UNIT 1

• Introduction to tissue engineering:Basic

definition; current scope of development; use
in therapeutics, cells as therapeutic agents,
cell numbers and growth rates, measurement
of cell characteristics morphology,number
viability,motility and functions.Measurement
of tissue characteristics ,appearance,cellular
component,ECM component,mechanical
measurements and physical properties
• The two broad types of mammalian
stem cells are:
• Embryonic stem cells -that are
isolated from the inner cell mass of
blastocysts.

 Adult stem cells that are found in

adult tissues.
• They maintain the normal turnover of
regenerative organs, such as blood,
skin, or intestinal tissues.
• Stem cell has two main properties:

• Self-renewal - the ability to go through

numerous cycles of cell division while
maintaining the undifferentiated state.

• Potency - the capacity to differentiate

into specialized cell types.
Pluripotent stem cells are the descendants of
totipotent cells and can differentiate into nearly
all cells.

Multipotent stem cells can differentiate into a

number of cells, but only those of a closely
related family of cells.

Oligopotent stem cells can differentiate into

only a few cells, such as lymphoid or myeloid
stem cells.

Unipotent cells can produce only one cell type,

their own.
Embryonic stem cell lines

Cultures of cells derived from inner cell mass (ICM)

of a blastocyst

ES cells are pluripotent and give rise during

development to all derivatives of the three primary
germ layers: ectoderm, endoderm and mesoderm.

They can develop into more than 200 cell types of the
adult body when given sufficient and necessary
stimulation for a specific cell .
• Human ES cells are grown on a feeder
layer of mouse embryonic fibroblasts
(MEFs) and require basic Fibroblast
Growth Factor (bFGF or FGF-2).
• Without optimal culture conditions or
genetic manipulation,embryonic stem
cells will rapidly differentiate.
• The transcription factors Oct-4, Nanog,
and Sox2 -suppression of genes .
• The cell surface antigens ,SSEA3 and
SSEA4 and the keratan sulfate antigens
Tra-1-60 and Tra-1-81.
Colony of differentiating Human Embryonic Stem Cells double-
stained with anti-Oct4 antibody (green) and Sox17 antibody
(red).
 Adult stem cells
 Have been successfully used for many years to treat
leukemia and related bone/blood cancers through bone
marrow transplants .

 Pluripotent adult stem cells are rare and generally small

in number but can be found in a number of tissues
including umbilical cord blood .

 The use of adult stem cells in research and therapy is

not as controversial as embryonic stem cells .

 An extremely rich source for adult mesenchymal stem

cells is tooth bud of the mandibular third molar.
Isolate Stem cells
 Umbilical cord blood stem cells
• Rich source of mesenchymal and
haematopoietic stem cells

• Precious than those found in bone marrow

• No risk to mother or baby-uncomplicated

normal delivery or CS /to be collected
properly
• low rate of viral contamination
 Human Stem Cells
Advantage Disadvantage
Embryonic High differentiation Ethical problem
stem cell capacity
In fertilized clinic or stillbirth
baby
Possibility of differentiation
to Tumor.
Umbilical cord Easy to obtain Small quantity of blood
blood stem No risk for donors
cells High capacity of
differentiation
Long telomere
Low risk of infection
Adult stem Relatively easy to obtain Invasion to donor
cell Plenty of cells Possibility of transmission of
infectious agents
 Cancer
Stem cell therapies may serve as potential treatments for cancer
Conventional chemotherapy treatments cannot discriminate between
cancerous cells and others.
Research on treating Lymphoma using adult stem cells is underway and
has had human trials.
 Possible Mechanisms for the Cell Therapy Using Cord Blood-Derived Stem Cells (V)

Cord blood
Diabetes mellitus

Stem cells

Injection of stem cells

(1) Growth factors release
: IGF-1, HGF, FGF, IGFBP

(2) Transdifferentiation of stem cells

into pancreatic ß cells (3) Activate endogenous
stem cell proliferation

Replacement of dysfunctional
pancreatic cells
 Wound healing

Stem cells can also be used to stimulate the growth of human tissues.
 In an adult, wounded tissue is most often replaced by scar tissue,
which is characterized in the skin by disorganized collagen structure,
loss of hair follicles and irregular vascular structure.
 In the case of wounded fetal tissue, wounded tissue is replaced with
normal tissue through the activity of stem cells.
[A possible method for tissue regeneration in adults is to place adult
stem cell "seeds" inside a tissue bed "soil" in a wound bed and allow the
stem cells to stimulate differentiation in the tissue bed cells.
This method elicits a regenerative response more similar to fetal
wound-healing than adult scar tissue formation.
 Induced pluripotent stem cells (iPSCs) 

• Adult cells that are genetically reprogrammed to an embryonic

stem cell–like state by being forced to express genes and factors
important for maintaining the defining properties of embryonic
stem cells
• Mouse iPSCs were first reported in 2006, and human iPSCs were
first reported in late 2007.  
• Capable of generating cells characteristic of all three germ layers.
• Useful tools for drug development.

