there
is false appearance that the epithelium is
multilayered. In fact some cells are short while the
others are tall but all the cells rest on basement
membrane e.g. epithelial lining of trachea.
Respirators patheliuan
PF

Croblet Cell

Basal hodics

\ ~. “ws
Loa,
al,
NA
analrg
Ou
peo
~
 B. STRATIFIED EPITHELIUM
Multilayered epithelium is known as stratified
epithelium.
i. Transitional epithelium- this kind of epithelium
lines an organ which is distensible e.g. urinary
bladder.
aes eal (OSD
say
5

RECO APE 9.
Oe 8
2
go
7
Welle’a” 4 ty%e@
ASS 8 8u0
12.9 ‘g
| 4

me
A v 04, fy ct, Se
at
43.8
_. =
| Distended bladder Empty bladder
|

)
ii. Stratified squamous’ non- keratinized
epithelium— it is multilayered epithelium in which
a

basal cells are columnar. E.g. epithelial lining of

esophagus.

@
oe. a
7
sa
a .~ &
Stratified squamous epithelum non keratme

a)
3
at
iii. Stratified squamous keratinized epithelium — It
is a multilayered epithelium just like Stratified
Squamous non- keratinized epithelium but here the
top most layer is that of keratin through which
water can neither be evaporated from the body as
in standing in sun nor can it be absorbed into the
body as while swimming e.g. epidermis of skin.
Stratified squamous epithelium
keratinizedtayers -

‘ws,
 CONNECTIVE TISSUE
As the name. suggests it connects different
components of our body. It is made up of cells, fibres
and matrix. Fibroblasts, fibrocytes, macrophages and
adipose cells are the example of some connective
tissue cells. Matrix is composed of ground substance
and fibres.
Functions:
i. Providing support to different parts of our body.
ii. Connects different components of our body.
iii. It is a medium for exchange of nutrients,
metabolites and oxygen between blood and cells.
 CELLS
a. i. Fibroblasts- the cells which synthesize collagen
fibres, elastic fibres and lays down matrix are
know as fibroblasts.
Fibrocytes- when the fibroblasts mature they are
ii.
known as fibrocytes.
>
Fibroblast Fibrocyte
Cytoplasm basophilic °
Cytoplasm acidophilic
because its contain more protein

producing organeties

‘
~

|
b. Adipose / fat cells- they are rounded cells but
they are usually present in group, they get mutually
compressed so may have variable shape. They have
fat globule in their cytoplasm which pushes the
nucleus to periphery giving the cell signet ring
appearance. Their function is to store fat in their
cytoplasm.

c. Plasma cells- rounded to ovoid basophilic cells

with cart wheel nucleus. They are rich in rER. Their
function is to synthesize immunoglobulins.
 FAT CELLS

PLASMA CELLS
CART WHEEL
ro 090
oO NUCLEUS

O00
[9000 PLASMA
CELLS
Te
090900
d. Mast cells- they are involved in inflammatory
reactions.
e. Macrophages- their function is to engulf various
foreign particles.
f. Leucocytes- White Blood Cells are known as
leucocytes. They are neutrophils, lymphocytes,
eosinophils, basophils and monocytes. They have
defensive action.
g. Pigment cells- import brown color to the skin when
they are present in epidermis. Their presence offers
protection against ultraviolet rays of the sunlight.
h. Mesenchymal cells- are undifferentiated cells. They
can differentiate into a variety of cell types.
 FIBRES
a. Collagen fibres- they are flexible but offer great
tensile strength. They found in tendons, ligaments
and all kinds connective tissues.
b. Elastic fibres- they can stretch and then come back
to their normal size when stretching force is
relieved. They are found in ligamentum nuchae,
ligamentum flava and in the wall of large arteries.
c. Reticular fibres- they are special type of collagen
fibres and can be seen only by special stain. They
are seen in the connective tissue framework of
spleen, liver lymph nodes.
 Collagen Fiber Elastic Fibers

Reticul “se
Fibers

Celis wv

tw Ff,
White Blood Cells
Connective tissue can be:
. General connective tissue:
D>

Loose connective tissue

®

. Dense connective tissue

Mucoid tissue
. Adipose tissue

B. Specialized connective tissue: this comprises of

bones, cartilages an hemolymphoid tissue.
a. Cartilage — Hyaline cartilage, elastic cartilage,
fibrocartilage
b. Bone- made up of organic & inorganic substances.
c. Hemolymphoid tissue- blood & lymphatics fall in this
group.
 MUSCULAR TISSUE
There are three types of muscles namely-
a. Skeletal muscle- they help in locomotion e.g.
biceps femoris.
b. Smooth muscles- they are present in the viscera
of our body e.g. muscle in the wall of stomach.
c. Cardiac muscle- it is seen only in the heart.
Contraction of this muscle helps in pumping of
blood throughout our body.
Types of Muscle Tissue
Sa
smooth muscle cells

striated muscle cells

cardiac muscle cells

 NERVOUS TISSUE
It consists of central nervous system, peripheral
nervous system and autonomic nervous system. It
helps in sending different kinds of information to
brain and bringing its responses back from brain to
the effector organ.
aed

Neuron cells

Bua: ~- FR
‘Contact wath
otner cols

microtubules
SS 5
|
.

Axon
STEM CELLS, CELL
LINEAGES AND
SOURCES
 1860 - 1920
"Stem cells" inferred from analysis of embryo development and
microscopy of bone marrow (Germany)

 1948 - 58
Stem cell mechanisms deduced for sperm development and
intestinal epithelium replacement (Canada)

 1956
First bone marrow transplants performed in human patients (USA)

 1958
Nuclei from adult frog cells reprogrammed to full embryonic
potential after transfer into frog eggs (UK)
1959
Experiments in mice prove the existence of resident blood stem cells in
marrow (England)

1961
The existence and properties of transplantable stem cells in mouse bone
marrow are established and the first colony methodology for counting them
is introduced. This discovery set the stage for all current research on adult
and embryonic stem cells (Canada)

1968
First allogeneic human marrow transplants achieved avoiding lethal
rejection reactions (USA)

1969
First application of cell separation technology to dissect marrow stem cell
hierarchy (Canada)

1974
Mouse embryonic cancer cells are shown to participate in the development
of normal tissues as well as teratomas (UK, USA)

1978
Transplantable stem cells are discovered in human cord blood (USA)
1981
Embryonic stem cells are first derived from the inner cell mass of mouse
blastocysts (UK, USA)

1982
Marrow stem cells measured by regenerative capacity in vivo are shown
to be distinct from progenitors measured by colony methods (Australia,
USA)

1984
Blood stem cells measured by colony formation in vivo are first
extensively purified (Holland)

1982 - 1986
First methodology developed for targeted genetic modification in
embryonic stem cells (UK, USA)

1990
Mouse marrow regenerating stem cells are first completely separated
from in vivo colony-forming cells (USA)

1992
Neural stem cells identified in the adult human brain (Canada)
1993
Pluripotency of embryonic stem cells is proven through the generation
of entirely embryonic stem cell-derived mice (Canada)

1994
• First separation of cancer stem cells from the majority of cells in a
cancer (Canada)
• Patients with damaged corneas are successfully treated with corneal
stem cells (Taiwan)

1995
First derivation of primate embryonic stem cell lines (USA)

1996
First cloning of a mammal: Dolly the sheep is born (Scotland)

1998
First human embryonic stem cell line derived (USA)

2000
Retinal stem cells identified in mice (Canada)
2001
• First collaborative stem cell research network - the Stem Cell Network - is
formed (Canada)
• Dermal stem cells identified in adult skin tissue (Canada)

2002
• The International Society for Stem Cell Research is formed.
• Creation of the International Stem Cell Forum (ISCF) to encourage
international collaboration, and with the overall aim of promoting global good
practices and accelerating progress in biomedical science

2003
• Cancer stem cells isolated in human brain tumours (Canada)
• Rare human breast cancer stem cells identified (USA)

