Unit 2 - 18BTE416T Tissue Engineering For Regenerative Medicine

Organotypic Models:
The cellular interactions that occur in the in vivo system are not possible with isolated cells. The recent
developments in the organ and histotypic cultures focus to create in vitro models comparable (as far as possible in
biology and functions) to the in vivo systems. The purpose of this organotypic models is to retain the original
structural and functional interactive relationships of the organ.
There are three broad approaches in this direction:
1. Organ cultures:
The whole organs or small fragments of the organs that retain the special and intrinsic properties are used in
culture.
2. Histotypic cultures:
The cell lines grown in three dimensional matrix to high density represent histotypic cultures.
3. Organotypic cultures:
In this case, the cells from different lineages are put together in the desired ratio and spatial relationships to create
a component of an organ in the laboratory.
1. Organ Cultures:
The use of organ cultures (organs or their representative fragments) with reference to structural integrity, nutrient
and gas exchange, growth and differentiation, along with the advantages and limitations is briefly described.
Structural Integrity:
As already stated, the isolated cells are individual, while in the organ culture, the cells are integrated as a single unit.
The cell to cell association, and interactions found in the native tissues or organs are retained to a large extent. As
the structural integrity of the original tissue is preserved, the associated cells can exchange signals through cell
adhesion or communications.
Nutrient and Gas Exchange:
There is no vascular system in the organ culture. This limits the nutrient supply and gas exchanges of the cells. This
happens despite the adequate care taken in the laboratory for the rapid diffusion of nutrients and gases by placing
the organ cultures at the interface between the liquid and gaseous phases.
As a consequence, some degree of necrosis at the central part of the organ may occur. Some workers prefer to use
high O2 concentration (sometimes even pure O2) in the organ cultures. Exposure of cells to high O2 content is
Growth and Differentiation:
In general, the organ cultures do not grow except some amount of proliferation that may occur on the outer cell
layers.
Advantages of Organ Cultures:
i. Provide a direct means of studying the behaviour of an integrated tissue in the laboratory.
ii. Understanding of biochemical and molecular functions of an organ/tissue becomes easy.
Limitations of Organ Cultures:
i. Organ cultures cannot be propagated, hence for each experiment there is a need for a fresh organ from a donor.
ii. Variations are high and reproducibility is low.
iii. Difficult to prepare, besides being expensive.
Techniques of Organ Culture:
The most important requirement of organ or tissue culture is to place them at such a location so that optimal
nutrient and gas exchanges occur.
This is mostly achieved by keeping the tissue at gas- limited interface of the following supports:
i. Semisolid gel of agar.
ii. Clotted plasma.
iii. Micro-porous filter.
iv. Lens paper.
v. Strip of Perspex or Plexiglas.
In recent years, filter-well inserts are in use to attain the natural geometry of tissues more easily.
Procedure for Organ Culture:
The basic technique of organ culture consists of the following stages:
1. Dissection and collection of the organ tissue.
2. Reduce the size of the tissue as desired, preferably to less than I mm in thickness.
3. Place tissue on a support at the gas medium interface.
4. Incubate in a humid CO2 incubator.
5. Change the medium (M199 or CMRL 1066) as frequently as desired.
6. The organ culture can be analysed by histology, autoradiography and immunochemistry.
Organ Culture on Stainless Steel Support Grid:
Small fragments of tissue can be cultured on a filter laid on top of a stainless steel grid (Fig. below).
Organ Culture on Filter-well Inserts:
Filter-well inserts have become very popular for organ cultures. This is mainly because the cellular interaction,
stratification and polarization are better in these culture systems. Further, the recombination of cells to form tissue —
like densities, and access to medium and gas exchange are better.
The four different types of filter wells for growing tissues in the form of cell layers are depicted in Fig.
i. Growth of cell layer on top of filter (Fig. A).