Advantages:
• Less probability to immune rejection.
• help researchers learn how to reprogram cells to repair damaged
tissues in the human body.
 Induced pluripotency : the two-stage process

Stage 1 Stage 2

Activation of auto-regulatory loop

Down regulation of lineage
genes
Activation of specific ES genes
Chromatin remodelling
Full reactivation of ES cell
transcriptional network
Completion of transgene silencing
 Draw back of Animal models in drug efficacy studies

• Toxicity,
• Cost,
• Time and ethical issues associated with in
vivo studies.
• Uncertainty of translatability to humans

The above points emphasize the need of

relevant in vitro models.
 Stem cells in Pharmaceutical technology - Beyond an improved in vitro model

• Helps to identify potential therapeutic compounds

• Better understanding of absorption, distribution, metabolism
and excretion properties.
Advantages:
 Convenience and scalability of screening process
 Normal phenotype and growth pattern
 Uniform physiological response towards drugs

U.S. Food and Drug Administration (FDA) has given importance

to in vitro testing, recommending the use of human cell lines to
characterize drug metabolic pathways
Companies like Pfizer, GSK [GlaxoSmithKline], Johnson &
Johnson and GE are in stem cells therapy and research.
Stem cell-based screening strategies and their potential applications
Types of stem cells and their potential to be
used in large-scale screens
Overview of stem cell-based drug discovery process
 ESCs in drug development studies
• ~230 human ESC lines are being used in research funded by
NIH.
• Unlimited and consistent source of cells for high-throughput
screening at a reduced cost.
• Used for chronic studies and provide a wide range of cell types.
• ESCs from Mouse, Rat, Cytomolgus monkey and Dog enables
cross-species comparisons, in vitro/in vivo correlations – Highly
valuable for drug development
• Diseases that can be treated - Parkinson's disease, diabetes,
traumatic spinal cord injury, Purkinje cell degeneration,
Duchenne's muscular dystrophy, heart failure, and
osteogenesis imperfecta.
Major Goals in the Development of Transplantation
Therapies from Human ES Cell Lines
 iPSCs in drug screening and development
• Offers premise of a “clinical trial in a dish”
• Helps in “personalized medicine” by deriving patient/disease
specific iPSCs.
• Differentiation of iPSCs obtained from early stage diseased
patients into differentiated cells represents a possible tool to
study the degeneration process.
• drive innovation in drug discovery via time-dependent stem
cell differentiation studies.
iPSC-derived disease models :
• neurological disorders like Parkinson’s disease and Reet
syndrome,
• blood diseases like Fanconi anaemia.
• cardiac syndromes , pancreatic type 1 diabetes and
• hepatic disorders like alpha-1 antitrypsin deficiency.
iPSCs- important factor in drug discovery
• iPSCs will be key for developing
clinically useful compounds.

• Drug screening assays using iPSCs

allow compound testing on
differentiated human cells that’s not
possible with traditional cell lines.

• iPSCs can be generated from

individuals that naturally harbour
disease-specific traits, which could
complement results from animal
model testing

• aid the development of more

effective compounds.
 Generation and lineage restriction of iPSCs derived from somatic cells can be
adapted for high-throughput screening.
Drug treatment increases nuclear gems in induced pluripotent stem cells
taken from a patient with spinal muscular atrophy.

• iPSCs derived from a healthy

individual show nuclear aggregates
of survival of motor neuron (SMN)
protein, termed gems, in the absence
or presence of drug treatment (a,c,e).
• Untreated iPscs derived from a
patient with spinal muscular atro-
phy (iPSC-SMA) generally lack the
SMN nuclear gems (b).
• Both tobramycin and valproic acid
will significantly increase the SMN
protein and sub-sequent gem
number (d,f), suggesting a possible
therapeutic application.

Gems are indicated by arrows. Scale bar

represents 50 μm. Images are courtesy of
V. Mattis, University of Missouri, USA.
Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human iPSCs

• Diabetic cardiomyopathy is a
complication of type 2 diabetes,
with known contributions of
lifestyle and genetics.

• Environmentally and genetically

driven in vitro models of the
condition will be developed
using human-iPSC-derived
cardiomyocytes.

• Helps in discovery and testing of

therapeutic strategies with ever-
increasing clinical significance.
Use of induced pluripotent stem (iPS) cells in
relation to Alzheimer’s disease (AD).
 NeuralStem Inc. -Spinal Cord Injury treatment with Neural Stem Cells

• Neuralstem's patented technology enables the production of

neural stem cells of the brain and spinal cord in commercial
quantities.
• Neuralstem's NSI-566 spinal cord-derived stem cell therapy is
in Phase II clinical trials for amyotrophic lateral sclerosis
(ALS)- Lou Gehrig's disease.
• Neuralstem has been awarded orphan status designation by
the FDA for its ALS cell therapy.
• In addition to ALS, the company is also targeting spinal cord
injury and ischemic stroke.
• The company has completed a Phase I safety trial evaluating
NSI-189, its first neurogenic small molecule product candidate,
for the treatment of major depressive disorder (MDD).
 Minerva Biotechnologies- Major breakthrough in Human Stem Cell Research