2004
• First derivation of dopaminergic cells from human embryonic stem cells, a
hope for Parkinson's disease treatment (USA)
• International Consortium of Stem Cell Networks (ICSCN) is initiated, which
aims to unify international efforts to make stem cell therapy a reality for a
broad range of debilitating diseases
2005
• First evidence for human bone cancer stem cells (USA)
• James Till and Ernest McCulloch win the Lasker Prize for experiments
that first identified stem cells and set the stage for all current research on
adult and embryonic stem cells

2006
• Normal mammary stem cells demonstrated in adult mice (Australia,
Canada, US)
• First induced pluripotent stem cells (iPS) generated by reprogramming
adult mouse skin cells. The altered iPS cells have characteristics similar to
embryonic stem cells (Japan)

2007
• Mario Capecchi, Martin Evans and Oliver Smithies win the Nobel Prize for
Physiology for Medicine for discoveries enabling germline gene
modification in mice
• First physical identification and localization of mammalian intestinal
stem cells (Holland)
• First evidence for human colon cancer stem cells (Canada)

2008
Sam Weiss is awarded the Gairdner Prize for the discovery of neural stem
cells
2009
• John Gurdon and Shinya Yamanaka win the Lasker Prize for discoveries
in nuclear reprogramming. Yamanaka is also awarded the Gairdner Prize
iPS cells created with minimal residual genomic alteration (Canada)

2010
• Adult cells reprogrammed directly to neurons, cardiac muscle and
blood cells (Canada, USA)
• iPS cells created by transfection of mRNA (USA)
• First clinical trial of human embryonic-derived stem cells for
treatment of spinal cord injury (USA)

2011
Isolation of multipotent human blood stem cells capable of forming all
cells in the blood system (Canada)

2012
John Gurdon and Shinya Yamanaka win the Nobel Prize in Physiology or
Medicine for the discovery that mature cells can be reprogrammed to
become pluripotent
 What is a stem cell? YO
replicate itself, or...

A single cell that can

differentiate into many

cell types.

C)
Image
prepored by Catherine Twomey for the Notional Academies,
ce"
Understanding Stem Cells: An Overview of the Science and Issues
from the Notional Academies, http://www.nationalacademies.org/stemcells.
Academic noncommercial use is permitted.
 All stem cells—regardless of their source—have three
general properties: they are

1. Capable of dividing and renewing themselves for long

periods

2. Unspecialized

3. They can give rise to specialized cell types

1. Capable of dividing and renewing themselves
for long periods
 A starting population of stem cells that proliferates for
many months in the laboratory can yield millions of cells.

 If the resulting cells continue to be unspecialized, like the

parent stem cells, the cells are said to be capable of long-
term self-renewal.

 Normally cell undergoes 50 cell division and loose self

renewal property

 But stem cells divides indefinitely by protecting telomerase

enzyme activity
 wo (@) |
Stem cell

=
YY GS = =
=>
“a
Cell division

(@) (@) caushter

cells
y
Specialization
(Differentiation)
2. They are unspecialized
 They do not display any specific structures associated with
carrying out specific functions in the body.

 For example, a stem cell cannot work with its neighbors to

pump blood through the body (like a heart muscle cell),
and it cannot carry oxygen molecules through the
bloodstream (like a red blood cell).

 However, unspecialized stem cells can give rise to

specialized cells, including heart muscle cells, blood cells,
or nerve cells.
Fertilised egg =>
Blastocyst
(S days old)

inner cell mass

Stem
celis
3. They can give rise to specialized cell types
 When unspecialized stem cells give rise to specialized
cells, the process is called differentiation.

 The internal signals are controlled by a cell's genes, which

are interspersed across long strands of DNA, and carry
coded instructions for all the structures and functions of a
cell.

 The external signals for cell differentiation include

chemicals secreted by other cells, physical contact with
neighboring cells, and certain molecules in the
microenvironment.
 Morula

Blastocyst

Inner cell

~ ESCs culture
mass

6eo
Muscle cells

oe @
Blood cells
:
we
\
/\

fee a
Liver cells

Pancreatic Islet cells

4

Neurons Intestinal cells

 Totipotent
1. Stem cells can differentiate into embryonic and
extraembryonic cell types. Such cells can construct a
complete, viable organism.

2. These cells are produced from the fusion of an egg

and sperm cell.

3. The only totipotent cells are the fertilized egg and

the cells produced by the first few divisions of the
fertilized egg are also totipotent.

4. Totipotent stem cells give rise to somatic

stem/progenitor cells and primitive germ- line stem
cells
 Pluripotent
1. Stem cells are the descendants of totipotent
cells.
2. It can differentiate into nearly all cells, i.e.
cells derived from any of the three germ layers.
3. These pluripotent cells are characterized by
self-renewal and a differentiation potential for
all cell types of the adult organism.
4. These are true stem cells, with the potential
to make any differentiated cell in the body.
Embryonic Stem Cells come under this category.
 @
hd
Piuripotent Stem Cells (PSC)

A Te 8
_ hee
boyd
Ectoderm

a
Gee" 3
Cartes Ware

|
tance Ca
a
teed
coh
ont
t
tr Sun Col
of tplderma
Reuren
Ca
nee
 FiUripureat Stem CLeus siulerenuate
into many Cell Types

growth factors
 Multipotent
1. stem cells can differentiate into a number of
cells, but only those of a closely related family
of cells.
2. These are true stem cells but can only
differentiate into a limited number of types.
3. For example, the bone marrow contains
multipotent stem cells that give rise to all the
cells of the blood but not to other types of cells.
4. Adult Adipose tissue is a source of
multipotent stem cells
MULTIPOVENYT STEM CELLS
CARTILAGE

BONE

Sz Chondrocytes MUSCLES
Cececserr

TENDON
LIGAME ~~ <== SKIN

\
Ase

Fibroblasts

CNS

Adipocytes MARRC
de hos:
Stromal cells
 Oligopotent
1. Stem cells can differentiate into only a few
cells, such as lymphoid or myeloid stem cells.

2. Haematopoeitic Stem Cells is an example of

oligopotency cell.
 (HSC)
@ @& a
Hematopoietic Stem Cell
—
‘-a®
Dendnitic
oat
Common lymphoid
progenitor
Dendnitic B cell Zz
cell, progenitor @)
@progenitor
cei
> ©.
(a
L£ \
Xt
Natural
killer cell
Common myeloid

yO
, 3 oot
ce
>:
\ OQ
|
T cell
VA
@
Erythroid
Megakaryocyte C)
Basophil
*.°
Eosinophil
progenitor progenitor
Granulocyte-monocyte
progenitor |

progenitor
»)
@
Yy
\4 ‘\

Erythrocytes Platelets Basophil Eosinophil Neutrophil Monocyte

 Unipotent
1. Stem cells can produce only one cell type,
their own, but have the property of self-
renewal.

2. Muscle stem cells is an example of unipotent.

3. Most epithelial tissues self-renew throughout

adult life due to the presence of unipotent
progenitor cells.
 ¢
aH
U

.
Unipotent
a} iDiferentation

Single Mature
Stem Call
Cell Type
 Embryonic stem cells
 Embryonic stem cells have the ability to form any
fully differentiated cell of the body

 During the early stages of embryonic

development the cells remain relatively
undifferentiated and possess the ability to
become, or differentiate, into almost any tissue
within the body

 Embryonic stem cells (ESCs) derived from the

early embryos
 Morula

Blastocyst

Inner cell

~ ESCs culture
mass

6eo
Muscle cells

oe @
Blood cells
:
we
\
/\

fee a
Liver cells

Pancreatic Islet cells

4

Neurons Intestinal cells

 ES cell lines are sometimes referred to as
immortal due to their ability to keep dividing
(self-renewing) over many generations.

 ES cells may have great potential in forming the

basis of long-term therapies, but issues regarding
their safety must be overcome first.