ii. Growth of cell layers on matrix (collagen or matrigel) on
top of filter (Fig. B).
iii. Cell layers grown on the interactive cell layers placed on
the underside of filter (Fig. C).
iv. Cell layer grown on the matrix with interactive cell layer
on the underside of the filter (Fig. D).
Filter well-inserts with different materials (ceramic,
collagen, and nitrocellulose) are now commercially
available for use in culture laboratories.
Filter-well inserts have been successfully used to develop
functionally integrated thyroid epithelium, stratified
epidermis, intestinal epithelium and renal (kidney)
epithelium.
2. Histotypic Cultures:
Growth and propagation of cell lines in three- dimensional matrix to high cell density represent histotypic cultures.
The advantage with this culture system is that dispersed monolayer cultures can be used to regenerate tissue-like
structures. The commonly used techniques in histotypic cultures use gel and sponge hollow fibers and spheroids.
Gel and Sponge Technique:
The cells (normal or tumor) in culture can penetrate gels (collagen) or sponges (gelatin) which provides a matrix for
morphogenesis of primitive cells. This approach has been used for the development of mammary epithelium, and some
tubular and glandular structures.
Hollow Fibers Technique:
In recent years, perfusion chambers with a bed of plastic capillary fibers have been developed. The advantage of using
hollow fibers in histotypic cultures is that nutrient and gas exchange is more efficient. As the cells attached to capillary
fibers grow, there occurs an increase in cell density to form tissue-like structures.
Many workers claim that the behaviour of high-density cells formed on hollow fibers is comparable to their in vivo
behaviour. For instance, choriocarcinoma cells grown in hollow fiber cultures release more chorionic gonadotrophin
than in a conventional monolayer. Hollow fiber culture techniques are regarded as ideal systems for the industrial
production of several biologically important compounds. Work is progressing in this direction.
Three Dimensional Cultures:
Spheroids in Histotypic Culture:
Spheroids represent the clusters of cells usually formed by the re-association of
dissociated cultured cells. It is known for some years that the dissociated embryonic
cells reassemble to form a specialized structure. The basic principle of using spheroids
in histotypic culture is that the cells in heterotypic or bomotypic aggregates are capable
of sorting out themselves into groups to form tissue-like architecture. The major
drawback of spheroids is the limitation in the diffusion and exchange of nutrients and
gases.
Multicellular Tumor Spheroids (MCTS):
Multicellular tumor spheroids provide an in vitro proliferating model for studies on
tumor cells. The three dimensional structure of MCTS allows the experimental studies
related to drug therapy, penetration of drugs, resistance to radiation etc.
Further, MCTS have also been used to study several biological processes:
i. Regulation of cell proliferation and differentiation.
ii. Immune responses.
iii. Cell death.
iv. Cell invasion.
v. Gene therapy.
The main advantage of three dimensional cell cultures (in the form of MCTS) is that
they provide a well-defined geometry of cells planar or spherical which is directly
related to the structure and function. It is now well accepted that the MCTS behave like
the initial avascular stages of solid tumors in vivo. However, beyond a critical size (≥
500 mm), most of the MCTS develop necrosis (death of cells) at the centre surrounded
by viable cells. A diagrammatic representation of MCTS in comparison with tumor is
depicted in Fig.
Technique of MCTS production:
Single-cell suspension obtained from trypsinized monolayer cells or disaggregated tumor is
inoculated into the medium in magnetic stirrer flasks or roller tubes. As the incubation is carried
out for about 3-5 days, aggregates of cells representing spheroids are formed. It is observed that
spheroid formation is more efficient under static conditions on stationary and non-adhesive
surfaces. For this reason, agar/agarose-coated culture dishes to which cells do not adhere are
frequently used to initiate spheroid formation.
Once the spheroids are formed, they are transferred to 24 well plates for analysis. Spheroid
growth is quantified by measuring their diameters regularly. This can be done by using a
microscope eyepiece micrometer or an image analysis scanner. Good growth of spheroids is
observed when grown in wells.
Transfectant mosaic spheroids:
It is now possible to produce spheroids from cells that have been transfected with different genes.
Mosaic spheroids are formed by mixing transfected and non-transfected spheroids in the desired
proportion.
MCTS co-cultures:
MCTS can be produced from heterogenous cells also, forming MCTS co- cultures. This is
comparable to heterologous spheroids (in short heterospheroids) consisting of tumor cells in
combination with host cells.
Some of the MCTS co-cultures are listed:
i. MCTS and immune cells.
ii. MCTS and fibroblasts.
iii. MCTS and endothelial cells.
Heterospheroids with heterotypic cell interaction serve as good models for studying several in