• Minerva Biotechnologies, a leading cancer and

stem cell development company made a major
breakthrough in human stem cell research.
• converted established human stem cells to the
elusive “naïve” state and maintained them there
indefinitely by newly discovered human growth
factor, called NM23-H1.
• Figuring out how to stably induce naïve
pluripotency in human stem cells is critical for
realizing the promise of human stem cell therapies.
 Pharmaceutical and biotech companies utilizing stem cell technologies
in their drug development processes.
Genentech is using ES cell and iPSC-derived cardiomyocytes
as high-throughput models to assess cardiotoxicity for drugs in development.
To investigate specific mechanistic cardiotoxicity findings during in
vivo studies or in the clinic.
Pfizer of New York and GE Healthcare of Chalfont St Giles, UK
Using stem cells in drug discovery with the California companies Novocell of
San Diego and Geron of Menlo Park, respectively.
GlaxoSmithKline teamed up with the Harvard Stem Cell Institute
Stem cell research in neuroscience, heart disease, cancer, diabetes,
musculoskeletal diseases and obesity.
AstraZeneca, London and Cellartis of Gothenburg, Sweden
Using stem cells to make human liver and heart cells for safety tests.

iPierian, San Francisco, California

has created motor neurons using iPSCs derived from people with and
without spinal muscular atrophy, a neurodegenerative disease.
 Stem cell therapy in India
 Currently over 40 institutions and hospitals involved in stem cell research.

 In 2008, Stempeutics launched its second stem cell laboratory on the Manipal
University campus for advanced stem cell research in human embryonic stem
cells.

 In 2009, a joint venture formed by StemCyte in India with Apollo Hospitals and
Cadila Pharmaceuticals to provide stem cell therapies.

 Major research institutes, like the NCBS in Bangalore, CCMB in Hyderabad,

NCCS in Pune and the NBRC near Delhi, are investigating the use of stem cells
to regenerate nerve, heart and adult muscle cells, and repair damaged bone
tissue.
Advantages and disadvantages of human cells available for drug screening
 Limitations
• Stem cells from different tissues are not the same
• Invitro culture of stem cells with growth factors is different
from the microenvironment in vivo.
• Stem cells have slower cell cycle than their progenitor cells in
vivo (so the degree of sensitivity of readouts obtained in vitro
may not be the same as in vivo.)
• Insufficient quantities of cells for screening,
• Don’t know if regulatory agencies will accept data derived
from them.
• Current knowledge of the signals controlling differentiation
falls short of being able to mimic conditions in vitro.
• Most drug companies remain to be convinced that new screens
can predict drugs' properties better than existing methods.
 Significant challenges
• Cost
• limited or no accessibility for particular cell
types—such as pneumocytes
• short life span and loss of function in vitro-
prevents chronic toxicity studies.
• limited availability of cells from one donor
• donor-to-donor variability
 Future directions

• In spite of various challenges, companies have already

invested in stem cell research and have identified central
applications, lifting the uncertainty of how successful stem
cell technology could be in drug discovery.
• Quick shift from early stage drug development to the clinical
space may occur in near future.
• Technology could be used on patients to study idiosyncratic
drug effects and heterogeneity in drug responses.
• Provide opportunity to drug development companies, the
ability to perform in vitro clinical trials with patient specific
iPSC-differentiated cells
 Conclusion
 Stem cells are a valuable tool in the drug discovery process.

 Enables rapid identification of therapeutically useful molecules

that can modulate stem cell behavior.

 Instead of ethical concern and teratoma formation, ESCs can still

be heavily exploited as a tool in various high content screens
directed towards self-renewal, multipotency, and differentiation.

 Tissue-specific stem cells and iPSCs offers an attractive opportunity

for developing drug screens aimed at various human disease
states.
 Drug screens and stem cell research have the potential to develop
novel cellular and gene therapies of various genetic and
degenerative disorders.
ECM
 Many animal cells are intrinsically linked to other cells and to
the extracellular matrix (ECM).
 Cell surface molecules bind to other cells, or to other
components of the ECM. They also play a role in mutual
recognition of similar cell types.
 Bone and cartilage are mostly ECM plus a very few cells.
Connective tissue that surrounds glands and blood vessels, is a
gelatinous matrix containing many fibroblast cells.
The ECM contains 3 classes of molecules:
 structural proteins (collagens and elastins)
 protein-polysaccharide complexes to embed the structural
proteins
(proteoglycans)
 adhesive
glycoproteins
to attach cells
to matrix
(fibronectins
and laminins).
 PROTEOGLYCANS
1. PROTEOGLYCANS are composed of a core protein to which glycosaminolycans
(GAGs) are attached. GAGs consist of repeating disaccharide subunits.
 One of the two sugars in the disaccharide is often an amino sugar (N-acetyl-
glucosamine or N-acetyl-galactosamine; usually with an attached sulfate
group) and the other is a sugar or sugar acid (galactose or glucuronate).
 Chondroitin sulfate, keratan sulfate, heparan sulfate and hyaluronate are the
most common GAGs.
 Proteoglycans

Proteins conjugated to
Glycoproteins saccharides lacking a Protein >> carbohydrate
serial repeat unit

Proteins conjugated to
Proteoglycans polysaccharides with Carbohydrate >> protein
serial repeat units