 Human embryonic stem cells open new avenues

for autologous cell-based therapy in
degenerative diseases, bypassing the ethical and
immunological problems
 Fetal Stem Cells
 Fetal stem cells are primitive cell types found in
the organs of fetus

 Fetal stem cells can be isolated from fetal blood

and bone marrow as well as from other fetal
tissues, including liver and kidney

 Most tissues in a fetus contain stem cells that are

pluripotent and drive the rapid growth and
development of the organs
 Cord blood stem cells
 Blood from the umbilical cord contains some
stem cells that are genetically identical to the
newborn

 They are multipotent stem cells, that are able to

differentiate into certain, but not all, cell types.

 The applications of cord blood are similar to

those of adult bone marrow and are currently
used to treat diseases and conditions of the
blood or to restore the blood system after
treatment for specific cancers.
 BLOOD
CORD STEM CELL
try
 stem ceil

Sry ox

an
20%

ed bicod celis Platelets

 Like the stem cells in adult bone marrow, cord blood
stem cells are tissue-specific.

 Blood can be collected from the umbilical cord of a

newborn baby shortly after birth.

 The umbilical cord blood is often banked, or stored,

for possible future use of stem cell therapy.

 The discovery that stem cells can be obtained from

umbilical cord blood instead of the more
controversial source of embryonic SC’s, has renewed
interest on the new, exciting therapeutic potentials
of this technology
 Adult stem cells
 Adult stem cells have been isolated from several
tissue sources, including the central nervous system,
bone marrow, retina and skeletal muscle

 It is also known as somatic stem cells

 Most adult stem cells are lineage-restricted i.e.,

multipotent and are generally referred to by their
tissue origin.

 These include mesenchymal stem cell, adipose-

derived stem cell, endothelial stem cell, dental pulp
stem cell, etc.
 ‘
Placenta and
Umblical Cord Adipose Tissue

Ha
on
Dent
Fo cle
Amniotic
Liver | | Marrow Pulp
Fluid

Gut Periodontal
N N
Epithelium Ligament

Neural

Tissue
™, /
Adult Stem

Cell \
Exfoliated
Deciduos

|
Tooth
 Adult stem cell treatments have been
successfully used for many years to treat
leukemia and related bone/ blood cancers
through bone marrow transplants.
 An extremely rich source for adult mesenchymal
stem cells is the developing tooth bud of the
mandibular third molar.
 Adult or somatic stem cells exist throughout the
body after embryonic development and are
found inside of different types of tissue
 These stem cells have been found in tissues such
as the brain, bone marrow, blood, blood vessels,
skeletal muscles, skin, and the liver.
 They remain in a quiescent or non-dividing state
for years until activated by disease or tissue
injury.
 Adult stem cells can divide or self-renew
indefinitely, enabling them to generate a range
of cell types from the originating organ or even
regenerates the entire original organ
 It is generally thought that adult stem cells are
limited in their ability to differentiate based on
their tissue of origin
 Induced pluripotent stem cells
 Induced pluripotent Stem cells (iPSC) are created
by inducing the specialized cells to express genes
that are normally present in embryonic stem
cells and that control cell functions
 Embryonic stem cells and iPS cells share many
characteristics, including the ability become the
cells of all organs and tissues, but they are not
identical.
 These are not adult stem cells, but rather
reprogrammed cells with pluripotent
capabilities.
INDUCED PLURIPOTIENT Stem Celt
4 factors
Oct3/4, Sox2, c-Myc, Kif4

— @
ES-like cells
Fibroblasts

IPS cells
(induced piuripotent stem cell)
 _ KLF4, SOX2, c-Myc, Nanog, Oct-3/4, LIN-28 ,

ttn | OAS
A. Td |

Adult Fibroblast Cell Reprogram Cells

Fe Se Nw
Hematopoietic
Progenitor Cells

~
Adipocytes + Neural
Cells Pancreatic f-Cells

Dopaminergic mn oea Oe
rig K Motoneurons
 Using genetic reprogramming with protein
transcription factors, pluripotent stem cells
equivalent to embryonic stem cells have been
derived from human adult skin tissue.
 Frozen blood samples can be used as a source of
induced pluripotent stem cells, opening a new
avenue for obtaining the valued cells
 iPSCs are useful tools for drug development and
modeling of diseases, and scientists hope to use
them in transplantation medicine.
 iPSCs are derived from somatic cells,
epigenetically reprogrammed to lose tissue-
specific features and gain pluripotency
 The concept of induced pluripotent stem cells
remains an important area of focus for future
research and has serious implications for the
stem cell cancer theory
 Induced pluripotent human cell lines are useful
in the production of new disease models and in
drug development as well as in transplantation
medicine
 Embryonic stem cells are pluripotent (can become any
cell types of the body).

 More stable. Can undergo many cell divisions.

 Easy to obtain but blastocyst is destroyed.

 Embryonic stem cells can be easily grown in culture.

 Embryonic stem cells from a donor introduced into a

patient could cause transplant rejection
 Adult stem cells are “Multipotent” (“can become many
but not any”). However adult stem cell plasticity may
increasing the number of cell types a given adult stem
cell can become.

 Less Stable. Capacity for self-renewal is limited.

 Difficult to isolate in adult tissue.

 Adult stem cells are rare in mature tissues and methods

for expanding their numbers in cell culture have not
yet been worked out.

 A potential advantage of using an adult stem cells is

that the patient's own cells could be expanded in
culture and then reintroduced into the patient.
OSTEOCYTES
CELLULAR COMPONENTS OF BONE

® Osteo-progenitor cells
© Osteoblasts
© Osteoclasts
® Osteocytes
© Bone lining cells
 INTERRELATIONSHIP
~
- Osteoblasts

y,

-
Cytokines
> RANKL
- M-CSF
J
¢
Osteoclast differentiation
¢ Stimulated osteoblasts >
Procollagenase

¢ Removal of collagenous
part of bone
Kobayashi and Udagawa
2007
¢ Osteoclasts > resorption of
mineralized part of bone
 REGULATION

Systemic Local produced

akeyanateyates factors

-PGE2
-Leukotrines
-Parathyroid hormone
-Vitamin D3 -Cytokines: IL2 IL3 & IL6
-Growth factors TNFa
-Calcitonin
:

TNFB TGFB PDGF

 BONE FORMATION
Multipotent stromal stem cells

Plating and attachment

Express Col |, fl, Ill and alkaline phosphatase & osteopontin

Increased synthesis of Coll and alkaline phosphatase

Diminishing of Col Il & Col Ill synthesis

Formation of collagen substratum

 Declining of bone osteopontin

Pre-osteoblast > osteoblast through interaction with azf1 receptor

Expression of bone sialoprotein

TaTheEhccelamelmaniatielipheeie
 Increased osteocalcin synthesis

Osteoblasts > osteocytes and bone lining cells

el

Xiao G, Wang D, Benson MD, Karsenty G, Franceschi RT

Role of the alpha2-integrin in osteoblast-specific gene expression
and activation of the Osf2 transcription factor. J
Biol Chem 1998: 273: 32988-32994
 Coeamised
Differentiated Cells
Precursor Cells
Osteoblasts
Precurser Cells osteocytes
wewew rer el
———> BONE
lining cells 0.4.0
fetetetetete
Mekipoan
Cell Cementoblasts
spin
“e-@36 ®,.-<<a>—-—> —
CEMPNEUM

Other Mesenchyma One

(cartilage. muscle. fat, fibrous tissues)
+
Fibroblasts
b

Chemotaxis Proliferation Differentiation Matrix Formation

ad
(decreasing)
ad
(increasing)
~ —>

Cytokine Concentration
 INITIAL OSTEOBLASTIC
DIFFERENTIATION

Regulatory Transcription
genes factors

Homeobox Runx2
genes Cbfal- Master
hoxa2, hoxal3, gene for
hoxd13, dix5, osteogenic
differentiation
msxi, msx2

eDucy P Zhang R, Geoffroy V, Ridall Al, Karsenty G (1997).