vivo processes e.g. inflammation. MCTS co-cultures are very useful in tissue modelling and tissue
engineering, the details of which are given later.
Applications of Spheroids or MCTS:
Spheroids have a wide range of applications. Some of the important ones are listed:
i. Serve as models for a vascular tumor growth.
ii. For the study of gene expression in a three dimensional configuration of cells.
iii. To determine the effect cytotoxic drugs, antibodies, radio nucleotides used for therapeutic
purposes.
iv. To study certain disease processes e.g. rheumatoid arthritis.
v. For the development of gene therapies for several diseases e.g. cancer.
vi. To evaluate radiation effects on target tissues.
vii. For the development of tissues and tissue models.
3. Organotypic Cultures:
Organotypic culture basically involves the combination of cells from different lineages in a determined ratio to create a
component of an organ. With the advances in the organotypic culture techniques, it is now possible to develop certain
tissues or tissue models.
i. Skin equivalents have been created by co-culturing dermis with epidermis with interviewing layers of collagen.
ii. Models for prostate and breast.
iii. Models for control of growth and differentiation of lung.
What is the organotypic culture?
Organotypic culture is defined as the culture of an organ collected from an organism. It is one method allowing the culture of
complex tissues or organs. It allows the preservation of the architecture of the cultured organ and most of its cellular
interactions.
Figure: Illustration of various organotypic culture methods
Several protocols have already been developed like agar methods, watch glass protocols, organ culture in
media, and filter well inserts.
Historical background
While cell lines are one of the most often used experimental models by scientists, surprisingly, the culture
of whole organs start a few decades before the first establishment of cell lines.
Figure: Time scale of the parallel development of both organotypic culture and cell lines
At the end of the 19th century, L. Loeb managed to cultivate the organs of rabbits. Quickly after, other
authors report the culture of different organs collected from different organisms. In the meantime, the first
establishment of cell lines has been done, slowing the development of organotypic culture. The HeLa cell
line marked the beginning of the intensive utilization of such a system by the scientific community. For
example, HeLa cells are now cited in more than 70000 articles and part of 11000 patents. MCF-7 cells
(another classically used cell line) are cited in more than 23000 articles. It reflects the intensive use of cell
lines in research.
Assets and drawbacks
The massive use of cell line by researchers comes from the need to have a robust, reproducible, cheap, and
fast experimental model, that are key features of cell line cultures. Despite all of these advantages, some
drawbacks appear over time. First, the genetic deviation/differences of cells from the same cell line
between two samples lead to the loss of reproducibility and robustness while using cell lines as models.
Second is the loss of certain cell functions because the culture conditions are driven by the growth and not
by the expression of a tissue-like phenotype. An illustration of this is the metabolism in the liver and liver
cell lines. This bias makes the extrapolation of cell lines data to human tricky or even unrealistic.
Figure: Schematic view of features for different culture model: from fractions to organotypic culture
For a few years, the pressure of regulatory agencies to improve the risk assessment for humans has to lead
to re-consider the use of organotypic cultures. Whereas they are more expensive, more complex, and time-
consuming, they offer better extrapolation possibilities and are more relevant by taking into account the
physiological environment. One of the best models is the culture of human organs, but the difficulties to
handle such a system and to obtain such organs push researchers towards new systems offering both the
advantages of cell cultures and organ culture.
From organotypic culture to 3D models
As mentioned above, there is now a need for developing new in vitro systems offering the advantages of
both cell lines and organ culture. One of the solutions is the 3D cell cultures, eg. spheroids. The development
of such a technique has to be based on the knowledge acquired with the two models.
Figure: A: representation of the derivation of human cell cultures to obtain human spheroids; B: synergy
and inter-dependency of different cultures approach. From Pamies and Hartun, 21st-century cell culture for
21st-century toxicology, Chemical Research in toxicology, 2016.
Introduction to Basic Wound Healing
WOUND
• It is a circumscribed injury which is caused by
external force and it can involve any tissue and
organ.
• A wound is a break in the integrity of the skin
tissues
or often which may be with
associated disruption of the structure and
function.
(SRB 4th edition)
• A cut or break in the continuity of any tissue,
caused by injury or operation.
(Baillière’s 23rd Ed)
CLASSIFICATION OF WOUNDS
Rank and Wakefield classification