Glycosaminoglycans Repeat unit

Mucopolysaccharides HexN and HexUA
 Glycopeptide bonds
CH2 OH O NH2

H
O HN C CH2 CH COOH Asn
H
Glc OH H

OH O H

H HN C CH3 NAc
Type I N-Glycosyl linkage to Asn

CH2 OH NH2 CH2 OH NH2

O O CH2 CH COOH O O CH CH2

H H
H H
Glc OH H
Ser Glc OH H CH2 HOLys
OH O H OH H CH2

H HN C CH3 NAc H OH H2 N CH COOH

Type II O-Glycosyl linkage to Ser (Thr) Type III O-Glycosyl linkage to 5-HOLys
 Glycosaminoglycans
b-1,3 b-1,4
COO - CH2 OH

H O O O
O H
H H
OH H H

H OH O H

H OH H HN C CH3

GlcUA GlcNAc

No protein link
Hyaluronate No sulfate
b-1,3 glycosidic linkage
 Glycosaminoglycans
b-1,3 b-1,4
COO - CH2 OH

H O - O O
O OSO3
H H
OH H H

H H O H

H OH H HN C CH3

GlcUA GalNAc

GlcUA-Gal-Gal-Xyl-O-Ser link
Chondroitin sulfate Sulfate at 4 or 6 C of GalNAc
b-1,3 glycosidic linkage
 Glycosaminoglycans
b-1,3 b-1,4
H CH2 OH

H O - O O
O OSO3
COO - H
OH H H

H H O H

H OH H HN C CH3

IdUA GalNAc

IdUA with some GlcUA

Dermatan sulfate Sulfate at 4 or 6 C of GalNAc
b-1,3 glycosidic linkage
 Glycosaminoglycans
a-1,4
COO - CH2 OSO3 -

H O H H
O H
a-1,4
H H
OH H OH H

O
O

H OSO3 - H NHSO 3-

GlcUA GlcNAc

GlcN and GlcUA or IdUA

Heparin N and O sulfate (C2,3,6)
a-1,4 glycosidic linkage

> NAc
Heparan sulfate < N and O sulfate
 Glycosaminoglycans
b-1,4
b-1,3
CH2 OH CH2 OSO3 -

OH O O O
H
H O H
H OH H

H H O H

H OH H HN C CH3

GlcUA GlcNAc

GlcNAc and Gal (no UA)

Keratan sulfate I Sulfate on C6 of Gal or HexN
b-1,4 glycosidic linkage

Keratan sulfate II GalNAc-O-Ser or Thr

• GAGs are highly negatively charged
molecules, with extended conformation
that imparts high viscosity to the
solution.

• GAGs are located primarily on the

surface of cells or in the extracellular
matrix (ECM).
• Along with the high viscosity of GAGs comes
low compressibility, which makes these
molecules ideal for a lubricating fluid in the
joints.
• At the same time, their rigidity provides
structural integrity to cells and provides
passageways between cells, allowing for
cell migration.
• All cellular processes that involve molecular
interactions
• at the cell surface, such as cell–matrix, cell–
cell
• and ligand–receptor interactions, likely involve
proteoglycans because these molecules avidly
bind proteins and are quite abundant at this
site
• The disaccharide units contain either of
two modified sugars, called amino
sugars N-acetylgalactosamine (GalNAc)
or N-acetylglucosamine (GlcNAc),
• And an acidic sugar uronic acid such as
glucuronic acid or iduronic acid.
• The amino group is usually
acetylated.
• This eliminates the positive charge.
• In some glycosaminoglycans, one or more of the
hydroxyls of the amino sugar is esterified with
sulfate.

• The combination of these sulfate groups and the

carboxylate groups of the uronic acid residues gives
the glycosaminoglycans a very high density of
negative charge.
• Keratan sulfate is an exception in which
galactose is present, instead of an acidic
sugar.

• Hyaluronic acid does not contain sulfate.

 Structure of Glycosaminoglycans
• GAGs in the body are linked to core proteins
( except hyaluronic acid), forming
proteoglycans (also called
mucopolysaccharides).
• The GAGs extend perpendicularly from
the core in a brush-like structure.

• E.g. in cartilage proteoglycan the GAGs

present are chondriotin sulfate and
keratan sulfate.
 Linkage

• The linkage of GAGs to the protein

core involves a specific trisaccharide
composed of two galactose residues
and a xylose residue (Gal-Gal-Xyl-O-
CH2-protein).
• The trisaccharide linker is coupled to
the protein core through an O-
glycosidic bond to a Serine residue in
the protein.
• Some forms of keratan sulfates are
linked to the protein core through an
N-glycosidic bond.
• The protein cores of proteoglycans
are rich in Serine and Threonine
residues, which allows multiple GAG
attachments.
 Proteoglycan Aggregates
• Proteoglycan monomers associate with a
molecule of hylauronic acid to form
proteoglycan aggregates.

• Association is not covalent but ionic between

hyaluronic acid and the core protein.