Osf2/Cbfal: atranscriptional activator of osteoblast differentiation.
Cell 1997: 89: 747-754
¢Rodan GA, Harada S. The missing bone. Cell 1997: 89:
677-680.
REGULATION OF BONE FORMATION

TGF-B
Calcium Insulin like
metabolism growth factor
hormones Bone
Glucocorticoids
morphogenetic
CALCITONIN protein
PARATHYROID
hoon
rmones
Estrogen
HORMONE FGF
VITAMIN-D PDGF
 FACTORS

sacelel
Ua dtelame)mey3c-1e)e)

r Val iT Yao)
mey-) (re) 8)
4 216
am

sYolal-ma=-Yols oh elaMmel lca th mel

me lalelie-ralthy
 PARATHYROI HORMONE
I
|

Anabolic effect Catabolic effect

|

Stimulates bone Stimulates bone

formation through resorption indirectly
TGF-8 & insulin like
growth factor (coupling)
increased serum Ca

Canalis E, Hock JM, Raisz LG. Parathyroid hormone: Anabolic and

catabolis effects on bone and interactions with growth factors. In:
Bilezikian JP, Marcus R, Levine MA, ed.
The parathyroids. New York: Raven Press, 1994: 65-82.
 VITAMIN D3
|

we
Anabolic effect Catabolic effect |

Primary function in Ca
absorption from Stimulates bone
intestine resorption
Stimulates synthesis of Suppresses collagen
osteocaicin & production
osteopontin /

DeLuca HF. Vitamin D revisited. Clin Endocrinol Metab 1980: 9: 1-26.

 | GLUCOCORTICOIDS
]
|

Anabolic Catabolic
effect effect

Promotes Prolonged t/t with C

differentiation of causes bone loss due
osteoblastic cells to inc PTH in
response to
Stimulates bone inhibitory effect on
matrix formation Ca absorption
 TGF-B

Anabolic effect Catabolic effect

induces osteogenic
differentiation of
mesenchymal cells Inhibits osteogenic
Decrease in matrix induction by BMP’
degradative activity
through inhibtion of
MMP’s

Reddi AH. BMPs: actions in flesh and bone. Nat Med 1997:
3: 837-839.
 INSULIN

Anabolic effect

Directly targets osteoblasts

Stimulates bone matrix formation and
mineralization
Stimulates insulin like GF produced in liver
 THYROID HORMONE |

Anabolic effect

Affects endochondral bone formation by its

action on cartilage formation
BONE MORPHOGENETIC PROTEINS |

Anabolic effect

ost profound effect on bone formation

 INSULIN LIKE GROWTH FACTO
|

Anabolic effect

Stimulates proliferation of osteoblast

precursors
Inhibits MMP’s

Hock JM, Centrella M, Canalis E. Insulin-like growth factor (IGF-I) has

|

independent effects on bone matrix formation and cell replication.

Endocrinology 1988: 122:254—260.
 PDGF AND FGF

Anabolic effect

increases proliferation of osteoprogenitor cells

Promote osteogenic differentiation
influence expression of other cytokines
Help in wound and fracture healing

Hock JM, Canalis E. Platelet-derived growth factor enhances

bone cell replication but not differentiated function
of osteoblasts. Endocrinology 1994: 134: 1423-1428.
STAGES IN THE LIFE CYCLE OF AN osTeoc.asT
OSTEOCE ASE FORMATION
im
Hematogenic Myeloid
Progenitor Progenitor

@xt M-CSF —o-Fos

(el
CFU-GM
e
Sec
teoblastic
tromal Cell

@ TGF-8
P-C-P Migration&
Activity

OS TORGALS
|
Pre-osteochast
Bioactive and Biomimetic material
in Dental and Bone applications
 Definition
Biomimetics, term was coined A bioactive material is one that
by Otto Schmitt in the 1950s . elicits a specific biological
Biomimetic is defined as the
response at the interface of the
study of the structure and material which results in the
function of biological systems as
formation of a bond between
models for the design and
the tissues and the material.
engineering of materials and
machines.

Karma M, etal Biomimetics in dentistry indian / Dent Edu Hench LL, Splinter Ri, Aen WC, Greenlee IK ir.; Banding
mechanismsat the
interface af ceramic prosthetic materials. / Biomed Mater Res,
1972; 2:117-141
 Applications of Bioactive Materials and Molecules
in Dentistry
» Root Canal Therapy
¢ Portland cement or MTA is a bioactive material used for
maintaining
pulp and periodontal tissue vitality as part of pulp-capping and
perforation repair procedures.
¢ Mineral trioxide aggregate is also used as an obturating material after
root canal therapy. It helps in apexification.
Tooth Repair and Regeneration
¢ Dentin extracellular matrix
proteins (ECMPs) contain growth factors
that can promote tooth healing and pulp regeneration.
° Dentonin
(peptide) can stimulate reparative mineralization of the
coronal pulp and occlusion of the lumen of the root canal.
° A novel biomaterial
(tissue regenerative gel), can promote the
regeneration of tooth tissue.
In Dental Surgery and Craniomaxillofacial
Reconstruction
¢
Biomaterials, such as Emdogain containing Porcine proteins play an
important role in periodontal regeneration after injury.
¢
Synthetic bone materials are used for maxillofacial and craniofacial
reconstruction.
¢ The ideal biomaterial for dental reconstruction should be
biocompatible and also able to maintain volume and can be easily
shaped.
°
n-Butyl-2-cyanoacrylate is widely used as a tissue adhesive. It is also
used for filling and repairing bone defects.
Coating of Implants
Dental implants are made from titanium alloys and have a coating of
hydroxyapatite to promote osteogenesis and bone healing.

Hypersensitivity
¢ A novel bioactive
glass-ceramic (biosilicate) is applied in
hypersensitivity cases.
° It is
capable of inducing HCA (hydroxyl carbonate apatite) deposition
in open dentinal tubules, and thus occluding the dentinal tubules.
¢
Hence, biosilicate may be a new way for treating dentin
hypersensitivity.
Biomedical

Stem Cell Therapy

¢ New methods of stem cells with bioactive materials renovate the

function of injured tissue by replacing dead or damaged cells with

new and healthy cells.
¢ Dental Tissue
Regeneration
¢ A combination of nanostructure materials, such as biomimetic
matrices and scaffold and stem cells, will certainly increase the
regenerative impact of dental pathological tissues
¢ Bone Graft
¢ Bioactive materials, such as osteoconductive matrix, which act as
scaffold to new bone growth, and osteoinductive proteins, which
support mitogenesis of undifferentiated cells combined with
osteogenic cells (mesenchymal stem cells), are capable of forming
bones.
° Cosmetic Surgery
Collagen is used in soft tissue repair for augmentation in cosmetic
surgery.
° Tissue
Engineering
Gelatin is used as a natural polymer, which is derivative of collagen.
Biomolecules released from gelatin are capable of maintaining their
biological activity and help them in tissue engineering
Various biomimetic and bioactive materials
are:

¢ Glass lonomer Cement (GIC)

¢ Resin Based Composite
¢ Smart Dentin Replacement (SDR)
¢ Ceramic
° Castor Oil Bean Cement
¢ Calcium Hydroxide
¢ MTYA1-Ca filler
¢ Calcium Sulfate
° Calcium Phosphate
¢ Calcium Enriched Mixture (CEM)
¢ Mineral Trioxide Aggregate (MTA)
¢ Calcium Aluminate Cement
¢ Doxadent

¢ Ceramir
¢ Biodentine™
¢ Theracal
°
Bioaggregate (BA)
¢
Endosequence Root Repair Material (ERRM) putty, ERRM paste RRM
putty fast set (FS) and iroot FS
¢
Bioceramic Sealers
¢
Bioceramic Gutta-Percha
¢
Bioactive Glass (BAG)
¢
Remineralizing Agents
¢
Casein phosphopeptide amorphous calcium phosphate (CPP-ACP)
¢
Demineralized dentin (dDM)
*
Enamel matrix derivative (EMD)
¢
Growth Factors
*
Bone Morphgenic Proteins (BMP)
Glass lonomer Cement (GIC)

¢ Glass ionomer cement (GIC) which was invented in 1969 is composed

of fluoroaluminosilicate glass powder and water soluble polymer
(acids).
¢ When powder and liquid is blended, it undergoes hardening reaction
that involves neutralization of the acidic group together with
~
significant release of fluorides.

mb
——/
¢ Bioactive formulation (such as 45S5, S53P4) has bioactive glass and
hydroxyapatite.
¢ The mechanical properties of GIC have been improved with
incorporation of metals such as stainless steel and bio inert ceramics
like zirconia.
¢ KT-308 (GC Corporation Company, Tokyo, Japan) a GIC sealers,
provides more resistance to coronal ingress of bacteria into the root
canal system.
Smart Dentin Replacement (SDR)
¢ Smart Dentine Replacement is a first flowable
composite material.
¢ Its characterized by low polymerization stress,
low polymerization shrinkage, high depth of cure
and bulk-fill material in increments of up to 4
mm in class i and ii cavities.