a) Tidy wounds
b) Untidy wounds
Classification based on type of wound
i. Clean incised wound
ii. Lacerated wound
iii. Bruising and contusion
iv. Haematoma
v. Puncture wound
vi. Abrasion
vii. Crush injury
viii. Injuries to bone and joint (maybe open or closed)
ix. Injuries to nerve (either clean cut or crush)
x. Injuries to arteries and veins
xi. Penetrating wounds
Classification based on thickness of wound
a) Superficial wound
b) Partial thickness
c) Full thickness
d) Deep wounds
e) Complicated wounds
f) Penetrating wound
Superficial
Partial thickness
Full thickness
Deep wound
Classification of surgical wounds
a) Clean wound
b) Clean contaminated wound
c) Contaminated wound
d) Dirty infected wound
HEALING
• Healing is the body’s response to injury in

an attempt to restore normal structure and
function.
The process of healing involves 2 distinct
processes:
A. REGENERATION
B. REPAIR
• Regeneration: Is when healing takes place by
proliferation of parenchymal cells and usually
results in complete restoration of the original
tissues.
• The goal of all surgical procedures should be

regeneration which returns the tissues to their
normal microstructure and function.
• Repair: It is a healing outcome in which tissues
do not return to their normal architecture and
function.
• Repair typically results in the formation of scar

tissue.
TYPES OF WOUND HEALING
• Healing by first intention (wounds with opposed

edges)
• Healing by secondary intention (wounds with

separated edges)
Healing by first intention (wounds with
opposed edges)
Healing of wound with following characteristics:
 Clean and uninfected
 Surgically incised
 Without much loss of cells and tissue
 Edges of wound are approximated by surgical sutures.
 Wounds with opposed edges
 Primary union
• The incision causes
death of a limited number of epithelial
cells and connective tissue cells
disruption of epithelial basal
membrane continuity
• The narrow incisional space immediately fills

with clotted blood containing fibrin and blood
cells; dehydration of the surface clot forms the
well known scab that covers the wound.
Within 24 hours
•Within 24 to 48 hours, spurs
• Neutrophils appear at margins of epithelial cells from the both
edges migrate and grow along
of incision, moving toward fibrin
the cut margins of the dermis,
clot
depositing BM components as
• Epidermis at its cut edges they move. They fuse in the
thickens as a result of mitotic midline beneath the surface
activity of basal cells scab, thus producing a
continuous but thin
epithelial layer.
By day 3,
• Neutrophils replaced by macrophages

• Granulation tissue progressively invades incision
space
•Collagen fibers are now

present in the margins of the
incision, but at first these are
vertically oriented.
•Epithelial cell proliferation

continues, thickening epidermal
covering layer
By day 5,
• Incisio
nal
space
is
filled
with
granul
ation
tissue
• Neova
sculari
sation
is
maxim
al
• Collag
en
During the second week
• Continued of collagen and

accumulation proliferation
of fibroblasts
• Leukocytic infiltrate, edema, and
increased vascularity have largely disappeared.
By the end of the first
month,
• Scar comprises a cellular connective tissue devoid of
inflammatory infiltrate, covered now by intact epidermis.
• Dermal appendages that have been destroyed in the line

of the incision are permanently lost.
• Tensile strength of the wound increases thereafter, but it

may take months for the wounded area to obtain its
maximal strength.
Healing by second intention
• Wounds with separated edges
• Secondary union
• When there is more extensive loss of

cells and tissue
• Regeneration of parenchymal cells
cannot completely reconstitute the
original architecture.
• Abundant granulation tissue grows in
from the margin to complete the repair.
Secondary healing differs from primary
healing in several respects:
 Inflammatory reaction is more intense
 Much larger amounts of granulation tissue are formed
 Wound contraction occurs in large surface

wounds
 Substantial scar formation and thinning
of the epidermis occurs
Difference between 1˚ & 2˚ union of
wound
FEATURES PRIMARY SECONDARY
CLEANLINESS CLEAN NOT CLEAN
INFECTION NOT INFECTED INFECTED
MARGINS SURGICALLY CLEAN IRREGULAR
SUTURES USED NOT USED
HEALING SMALL GRANULATION LARGE GRANULATION