• Stabilized by link proteins

 Classification of Glycosaminoglycans

The classification is based on:

OR the GAGs differ from each other:
Monomeric (acidic & amino sugar) composition
Degree & location of sulfation
Type of glycosidic linkages
Chain length of the disaccharides
Nature of the core protein
Their tissue distribution
Their biologic functions
The specific GAGs of physiological significance are:

Hyaluronic Acid
Dermatan Sulfate
Chondroitin Sulfate
Heparin
Heparan Sulfate
Keratan Sulfate
 Characteristics of GAGs

• Although each of these GAGs has a

predominant disaccharide component ,
heterogeneity does exist in the sugars present
in the make-up of any given class of GAG.
 Hyaluronic acid

Hyaluronic acid is unique among the

GAGs in that it does not contain any
sulfate and is not found covalently
attached to proteins as a proteoglycan.
It is, however, a component of non-
covalently formed complexes with
proteoglycans in the ECM.
• Un sulfated
• Only GAG present both in animals
and bacteria.
• Found in synovial fluid,
• vitreous humor,
• ECM of loose connective tissue
• Umbilical cord
• Cartilage
Specific function:
1. Hyaluronic acid is especially high in
concentration in embryonic tissues and
is thought to play an important role in
permitting cell migration during
morphogenesis and wound repair.
2. Act as lubricators and shock
absorbers.
Association with major diseases:

• Hyaluronic acid may be important in

permitting tumor cells to migrate through
the ECM. Tumor cells can induce fibroblasts
to synthesize greatly increased amounts of
this GAG, thereby perhaps facilitating their
own spread
 Chondroitin sulfate

• most abundant GAG

• Cartilage (bind collagen and hold the
fibers strongly)
• Tendons
• ligaments
• Heart valves
 Heparan sulfate

• Extracellular GAG
• contains higher acetylated
glucosamine than heparin
• And less sulphated groups
• Found in the basement membrane of the
kidney along with type IV collagen and
laminin where it plays a major role in
determining the charge selectiveness of
glomerular filtration
• Are associated with the plasma membrane
of cells, with their core proteins actually
spanning that membrane.

• In it they may act as receptors and may

also participate in the mediation of cell
growth and cell-cell communication.
Association with the disease:
• Some tumor cells have less heparan
sulfate at their surfaces, and this may
play a role in the lack of adhesiveness
that these cells display.
 Heparin

• It is an intracellular GAG.
• Component of intracellular granules
of mast cells lining the arteries of
the lungs, liver and skin
• more sulfated than heparan sulfate
• Heparin is an important anticoagulant. It
binds with factors IX and XI, but its most
important interaction is with plasma
antithrombin III.
• Heparin can also bind specifically to
lipoprotein lipase present in capillary
walls, causing a release of this
enzyme into the circulation.
Specific function:
• Heparin and warfarin are widely used in the
treatment of thrombotic and thromboembolic
conditions, such as deep vein thrombosis and
pulmonary embolus.
• Heparin is administered first, because of its
prompt onset of action, whereas warfarin
takes several days to reach full effect.
• Their effects are closely monitored by use of
appropriate tests of coagulation because of
the risk of producing hemorrhage.
 Dermatan sulfate

• Sclera- gives shape to the eye.

• Binds LDL –plays a role in the
development of atherosclerosis.

• skin, blood vessels, heart valves

 Keratan sulfate

• cornea,
• bone,
• cartilage aggregated with
chondroitin sulfates
• Both keratan sulfate I and dermatan
sulfate are present in the cornea. They
lie between collagen fibrils and play a
critical role in corneal transparency.
• In various types of arthritis,
proteoglycans may act as autoantigens,
thus contributing to the pathologic
features of these conditions.
• The amount of chondroitin sulfate in
cartilage diminishes with age.
• Whereas the amounts of keratan sulfate
and hyaluronic acid increase.
• These changes may contribute to the
development of osteoarthritis.