Jad
Calcium Hydroxide
¢ In1928, Calcium Hydroxide was introduced by
Hermann in dentistry.
¢ It has beenwidely used as a mineralizing agent
and antimicrobial agent.
¢ Calcium hydroxide causes release of extracellular
matrix molecules (like dentine phosphoproteins,
dentine sialoproteins), raises expression of
biomolecules, like BMP, and has antimicrobial
and anti-inflammatory action
MTYA1-Ca filler
>it is a resin-based direct pulp-capping agent.
>»Composition:
® Powder
:89.0% microfiller, 10.0% calcium hydroxide, and 1.0% benzoyl
peroxide
®
Liquid : 67.5% triethyleneglycol dimethacrylate, 30.0% glyceryl
methacrylate, 1.0% O-methacryloyl tyrosine amide, 1.0%
dimethylaminoethylmethacrylate, and 0.5% camphorquinone
>»MTYA1-Ca developed dentine bridge formation without formation of a
necrotic layer, revealed to have good physical properties, and was not
inferior to Dycal histopathologically.
Niinuma A. Int Endod J. 1999
Calcium Sulfate
¢ Calcium sulfate (CS) has been shown to be SAYIN
-
completely bio absorbable, osteoconductive, graft2set
allow fibroblast migration, do not cause an
inflammatory response _@
|

SauyN
° It also do not elevate serum calcium levels.
e
Recently, it has been shown that CS can be
manufactured into a granular composite of CS and
poly-l-lactic acid to decrease the degradation rate.
 Calcium Phosphate

¢ Calcium phosphates plays important role in

biological and pathological mineralization.
¢ Most commonly used in the form of paste,
cement, ceramics and scaffold.
° It
helps in induction of bridge formation with
no superficial tissue necrosis and significant
absence of pulpal inflammation.
E
Bio ceramic based material/tricalcium silicate
(calcium silicate based materials)
* MTA which was developed by Mahmoud Torabinejad
at Loma Linda University, consists of 50-75 % (wt.) he MIA
oh,
nwt é

calcium oxide and 15-25 % silicon dioxide.

> It has high pH (12.5),
° causes regeneration of the periodontal ligament (PDL)
¢ dentinal bridge formation
¢ biomineralisation and stimulation of cell differentiation
e has antimicrobial activity.
> However, difficulty in manipulation and longer setting
time are its limitations
 Biodentine™
°
(Septodont, Saint-Maur-des-Fossés, France),
introduced in 2011.
bioactive dentin replacement material Biodentine

r
¢ It is a
having similar properties of dentin and has a

septodont
positive effect on vital pulp cells stimulating
tertiary dentin formation

?7 @
¢ When biodentine comes in contact with dentine it results into
formation of the tag-like structures and is called “Mineral Infiltration
Zone,’ which may contribute to adhesive properties.
¢ It has improved- physical properties,
-reduced setting time (12 min)
-induces odontoblast-like cell differentiation
e . . =. -
@p
-mineralization 9 Biadentine’

=< iM

8
Theracal
¢ It is a light-cured, resin-modified calcium silicate—filled liner insulating
and protecting the dentin—pulp complex.
¢ It can be used in direct and indirect pulp capping, as a protective
base/liner under composites, amalgams, cements, and other base
materials.
¢ When this material was compared with ProRoot MTA and Dycal, it
was found that calcium release was higher and solubility was low.

_
Polyhedral Oligomeric Silsesquioxanes (POSS)

¢ Biomaterials such as polyhedral oligomeric silsesquioxanes (POSS)

and polyhedral oligomeric silicates (POS) may be fabricated by the
incorporation of POSS molecules to provide a nanoscopic topology
which favors cellular modulation, bioavailability and differentiation.
¢ Advent of POSS has led to formulation of dental adhesives and
composites with improved mechanical and physical properties.
Implant Biomaterials/Biomimetic Coatings on
Implants
¢ Itincludes metals and metallic alloys,
ceramics, natural materials and synthetic
polymers.
@
¢ Metals and metallic alloys include titanium,
tantalum and alloy of Ti-Al-Va, Co-Cr-Mb, Fe-
Cr-Ni. Devices made from zirconium,
hafnium and tungsten have been evaluated
recently.
¢
Hydroxyapatite-coated metal’s coating is
done using robotic techniques for
osteoconductivity.
Polymers

°
Polymers like polymethylmethacrylate (PMMA), polyethylene (PE),
polyurethanes are used in dental surgery since decades.
¢
Polyglycolic acid (PGA), polylactide (PLA), polydioxanone (PDS) are
mainly used as resorbable bone fixation devices or as suture
materials.
¢ For surgical wound repair fibrin glue is used as tissue adhesive.
¢
Alginate can be used as growth factor delivery system.
Regenerative endodontics and tissue engineering

¢ Dental regeneration is a process in humans by which specialized

dental tissues are replaced by the recruitment, proliferation,
migration, and differentiation of dental stem cells.
¢ Stem cells, scaffolds, and growth factors are 3 key elements for tissue
regeneration.
¢ Root Canal Revascularization via Blood Clot
In the Root canal revascularization via blood clot in apical region, the
bioceramic material is placed as mid-root/coronal plug, thereby
providing permanent and superior quality seal.
Conclusion
¢ Biomimietic materials functions as root canal sealer, filling materials,
cements and root and crown repair material and possesses features
as like strengthening the root following obturation, good sealing
ability, enhanced biocompatibility and antibacterial properties.
¢
Contemporary biomaterials have shown ability to overcome the
limitations of traditional materials.
Drug Delivery
Nano medicine had viewed
countless breakthroughs in drug
delivery implementations.
Nanoparticles are defined as
particulate distribution or solid
particles with a size in the range
of 10-100 nm. Na)

Controlled nano drug delivery

seeks the development of
suitable drug carriers that can
transmit a sufficient dose of drug
to diseased lesions.
Various nanostructures including liposomes,
polymers, dendrimers, micelles and magnetic
nanoparticles have been tested as carriers in
drug delivery.