TISSUE TISSUE
OUT COME LINEAR SCAR IRREGULAR WOUND
COMPLICATION NOT FREQUENT FREQUENT

STAGES OF WOUND HEALING
1. Stage of inflammation.
2. Stage of granulation tissue formation and organisation.
3. Stage of epithelialisation.
4. Stage of scar formation and resorption.
5. Stage of maturation.
PHASES OF WOUND HEALING
•For soft tissue wound healing:

1. Inflammatory phase: It can be broken down into further
a) Clot formation
b) Early inflammation
c) Late inflammation
2.Proliferative phase 3.Maturation phase

Inflammatory phase
a) Clot formation:
- Begins with three events:
i. Blood vessel contraction initiated by
platelet
degranulation of serotonin, which acts on endothelial
cell and increases the permeability of the vessel,
allowing a protein rich exudate to enter the wound
site
ii. A platelet plug formation
iii. Activation of extrinsic and intrinsic clotting
mechanism
CONTACT ACTIVATION
PATHWAY
TISSUE ACTIVATION
PATHWAY
• These events stabilize hemostasis, begins production of
chemoattractants and initiate the process of wound
decontamination.
• This result in formation of a coagulum.

• Compression of surgical flap with sterile iced
gauze immediately after surgery is designed to
minimize the thickness of fibrin clot and thereby
accelerate optimal wound healing.
b) Early inflammation:
• Characterized by of polymorphonuclear
production neutrophils (PMNs)
• Begin to enter the wound site within 6 hours of

clot
stabilization.
• The number of PMNs increases steadily, peaking

at
about 24 to 48 hours after the injury.
• Three key steps mark PMN migration into the
wound site: a) Pavementing
b) Emigration
c) Migration
• Main role of PMNs is wound decontamination
by
phagocytosis of bacteria.
• The number of PMNs drop rapidly after the third day.
c) Late inflammation:
• Presence of macrophages.
• Reaches peak concentration by

approximately third or fourth day.
• They have longer life than PMNs and remain in

wound till healing is completed.
• Are more bioactive then PMNs – secrete a vast array of
cytokines – leads to initiation of proliferative phase of
wound healing.
• Major functions of macrophages:
 wound decontamination through phagocytosis

and digestion of microorganisms and tissue debris.
 ingestion and of antigens for
processing
presentation to T lymphocytes
 regulation of wound healing
Proliferative Phase
• Characterized by formation of granulation tissue in the wound.

• 2 key cell types are present in this phase:
a) fibroblasts
b) endothelial cells
• Granulation tissue:
It is a fragile structure composed of an extracellular
matrix of fibrin, fibronectin, glycosaminoglycans,
proliferating endothelial cells, new capillaries, and
fibroblasts mixed with inflammatory macrophages and
lymphocytes.
• Epithelial cells are active during this phage and
are responsible for initial wound closure.
• GTR procedures are based on control of

the epithelial cell growth rate during this phase.
a) Fibroblasts: Fibroplasia
– They migrate into the wound site on the 3rd day after
injury and achieve their peak numbers by
approximately 7th day.
– This action is stimulated by combination of cytokines

produced initially by platelets and subsequently by
macrophages and lymphocytes.
– As the number of macrophages declines and
fibroblasts population increases the wound transforms
from granulomatous tissue to granulation tissue.
– Fibroblasts produces collagen (first detected in the

wound about the third day of injury) .
– Fibroblasts produce type III collagen initially and as

the wound matures type I collagen is formed.
– As wound healing progresses, the collagen fibers become
organized by cross-linking.
– A focused type of fibroblast known as a myofibroblast plays a
significant role in wound contraction, particularly in incisional-
type wounds.
– Myofibroblasts align themselves parallel wound

surface with the then contract,
and together. drawing the wound edges
– These cells are eliminated by apoptosis after wound closure.