• Changes in the amounts of certain GAGs

in the skin are also observed with aging.
 Mucopolysaccharidosis

• Several genetically inherited

diseases, for example the lysosomal
storage diseases, result from defects
in the lysosomal enzymes
responsible for the metabolism of
complex membrane-associated
GAGs.
• These specific diseases, termed
mucopolysaccharidoses (MPS) lead
to an accumulation of GAGs within
lysosomes of affected cells.
• There are at least 14 known types of
lysosomal storage diseases that
affect GAG catabolism.
GLYCOPROTEINS
 Glycoproteins are globular
proteins to which shorter,
GLYCOPROTEINS
branched oligosaccharide
chains are covalently bound.
 These so-called adhesion
glycoproteins mediate
attachment of cells to their
matrix, influence the state of
differentiation of cells, and
organization of their
cytoskeleton.
 Examples are fibronectin,
laminin, thrombospondin,
chondronectin and fibrillin.
 FIBRONECTINS, a family of closely related
glycoproteins, are soluble in body fluids
(blood), insoluble in the ECM and partially
soluble at the cell surface.
 The fibronectins bind cells to the matrix and
guide cellular movement.
 The RGD (arginine-glycine-aspartate)
sequence binds to the integrin fibronectin
receptor.
 The fibronectins bind cells to the ECM by
bridging cell-surface receptors to the ECM.
 The intracellular cytoskeleton will align with
the extracellular fibronectin to detemine
cell shape.
 In many kinds of cancer, cells unable to
make fibronectins loose shape and detach
from the ECM to become malignant.
 During cell movement (as
during embryogenesis),
pathways of fibronectins guide
cells to their destinations.
 Soluble plasma fibronectin
promotes blood clotting by
direct binding of fibrin.
 Fibronectins guide immune
cells to wounded areas and
thus promote wound healing.
 LAMININS bind cells to
the basal lamina of
epithelial and connective
tissues, and to their
surrounding muscle cells,
fat cells, and Schwann
cells.
 The basal lamina serves
as a structural support for
tissues and as a
permeability barrier to
regulate movement of
both cell and molecules.
 Laminin is a very large
protein comprised of three
proteins that form a cross.
The domains of laminin
bind type IV collagen,
heparin, heparin sulfate,
entactin and laminin
receptor proteins in
overlying cells to allow
bridging between the cells
and the ECM. Progeria
(early onset of aging), is
possibly due to a defective
laminin.
 THROMBOSPONDIN - activated platelet-product. It
binds to fibrinogen, plasmalogen and its activator; a
participant in blood clotting. Its function is poorly
understood.
 CHONDRONECTIN- a component of cartilage matrix
that mediates attachment of chondrocytes to their matrix
 FIBRILLIN- a nonsulfated glycoprotein speculated to be
essential for normal development. It is often associated
with elastic fibers or with epithelial basal laminae.
Marfan syndrome is due to a defective fibrillin gene
on chromosome 15, characterized by excessively long
arms and legs and progressive dilatation and fatal
rupture of the ascending aorta .
Structural proteins
 Principal producers of collagen fibers are fibroblasts; epithelial
and smooth muscle cells also secrete their own type-IV collagen.
 Most numerous CT matrix, running in all directions in a wavy
course; dull and opaque in appearance.
 Fibers bundled together branch and anastomose; individual fibers do
not branch.
 With the EM, unit fibrils of collagen show periodic cross striations
every 67 nm of their length.
 (a) In tendons, type I
fibrils are all oriented in the Interactions of fibrous collagens
direction of the stress applied to with nonfibrous fibril-associated
the tendon. Proteoglycans and type
VI collagen bind noncovalently to
collagens.
fibrils, coating the surface. The
microfibrils of type VI collagen,
which contain globular and triple-
helical segments, bind to type I
fibrils and link them together into
thicker fibers. (b) In cartilage, type
IX collagen molecules are covalently
bound at regular intervals along
type II fibrils. A chondroitin sulfate
chain,
covalently linked to the 2 type IX
chains at the flexible kink, projects
outward from the fibril, as does the
globular N-terminal region.
 Because collagen synthesis depends on the expression of
several genes and on several post-translation events, many
human diseases are associated with faulty collagen
synthesis.
 Progressive systemic sclerosis- excessive accumulation of
collagen (fibrosis) in almost all organs
 Keloid- local swelling caused by abnormal amounts of collagen
that form in scars of skin
 Ehlers-Danlos type IV- aortic/ intestinal rupture due to faulty
transcription of collagen type III
 Ehlers-Danlos type VII- increased articular motility due to
decreased procollagen peptidase activity
 Scurvy- ulceration of gums, hemorrhages due to lack of Vit. C, a
cofactor for proline hydroxylase
 Osteogenesis imperfecta- spontaneous fractures & cardiac
insufficiency due to mutation in collagen type I
 YELLOW or ELASTIC FIBERS
 Form gentle curves or spirals at their free ends when
released from tension
 Do not form bundles; individual fibers branch and
anastomose to form networks
 They can be stretched to 150% of their length without
breaking, but lose their resiliency with advancing age.
 Appears yellowish, highly refractile, homogenous and are
not made up of fibrillar subunits that are visible with the
light microscope.
 Each fibril is made up of still smaller fibrils united by a
small amount of ground substance. These smaller
“microfibrils” have periodic cross bandings.
 Synthesis and Assembly of Elastin:
1. Intracellular- microfibrillar proteins containing mostly
hydrophilic amino acids, and proelastin (contains large
amounts of the hydrophobic amino acids glycine, proline and
valine, thus accounting for elastin’s insolubility) are
synthesized on rER and secreted separately.
2. Extracellular- proelastin molecules polymerize extracellularly
to form elastin chains.
 Lysyl oxidases then catalyze the conversion of certain lysine
residues of elastin to aldehydes, 3 of which condense with a 4th
unaltered lysine residue to form desmosine and isodesmosine.
 These very rare amino acids found in elastin cross-link
individual chains, which then associate with numerous
microfibrils to form a branching and anastomosing network of
elastic fibers.
 ARGYROPHYL or RETICULAR FIBERS
 Fibers are not branched, and are not so wavy as the collagenous
fibers when released from tension.
 They are chemically identical to collagen, hence these fibers are
considered as precursors of type I and III collagen; however,
they are thinner and form delicate networks instead of thick
bundles
 Chemical characteristics- show affinity to silver (black) stains,
do not yield gelatin on boiling; not easily dissolved by dilute
acids and alkali; not so easily digested by gastric juice; not so
resistant to solutions of alkaline pancreatic juice.
 Distribution- abundant in regions around blood vessels, muscle
fibers, fat cells, basement membrane of epithelia, endoneurium,
lymphoid organs and red bone marrow.
Collagen
The connective tissue protein
Why study collagen?
 To understand normal body functions .
 In development
 In inflammatory states
 Aging process.
 To identify the cause of various genetic and metabolic disorders related to
tissue proteins
 In the spread of cancer cells.
 Several diseases (eg:Osteogenesis imperfecta and a number of types of the
Ehlers-Danlos syndrome) are due to genetic disturbances of the synthesis of
collagen.
.
 To use and apply them in the medical, industrial, commercial fields.
 In food products, cosmetic surgery.
 The gelatin used in food and industry is derived from the partial hydrolysis of
collagen.
The Extra cellular matrix
 Collagen
Most abundant insoluble fibrous protein in the connective tissue of mammals.
Makes up about 25% to 35% of the whole-body protein content.
Scleroprotein secreted from the cells called fibroblasts.
In greek ‘kolla’ means ‘glue’.Collagen is also called as glue-producer.
Distribution of collagen varies in different tissues.