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Nanoparticles can be prepared from different
types of materials such as proteins,
polysaccharides and synthetic polymers
The selection of matrix depends on many
factors including requisite size of
nanoparticles, underlying properties of the
drug such as solubility, stability, surface
characteristics (charge and permeability),
degree of biodegradability, biocompatibility
and toxicity, drug delivery profile desired,
antigenicity of the final product.
Why is there a need to controlled
drug delivery?
To overcome the limitations and drawbacks of
conventional drug administration like limited
effectiveness, poor biodistribution, toxicity
and lack of sensitivity, controlled drug delivery
system is utilized.
In controlled drug delivery systems, the drug is
transported at the place of action, increasing
its influence on the vital tissues and
minimizing its undesirable side effects.
 Delivery duration

Nature of
a a we Targetabitity

Designing of
nanodelivery
Blocompathdity system (hug release
\ JS

Rerate of
Drug properties achapisriets ation

Figure showing aspects of controlled drug delivery applications

 1) improve delivery of poorly water
soluble drugs
The poor solubility of drug is major problem
a

which limits the development of highly potent

pharmaceutics.
nanotechnology based drug delivery system has
potential to overcome the poor aqueous
solubility and poor enzymatic/metabolic stability
of drugs associated with the oral route of
administration.
nanoparticles improve delivery of poorly
water soluble drugs in water by delivering
drug of small particle size allowing faster
dissolution in blood stream leading to
targeted drug delivery ina cell- or tissue-
specific manner.
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 Fig. 1

Compoede of tow-solitity drugs and sstactarés (Sobc:

Highly lipophilic
S$)

drugs can also be r— Hydropnec

at?e
S (Hydro)

_
employed inside
Camposie of low-sokbility
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(Solid 8) Low-sokoly drugs

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the hydrophobic 5 (LipoS}

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core of Hydrophikc Lipophaic

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biocompatible
polymer or
surfactant.
 2) protect the therapeutic agents
trom physiological barriers
A) blood-brain-barrier (BBB):
nanoparticles can be employed in delivering
therapeutic agents in brain tumor as they can
cross blood brain barrier through opening of
tight junctions eg. (hyper-osmotic mannitol).
Tween-80 coated nanoparticles have been
shown to cross the blood-brain barrier.
B) enzymatic degradation in GIT:
Three main factors have been reprted to
destabilize oral delivery.
These include bile salts, pH, pancreatic and
gastric enzymes (which would mainly impaire
delivery of protein based drugs like insulin).

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To protect the drug from these harsh
condition drug is better coated with a
biocompatible carrier eg. (liposome, poly
saccharide ...,etc).

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orug e
 For example, using liposomes as a suspension
or solubilizing agent for highly lipophilic drugs
to be delivered as micro-emulsion in soft gel
capsule for oral dosage.

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C) immunogenic clearance:
When nanoparticles are adminstered
intravenously, they are treated as foreign
particles by the body immune systems, and
they are phagocytosed from the circulation.
The surface lipophilicity of nanoparticles
decides the amount of adsorbed blood
components as proteins (opsonins) in
circulating blood influence the in vivo fate of
nanoparticles.
Binding of these opsonins on the surface of
nanoparticles called opsonization, which acts
as a bridge between nanoparticles and
phagocytes.
OPSONIZATION
Binding of Fc recoptor K
Opsonization opsonized microbes signais Phagocytosis ot
to phagocyte of microbe ingested
by oe Fc receptors (FcyRl)
activate
phagocyte

re body

Feu
Phayocyle

Phagocytosis is ineffective/siow without opsonization

in order to prevent drugs from getting ingested by
phagocytes, the surfaces of the nanocarriers are
typically coated with the biocompatible synthetic
polymer poly(ethylene glycol) (PEG) which is called
stealth effect.
So these nano carriers prolonged the retention
time of drug (T1/2 in circulation) and decreased its
clearance by spleen....etc.

f~

meacr
 Cross BBB
Gene delivery
mm
pNP,
w

.° Oral bioavailability
betterment

Cancer therapW”
>
? |

a
and targeting

oad OY

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 3) Targeted drug delivery
controlled drug delivery system enhances drug
concentration in diseased tissues, therefore,
lower doses of drugs are required so it improved
efficacy, reduced toxicity and improved patient
compliance and convenience.

C.eneoer

|
serious side effects associated with
medications as chemotherapeutic agents like
bone marrow inhibition, destruction of gut
mucosa,..etc are sometimes more dangerous
than the disease itself.

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BRUISING AND BLEEDING MORE EASILY
Cell-specific targeting is generally achieved by
attaching drugs to individually designed nano
Carriers.
There are two main mechanisms of targeted
drug delivery:
A) passive targeting. 8B) active targeting.
Paaaive targeting Active targeting
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A) Passive targeting:
passive targeting is achieved through a process
called enhanced permeability and retention
(EPR) effect.
As tumor cells tend to grow quickly, they must
Stimulate the production of blood vessels.
These newly formed tumor vessels are usually
abnormal in form and architecture.
They are poorly aligned defective
endothelial cells with wide
fenestrations, lacking smooth muscle a

layer, or innervation with a wider

lumen, and impaired functional
receptors for angiotensin Il.

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The enhanced permeability and retention
(EPR) effect is controversial concept by a

which molecules of certain sizes (typically

liposomes or nanoparticle) tend to
accumulate in tumor tissue much more than
they do in normal tissues.
Arigornyores Lluced -osaute in Qonare

Oot 1
-
Mes ation tyoctedimn theed¢ en enter
nn tuesces paserety the EPR oftect Cantimassus ard well
| on dathatel :eB
~S Tumor
a MNOS
vreec.eeture
a

Conaveetian ard reterenr

af mms o6e@0 in Ihe han ore
e

Lewenate
tumor tissues usually lack effective lymphatic
drainage.
All of these factors lead to specific
accumulation and prolonged retention of the
nano particulate drug.
Normal endothelia Leaky vasculature
“-> :
<=> —=_

[= od
[o> @ @ @
@
Nornal tieeuwe

vr Ym
Enooc yto

oe
5
Tumor coil

Non-Aargeted

EMCO Voters ett: rege «4 ome ot overs: of rrmsetetty ancl swtmrems: oftert 197M, 0: che bebee 9
i
’
5
be gros
YT web poland ruses tee ow
'

He bier wes tered ter germ ce tor @tte eres woes ee

B) Active targeting:
Active targeting can be referred to as
receptor mediated drug delivery.
In the most basic sense, drug design involves
the design of molecules that are
complementary in shape and charge to the
biomolecular (cell receptor) target with which
they interact and therefore will bind to it.
Drug(Ligand) «<> Receptor interaction
Lawey (178

@ ove Orug-Receptor
Complex

="y euman .
Receptor Ly enect
the ligand is usually molecule which
a

produces a signal by binding toa site ona

target receptor.
Ligand binding to a receptor protein is named
“docking”.
Ligands might be antibodies, polypeptides,
oligosaccharides (carbohydrates), viral
proteins, fusogenic residues, and molecules of
endogenous origin. ligand ©
\,
Ligand-mediated active targeting has emerged
as a novel strategy in targeting diseased tissue
leaving the normal.
the receptors act as molecular targets or
portals, and ligands being added to the
surface of nanoparticles, with receptor
specificity and selectivity, are trafficked en
route to the target site.

wit

Crd
 Receptor-mediated endocytosis

pe.

Actrve

Nanoparticie-mecated
targeted drug delivery system

Ligands: sacchandes potysecchardes folate. botn. retinoic acid. dehydroascorbic acid. arebodies
peptides. eptamers. transferrin. gtycyrrhetinic acid. growth factors. lipoproteds. nest-tebde
enterotoxmn subunt B serotonin. etc

Y Receptors ASGPR
isoform |
folate receptor biotin receptor. retinoic acid receptor glucose transporter
surfece antigens. integrins. growth fector receptors. transfernn receptor.
glycytrhetinic acid receptor. low-density bpoproten receptor. scavenger receptor type B-!
serotonin receptor etc

Therapeutics chemotnerspeutic drugs. genes. photosensitizers

features of using nanoparticles as drug
delivery systems include easy manipulation of
particle size and its surface characteristics to
achieve both active and passive drug
targeting, controlled and sustained discharge
of the drug both during the transportation
and at the target site increasing drug’s
therapeutic efficacy and reducing side effects.
Nanoparticles (NPs), in contrast to molecular
probes, often are less cytotoxic and do not
suffer from nonspecific binding by cellular
biomacromolecules or unwanted
sequestration.
Nanoparticles have several advantages over
small molecule traditional imaging agents,
such as long blood-pool residence times, and
the potential for cell tracking and targeted
imaging applications.
c) ligand for targeting specific receptor ona
diseased tissue as discussed before.