b)Endothelial cells: Angiogenesis
–Formation of new blood vessels at the site of injury

takes place by proliferation of endothelial cells from the
margins of severed blood vessels.
–The newly formed blood vessels are more leaky

accounting for the more edematous appearance of new
granulation tissue.
Epithelium:
• The first step is formation of an epithelial seal on the surface
of the fibrin clot. It begins at the edge of the wound, where the
basal and suprabasal prickle cells rapidly undergo mitosis.
• Migration stops as a result of contact inhibition of the
epithelial cells from the opposing wound edge.
• In wounds healing by primary intention, formation of an
epithelial seal typically takes 21 to 28 hours after
reapproximation of the wound margins.
Maturation Phase
• Begins 5 to 7 days after injury.
• There is conversion of granulation tissue to fibrous

connective tissue and decrease parallelism of collagen
to the plane of the wound.
• Maturation of the epithelial layer quickly follows

formation of the epithelial seal.
• Scar strength is:
– 3% in 1 week
– 20% in 3
weeks
– 80% in 12
weeks
Growth Factors and Cytokines:Affecting Various Steps in
Wound Healing
Monocyte chemotaxis PDGF, FGF, TGF-ß
Fibroblast migration PDGF, EGF, FGF, TGF-ß, TNF,
IL-1
Fibroblast proliferation PDGF, EGF, FGF, TNF
Angiogenesis VEGF, Ang, FGF
Collagen synthesis TGF-ß, PDGF
Collagenase secretion PDGF, EGF, FGF, TNF, TGF-ß
inhibits
PDGF- platelet-derived growth factor IL- interleukin

FGF- fibroblast growth factor TNF- tumor necrosis factor
TGF- transforming growth factor VEGF- vascular endothelial growth factor
EGF- epidermal growth factor
HARD TISSUE HEALING
• The inflammatory and proliferative phases

are similar to those for soft tissue.
• The maturation phase differs markedly from that

for soft tissues because of the tissue involved.
Osteoblasts: Osteogenesis
• A major difference lies in the role of the osteoclast.
• Osteoclasts acts as an organizational unit to debride

necrotic bone from the wound margin similarly as
macrophages remove tissue debris from the clot.
• New bone formation is apparent about 6 days after
surgery.
• Bone formation is categorized into two types:
a) Matrix vesicle – based process
b) Osteoid secretion
a) Matrix vesicle – based process
• Woven bone formation occurs by this process.
• In this process osteoblasts produce matrix vesicles through

exocytosis of their plasma membrane.
• It begins with the deposition and growth of hydroxyapatite
crystals in the pore regions.
• The crystals amalgamate to form spherulites whose union
results in mineralization.
b)Osteoid secretion
• Lamellar bone formation occurs by this process.
• Osteoblasts an organic matrix composed of

secrete
longitudinally arranged collagen matrix fibrils.
• Mineralization by mineral deposition directly

occursthe collagen fibrils.
along
• In this stage alkaline phosphatase plays an
important role in mineralization.
• Several growth factors identified as a key components in
the production of osseous tissue are:
a. TGF- ß
b. BMP
c. PDGF
d. FGF
e. IGF
• The osseous defect is filled with bone tissue by
16 weeks after surgery.
Cementoblasts: Cementogenesis:
• Begins 10 to 12 days after root end resection.
• The exact sequence leading to the formation of

new cementum remain unidentified.
• Cementum covers the resected root end

in
approximately 28 days.
FACTORS AFFECTING WOUND
HEALING:
1) Local factors:
i. Infection
ii. Presence of necrotic tissue and foreign body
iii. Poor blood supply
iv. Venous or lymph stasis
v. Tissue tension
vi. Hematoma
vii. Large defect or poor apposition
viii. Recurrent trauma
ix. X-ray irradiated area
x. Site of wound, eg.wound over the joints and back has
poor healing
xi. Underlying diseases like osteomyelitis and
malignancy
2) General factors:
i. Age, obesity,smoking
ii. Malnutrition, zinc, copper
iii. Vitamin deficiency (vit C, vit A)
iv. Anemia
v. Malignancy
vi. Jaundice
vii. Diabetes
viii. HIV and immunosupressive diseases
ix. Steroids and cytotoxic drugs
COMPLICATION:
1. Deficient scar formation:
a) Wound dehiscence
b) Ulceration
2. Excessive formation of the repair components:
a) Aberrations of growth: -
hypertrophic scar
-keloid
b)Excessive amount of granulation
tissue formation
c) Exuberant proliferation of fibroblasts and other
connective tissue elements: Desmoids or Aggressive
fibromatoses
3. Formation of contractures
SYSTEMIC MEDICATION
AFFECTING WOUND HEALING:
I. Bisphosphonates
II. Glucocorticoids
III. NSAIDS
IV. Cyclooxygenase -2 inhibitors
HEALING OF SINUS TRACT:
• Sinus is a tract leading from an enclosed area of
inflammation to an epithelial surface, and is one of the
sequelae of inflammatory disease.
• A sinus tract is a drainage duct for the suppuration