Also found in mucous membranes, nerves,Blood vessels, and organs.

bones tendons
skin liver
4%
70% 90%

85%

Collagen fibers in muscle

tendons
Functions Of Collagen
 It imparts strength, support, shape
and elasicity to the tissues.
 It accounts for 6% of the weight
of strong, tendinous muscles
 It provides flexibility, support, and
movement to cartilage.
 It encases and protects delicate
organs like kidneys and spleen.
 It fills the sclera of the eye in
crystalline form.
 Teeth(dentin) are made by adding
mineral crystals to collagen.
 Collagen contributes to proper
alignment of cells for cell proliferation
and differentiation.
 When exposed in damaged blood
vessels, it initiates thrombus
formation
 Types Of Collagen
 In humans, there are at least 19 distinct
types of collagen made up of 30 distinct
polypeptide chains (each encoded by a
separate gene).
 They are subdivided into a number of
classes based primarily on the structures
they form
 However, 90% of the collagen in the body
are of type I, II, III, and IV.
 These types determine the physical
properties of specific tissues and perform
their specialized function.
Structure Of Collagen
The basic structural unit of a collagen
Amino-
molecule is a triple helix.
Triple helical structure may occur acids
throughout the molecule or only in a part
of it.

Structure Of type I mature Collagen:

 Alpha chain Triple Helix
Triple helical structure occurs
throughout the molecule.

 This triple helix is composed of 3

polypeptide chains twisted around
each other.

 Each polypeptide/alpha chain is in

turn a left handed helix with 3 amino
acids per turn totally containing
approximately 1000 amino acids per
chain.
 Each alpha chain has an unusual abundance of
3 amino-acids glycine, proline and
hydroxyproline.

 Glycine occurs at every third position in the

amino acid sequence which can be
represented as (Gly-X-Y)n.

 X and Y are other amino acids of which

proline and hydroxyproline occupy 100
positions each.

 Glycine occupies the crowded center of the

triple helix as it has a small side chain[ H
atom] where as Hydroxyproline and proline
point outwards imparting rigidity to the triple
helix.

 The alpha chains are wound around each

other in a right handed super helix to form a
rod like molecule 1.4nm wide and 300nm long [Gly-X-Y]n
 These triple helical molecules pack together side
The triple helices are stabilized by
by side to form elongated fibrils.
Hydrogen bonds, covalent cross-links,
electrostatic and hydrophobic interactions Fibrils are displaced longitudinally from each other
and van der waals forces. by 67 nm [one quatter of its length] to form a quarter
staggered arrangement.
The covalent cross links within and
between the helices are formed by copper Fibrils bundle up to form fibers making up tissues.
dependent enzyme lysyl oxidase
between the lysine and hydroxylysine
residues. Cross link formation
Structure of collagen fibers
Biosynthesis of Collagen
 Collagen synthesis occurs in the fibroblasts, osteoblasts in bone,
chondroblasts in cartilage and odontoblasts in teeth.
 First synthesized in precursor form of preprocollagen polypeptide chain in
the ribosomes during translation
 The leader sequence of amino acids[signal peptide] in the
preprocollagen directs it to enter the lumen of E.R
 In the lumen of E.R, the Signal peptide is cleaved to form procollagen.
 The proline and lysine amino acids in the procollagen chain undergo
hydroxylation and glycosylation known as post translational modifications.
 Disulfide bonds are formed between three procollagen chains which twist
around each other to form a triple helix molecule.This step is called
registration.
 This Procollagen molecule is secreted into the extracellular matrix from
the golgi compartment of the E.R.
 Here, the procollagen aminoproteinase and carboxy proteinase enzymes
remove extra terminal amino acids from the procollagen molecule to form
collagen .
 The collagen molecules assemble into fibrils and inturn fibers being stabilized by
the covalent cross-links.
Biosynthesis of
collagen from
Preprocollagen
Synthesis Of Collagen
 Abnormalities associated with collagen
 Collagen-related diseases arise from
 genetic defects
 nutritional deficiencies
 They affect the biosynthesis, assembly, postranslational modification, secretion, or
other processes involved in normal collagen production.
 These include :
 Ehler Danlos syndrome
 Alport syndrome
 Epidermolysis bullosa
 Osteogenesis Imperfecta
 Chondrodysplasias [affects cartilage]
 Scurvy
 Osteolathyrism
Ehler Danlos Syndrome
 Ehlers-Danlos Syndrome is a group of inherited connective tissue disorders.
 CAUSE
 abnormalities in the synthesis and metabolism of collagen
 Mutations in the collagen genes: COL1A1, COL1A2, COL3A1, COL5A1, COL5A2
 a deficiency of enzyme lysyl hydroxylase.
 A deficiency of procollagenN-proteinase, causing formation of abnormal thin,
irregular collagen fibrils
 EFFECT
 Mutations alter the structure, production, or processing of collagen or proteins that
interact with collagen
 WeakenS connective tissue in the skin, bones, blood vessels, and organs causing-
 Skin hyperextensibility
 Joint dislocations
 Tissue fragility
 Poor wound healing.
Ehler-Danlos Syndrome