Targeting
antibody
=
Drug
PEG/spacer
yUf

Drug payload
Fluorescent
probe
rr

Targeting *
+ '

peptide
Cationic Targeting
molecules aptamer
 PROTEIN AND PEPTIDE DRUGS

Management of illness through medication is

entering a new era in which a growing number of
biotechnology produced peptide and protein drugs
are available for therapeutic use.

Ailments that can be treated effectively by this new

class of therapeutic agents include cancers, memory
impairment, mental disorders, hypertension.

BY VGHAL SHARMA
 MARKETED PROTEINS FREEZE DRIEL
IN
FORMULATIONS
—
Product Formulation Route Indication

Metrodin FSH 75 IU im. Induction of

ovulation
Pergonal FSH and LH im. infertility

Profasi HCG im. Infertility

Elspar Asparginase i.m. iv. Leukemia

Glucagon Glucagon im. i.v. S.C. Hypoglycemia

Acthar Corticotropin im. i.v. S.C. Hormone

Deficiency
BY VGHAL SHARMA
 MARKETED PEPTIDES IN READY TO USE
FORMULATIONS
iil
Product Formulation Route Indication
Pitressin 8-Arginine i.m. S.C. Post operative
Vasopressin abdominal
distension

Lupron Leuprolide S.C. Prostatic cancer

syntocinon Oxytocin i.m. i.v. Labour

induction

Sandostatin Octreotide S.C. Intestinal

tumour
Calcimar Salmon S.C. hypercalcemia
ey vena GaeGitonin
 SUSTAINED RELEASE
DOSAGE FORMS
Product Formulation Route Indication

Lupron Leuprolide i.m. Prostatic

cancer

H.P.Acthar gel ACTH im. S.C. Antidiureti

Cc

Pitrressin tannate Vasopressin i.m. Endocrine

in oil tannate cancer

BY VGHAL SHARMA
 PROTEIN AND PEPTIDE DRUGS

They are therapeutically effective only by

parenteral route.

Repeated injections are required.

Therapeutic applications of these drugs rely on

successful development of viable delivery systems
to improve their stability and bioavailability.

BY VISHAL SHARMA
 APPROACHES

“e SEVERAL EXTENDED RELEASE

PRODUCT OF PGLA R AVAILABLE
e¢
CHEMICALLY ALTERD ETC

‘e THREE NASAL PRODUCTS &

TWO ORAL PEPTIDE PRODUCTS
R US FDA APPROVED
e SEVERAL PULMONARY R UNDER
CLINICAL TRIALS
|

BY VSHAL SHARMA
12 CHALLENGES

Large molecular size

Susceptibility to enz. Degradation

Short plasma half life

lon permeability

Immunogenicity

Aggregation

Denaturation etc

BY VISHAL SHARMA
13
ABSORPTION OF PROTIENS
FOLLOW

BY VSHAL SHARMA
15

Considerations are to be given for following

aspects:
barriers to oral absorption

Preformulation and Formulation

considerations

Pharmacokinetic considerations

Analytical considerations

Regulatory considerations
BY VISHAL SHARMA
26
PROTEASE INHIBITORS

Coadministration of protease inhibitors provides a

viable means to circumvent the enzymatic barrier in
achieving the delivery of peptide and protein drugs.

Th e choice of protease inhibitors will depend on the

structure of these therapeutic drugs, and the
information on the specifi city of proteases is essential
to guarantee the stability of the drugs in the Gl tract.

A number of inhibitors including aprotinin (trypsin

/chymotrypsin inhibitor), amastatin, bestatin,
boroleucine, and puromycin (aminopeptidase
inhibitors) have been reported for this purpose

BY VSHAL SHARMA
29 PERMEATION ENHANCER

. Without permeation enhancers’ lower

bioavailability is achieved when these routes are
used.
*
Lower bioavailability is due to poor mucosal
permeability.
*
Sodium tauroglycocholate is commonly used
penetration enhancer.

BY VGHAL SHARMA
 PREFORMULATION AND
31 FORMULATION
CONSIDERATIONS

Denaturation stabilizers

Maximising oral protein and peptide absorption

Chemical Modifications

“Amino acid Modification

*
Hydrophobization

Conjugation with polymers

BY VISHAL SHARMA
32
DENATURATION

Specific confirmation is required for proteins to exert

pharmacological and physiological activities.
Denaturation is a process of altering protein
confirmation. Heat, organic. solvents, high salt
concentration, lyophilization can denature proteins.

Protein confirmation refers to the specific tertiary

structure, which is determined by the primary and
secondary structures and the disulfide bonds and is held
together by three forces : hydrogen bonding, salt
bridges, and hydrophobic interactions.

BY VGHAL SHARMA
 33 COMMON STABILIZERS
____ail
SERUM ALBUMIN :

It can withstand heating to 60°C for 10 hours.

At pH 2 albumin molecule expands and elongates but

can return to native confirmation reversibly. Also, it
shows good solubility.

BY VISHAL SHARMA
AMINO ACIDS

Glycine is most commonly used stabilizer.

Mechanism of action of amino acids as stabilizers may
be one of the following:

Reduce surface adsorption.

Inhibit aggregate formation.

Stabilize proteins against heat denaturation.

BY VISHAL SHARMA
35
SURFACTANTS

They cause denaturation of proteins by hydrophobic

disruption. However judicious use of surfactants can
protect proteins from other denaturants. Proteins have
tendency to concentrate at liquid/liquid or liquid/air
interface. Due to this proteins may adopt non native
confirmation and such confirmation is having less
solubility.

Optimal concentration of surfactants for stabilization

should be greater than cmc. lonic surfactants are more
effective stabilizers than non ionic surfactants.

Various surfactants used are : poloxamer 188,

polysarhate.
36
POLYHYDRIC ALCOHOLS AND
CARBOHYDRATES :

They contain -CHOH-CHOH- groups which are

responsible for stabilizing proteins. They stabilize
proteins against denaturation caused by elevated
temperature or by freeze drying or by freeze thaw
cycles.

Many important therapeutic proteins and peptides are

derived from blood such as immune globulin,
coagulation factors. For viral destruction
pasteurization at 60°C for 10 hours is needed. Hence
thermal stability is needed. Long chain polyhydric
alcohols are more effective as stabilizers. e.g. sorbitol,
xylitol.
BY VGHAL SHARMA
37

Mechanism of action as stabilizers for polyhydric

alcohols is that they have effect on structure of
surrounding water molecules' which strengthens
hydrophobic interactions in protein molecules.
Mechanism of action as stabilizers for carbohydrates is
that they provide dry network that provides significant
support for protection.

Polyhydric alcohols used are sorbitol, mannitol, glycerol,

PEG.

Carbohydrates used are glucose, mannose, sucrose,

ribose.
BY VISHAL SHARMA
 MAXIMISING ORAL PROTEIN
39 AND PEPTIDE ABSORPTION

1. Amino acid modifications

Metkephamid, an analog of methionine

enkephalinwith substitution of glycine, by I-
alanine and modified methionine, readily
penetrated across the nasal mucosa with 54%
bioavailability relative to subcutaneous
administration but was orally inactive.

BY VGSHAL SHARMA
40

ee2.
Hydrophobization

Hydrophobization of peptides may be attempted by

two approaches. The first ispeptide backbone
modification to include more of hydrophobic amino
acids; the second would be covalent conjugation of a
hydrophobic moiety—for example, a lipid
orpolymeric tail.

Increasing the hydrophobicity of a peptide or protein by

surface modification using lipophilic moieties may be of
particular benefit to transcellular passive or active
absorption by membrane penetration or attachment,
respectively; or it may simply aid in the increased
Stability of the protein.
BY VGHAL SHARMA
43
BENEFITS

increase clearance half life

Provide possibility of drug to stay more in the

circulation.

Increase molecular stability

Change the vol. of distribution

Reduce immune response
Application of Biomaterials
Healthcare
Organ/Tissue Examples

heart pacemaker, artificial valve, artificial heart

eye contact lens, intraocular lens

ear artificial stapes, cochlea Implant

bone bone plate, intramedullary rod, joint

prosthesis, bone cement, bone defect
repair

kidney dialysis machine

bladder catheter and stent

muscle sutures, muscle stimulator

circulation artificial blood vessels

skin bum dressings, artificial skin

endocrine encapsulated pancreatic Islet cells

 &~ 4~ G4]04.WQMOJl.Q,

x Heart valve
 Heart Valve
• Fabricated from carbons,
metals, elastomers, fabrics, and
natural valves.

• Must not React With Chemicals

in Body.

• Attached By Polyester Mesh.

• Tissue Growth Facilitated By

Polar Oxygen-Containing
Groups.
 Heart Valve
• Almost as soon as valve
implanted cardiac function is
restored to near normal.

• Bileaflet tilting disk heart valve

used most widely.

• More than 45,000 replacement

valves implanted every year in
the United States.
x dental implants
Dental Implants
• Small titanium fixture that serves as the
replacement for the root portion of a
missing natural tooth.

• lmplant is placed in the bone of the

upper or lower jaw and allowed to bond
with the bone.

• Most dental implants are: pure titanium

screw-shaped cylinders that act as roots
for crowns and bridges, or as supports
for dentures.
A titanium dental implant. (Photograph courtesy
of Or. A. NotrNn Cranin, Brookdale Hospital
MeditalCtnte, Brooklyf\. Ht'.)
Dental Implants

• Capable of bonding to bone, a

phenomenon known as
"o sseointegrati on".

• Bio-inert, there is no reaction

in tissue and no rejection or
allergic reactions.
x hip replacements
Hip-Replacements
• Most Common Medical Practice Using
Biomaterials.

• Corrosion Resistant high-strength

Metal Alloys.

• Very High Molecular Weight Polymers.

• Thermoset Plastics.
Intra ocular lenses
Intraocular Lenses
• By age 75 more than 50% of
population suffers from
cataracts

• Made of PMM, silicone

elastomer, and other materials.

• 1.4 million implantations in the

United States yearly.

• Good vision is generally

restored almost immediately
after lens is inserted.
Artificial tissue
vascular grafts
 Vascular Grafts
• Must Be Flexible.
• Designed With Open Porous
Structure.
• Often Recognized By Body As
Foreign.
• Achieve and maintain
homeostasis.
• Good structure retention.
• Adequate burst strength.
• High fatigue resistance.
• Poly(ethylene terephthalate)-PET
or Dacron
• Good handling properties.
• Biostable.
 TAILl I Some Apphca11ons oí Syn,bcuc t\1:ucnal~ :tnd
;\1odifiC'd Na,ur.tl ,\111,en:d, tn ~1echein(

Skder-.1.I a;ri,1t111
Jvint repla...c1ncnb lhjp, kn«) Tir;anmm, Ti-Al-V ilk>)-, \.t.Jink» '\tttl, polycth)'knr
Bone plate for ír,1,,vrc lix:irinn St-amie... steel, eòbilt-dirom1urn~
6oße C'(OU''Ot Poly,m,rthri ll'Kth.l\.'.ryl:&tc-;
l\ony defecr f~1';11r H,Jro,ryl-apa.t11K
Art1fi~·i.llt<•nJun anJ hg_An1c:1u reûon, Dl..:ron
llt:nul unrl.un for too1h fixation ·1i,.,.n111n1,11lun11n.J. ale1um ~piure-
C.'.Ahhu~.,,,,:ulur,y,11::n\
IUnod vecel prrnoth,r1i~ DJ(fOH, 1 c:ilon, potyutt,h.rinc
1 •c.11r1 ¥':ih,· k('prl.K'C"•M"d useue, •t.11nlcn-•red, orbt,n
( .11h11u:r .;,liMnc- rubl,ç-r, 'l"rflon. pc.1h•utr1h..,ic
()~!!\.
Anlfi ...l:111~.An l\.tyurrth11n,
~k111 rc,uu 1t~n1.1IJtt ~1ll<flnc--('l)lbJC1,,,ornpo,1tc
AnWu"I kl,Jn1•)' (l1e1nodrnly1t't) (,rllulot(', polya ... ,ylnru,nk
I J<,,1r1-I """"'"'hint .;.i1,1l"C' ruhht't'
''"""•'"
C..n~hl1.•arn:plac-cnirllt Pbunum elecrrodes
luu.aocular lt·n~ Poly(mc-thyl mC"fhKrybtcl, silicooc ni.bbt'f. hydrogc-1
(.onta,·, lt1ts Sdkonc--.1,,yb.rc, hydrog«I
C.ornç,1I b:and.JJl.C'. Col13Rt'O, h}"d.JUl(:t'I
 Cr.1.ru:;e..l J.161. S.~. T, =ic-,.hc. I-IA, TCP

,tn,o,:1lktlx1:.1.I tceCK'l-""'n,cn~ \l:0:1 li,\,

lklual~ ccevne, gold. 31<:tL :sS.. C'°"C..r-!\llo. TCP. 11.,, J>l-A. B,ugl~. Ti T1-..-'-I·''
Ti. I ,-.°'I-V. Atz<>,. J LA. 1:1~..._.._.Ja:,.,
D~rud:ahlc Sun.are...~I, men. of
PL..,,. P.;...A.,l"'<•t •• P~tC. í'Ol)

Jtcan.. C..:~í-lvl.o.-¡ •-Ai-, r, ~ .--o,~t,c

C.cl~I Pl-T f"t'R
P'acçnli&l.C'f" 3 I 6L SS.. Pl. Pt 1R
,,lit,¡,:.-,.nc, Pl f

Zu"'·o1ua. 316L SS. "re, ·r-,•.'\_... V.

<.:...,_<..,-~Jo. UJl.,I\\ l'I:.

Tendon & l.11,!'n,c-n,1~ Pl./\(._

PL,\. pol"\. bctl"C" fihc,-. cr-n r-:... PET l 1flf\.lY•t•I
POA pul,.a,IJt:ol,Jc
l"T,t<-' p,."lh.1nn:'lel.h, lc:nc.:nd,,,.-;ru1.e
PIX>-pvh f p,<iaU..'(:U'IO.llCt fJ..:.ric r,~u,o,n 3 lt.L s~. Co-C,-!>.t••·
Pl.i K l"'C)I) Wclhanc T,. y.,._.,1.,.r. Pl.,'- I LA.. PL\.. PG.'\ .
..:-PTFF <.'-J'Q.:111.kJ
......._." 1e,mn~~·" k.""f'loe"
UJU\l"'-'P! -u111,sh1¡;:h mal ,..,-.
p:,1) ctJ,:. lcnc
PET pol)-dh) 1- l.:!1'1:'J)lululalt:'
I-ti\ b).JrO."\.\.S~lt~
,~ - ,-u;unl¢C,1'1i, ._u;.,c:t
 Materials for Use in the Body

Mot<rlJls i\dV'Jlltag<S Olsadvuntag<S Examples

Polym<rs (nylon, siIkone rubber, R<Silicnt Not strong Sutures, blood vessels,
polyesttr, ¡>olyt<trafuor0<1hylene, erc.) Easy to fabric"t< Deíonnswith ilmc hip socker.ear, nose,
May degrade other soíl tissues.
SUI Ures
MeL1is (Ti and its alloys, C:0-Cr alloys. Strong, tough, ductile fi.iay corrode Joint replaœments, bone plates
stainless S1eels, /\u, i\g, Pt. erc.) Dense and screws. dental root
Oiflicuh 10 nuke implants, pacer and sururewnes
Ceramics (aluminum oxide, calcium Very bíocompatible, ßrinle D<ntal; femoral head of hip
phosphate< including Inert Notröilient replacement, coating of d('nlul
hydroxy.apatite, carbon) S1rong in Oiflicult 10 make and orthopedic implants
conipression
Composites (carbon-carbon, wire or Strong, tailor-made Difficuh to make Joint implants, heart valves
fib« reinforced bone cement)