•Hyperextensibility of
skin

•Hypermobility of joints
 Alport Syndrome
 Alport syndrome is a genetic disorder
characterized by glomerulonephritis,
endstage kidney disease, and hearing loss.
 It also affects the eyes.
 The presence of blood in the
urine[hematuria] is almost always found
in this condition.
 CAUSE
 Mutations in COL4A3,COL4A4,COL4A5
collagen biosynthesis genes.
Alport syndrome affecting eyes  These prevent the production or
assembly of the type IV collagen
network in the basement membranes.
 kidneys are scarred and unable to filter
waste products resulting in hematuria
and renal disease.
 Epidermolysis Bullosa
• Epidermolysis bullosa refers to a group of
inherited disorders that involve the
formation of blisters following trivial trauma.
• CAUSE Blister
 mutations in COL7A1, affecting the formation
structure of type VII collagen.
 Type VII collagen forms delicate fibrils
that anchor the basal lamina to collagen
fibrils in the dermis.
 These anchoring fibrils are reduced in
this form of the disease, causing friction
and blistering.
• EFFECT
 Blistering and painful sores like third
degree burns
Osteogenesis Imperfecta
 Osteogenesis imperfecta or Brittle Bone Disease is a
genetic bone disorder due to decreased collagen
formation.
 CAUSE
 Mutations in the COL1A1 andCOL1A2 genes coding
for procollagen chains.
 Replacement of glycine by another bulkier amino acid
resulting in decreased collagen or improper
procollagen structure forming abnormal fibers.
 Mutations also cause ‘procollagen suicide ‘
 All these cause brittleness.
 EFFECT
 Thin,t ransclucent, blue scleras.
 Affected infants may be born with multiple fractures
and not survive.
 weak muscles, brittle teeth, a curved spine and
hearing loss.
 Chondrodysplasias
 Chondrodysplasias are a mixed
group of hereditary disorders
affecting cartilage.
 One example is Stickler
syndrome, manifested by
degeneration of joint cartilage
and of the vitreous body of the
eye.
 CAUSE
 Mutations in the COL2A1
gene, leading to abnormal
forms of type II collagen.
 EFFECT
 shortlimbed dwarfism
 skeletal deformities.
Osteolathyrism
 Osteolathyrism is a collagen cross-linking
deficiency caused by dietary over-reliance
on the seeds of Lathyrus sativus (kesari
dal) in some parts of India.
 CAUSE
 Osteolathyrogenic compounds like Beta-
aminopropionitrile(BAPN) and Beta-oxalyl
aminoalanine [BOAA] found in Kesari dhal
inhibit enzyme lysyl oxidase required for
the formation of cross links in the triple
helices
 EFFECT
 weakness and fragility of skin, bones, and
blood vessels
 Paralysis of the lower extremities
associated with neurolathyrism
 Scurvy
 Scurvy is a disease due to deficiency of
vitamin C
 It is not a genetic disease.
 It is related to improper collagen
formation
 CAUSE
 Vitamin C [ascorbic acid ]is required
as a cofactor for hydroxylase
enzymes during the hydroxylation
of proline and lysine in the
synthesis of collagen.
 Deficiency causes impaired collagen
synthesis due to deficiency of
hydroxylases.
 EFFECT
 Bleeding of gums
 Poor wound healing
 Subcutaneous hemorrhages
Uses Of Collagen
 Industrial Uses
 Collagen is used as temporary thermoplastic glues in musical instruments like violin
and guitar .
 Recently used as a fertilizer
 Gelatin derived from the partial hydrolysis of collagen is used in food products like
desserts, jellies.
 It is also used in pharmaceutical, cosmetic, and photography industries.
 Medical uses
 Mild benefit to rheumatoid arthritis patients.
 Keeps the valvular leaflets of heart in shape.
 Helps in the deposition of calcium during aging.
 Used in cosmetic surgery, for burn patients for reconstruction of bone and a wide
variety of dental, orthopedic and surgical purposes.
 Main ingredient of cosmetic makeup.
 Human collagen is used for immunosuppression during transplantation.