produced by abscesses.
• Suppuration from the periapical inflammatory process
may be resorbed by the host organism, otherwise it will
flow through the less resistant tissue area.
• It drains onto the epithelial tissue through either a

mucosal, or occasionally, a cutaneous sinus tract.
• Sinus tract adjacent to teeth or near the apex of the tooth
is usually considered to be of endodontic origin and root
canal therapy is the primary treatment to achieve its
healing.
• The presence of a sinus tract in the oral cavity is usually

considered of pulpal origin, but it can also be caused by
periodontal disease.
Draining sinus Healed sinus
Sinus is active with pus drainage Sinus has healed with pus discharge
absent
Surrounding mucosa reddish pink in Surrounding mucosa presents normal
color tissue coloration
Gutta- percha point can be inserted Gutta- percha cannot be inserted
Treatment:
• Treatment is directed towards elimination of the source
of infection.
• The offending tooth is removed if it is too badly decayed,
or if there is extensive loss of the surrounding alveolar
bone.
• In most cases, the sinus tract heals spontaneously if the
infected pulp is removed, and the root canal debrided
and filled.
• Some chronic sinus tracts heal leaving behind a
small residual scar, excision may not be
required unless its appearance is of concern to
the patient.
• If there is fibrosis of the sinus tract trajectory

then surgical removal is necessary.
WOUND HEALING AFTER
PULPECTOMY
• The healing pattern following pulpectomy is characterized by an initial

inflammatory reaction in the apical tissue due to the trauma induced by
the cutting procedure.
• In the absence of wound infection, reorganization soon occurs. This
involves replacement of the injured tissue by connective tissue derived
from the periapical region.
• Materials used to fill root canals may compromise the
normal healing pattern, owing to their irritating capacity,
and result in a longstanding inflammatory lesion.
• Inflammatory cells accumulate close to the root filling

material and remain for as long as toxic components are
released. Eventually the material will be lined off by
fibrous connective tissue.
• When overfilling occurs, the process of phagocytosis

may eliminate the excess root filling material and
occasionally also material inside the canal.
WOUND HEALING OF APICAL
PERIODONTITIS
• Follows the general principle of wound healing of
connective tissues elsewhere in the body, with the
formation of fibrovascular granulation tissue, removal of
necrotic tissue and dead bacteria by activated
macrophages, and finally repair and/or regeneration of
the wounded tissue.
• Healing is largely accomplished by regeneration and

to some degree by fibrosis.
• Local tissue resident cells involved in periapical wound
healing are osteoblasts and bone marrow stromal cells in
alveolar bone and multipotent stem cells in periodontal
ligament.
• The extracellular matrix and growth factors of cementum

(i.e., IGF-1, FGFs, EGF, BMP, TGF-β, PDGF) are
capable of inducing proliferation, migration, attachment,
and differentiation of multipotent stem cells in the
periodontal ligament into cementoblast-like cells and
produce cementoid tissue on the root surface denuded
of periodontal ligament.
During periapical wound healing, the osteoblasts or
mesenchymal cells lining the surfaces of endosteum
Stimulated by TGF-β, BMPs, IGFs, PDGF, VEGF, and

cytokines released by stromal cells, osteoblasts,
platelets, and bone matrix after bone resorption
Undergo proliferation and differentiation into

osteoblasts
Produce bone matrix

• When one of the cortical bone plates (buccal or
lingual/palatal) is destroyed
• Mesenchymal cells in the inner layer of

periosteum beneath the oral mucosa –
stimulated by TGF-β, BMPs,IGFs, PDGF, and
VEGF
• Undergoes proliferation and differentiation into

osteoblasts and produces bone matrix
• If both cortical bone plates are destroyed by large apical
periodontitis lesions, it is possible that the lesion may be
repaired with fibrous scar tissue because of extensive
destruction of the periosteum beneath the oral mucosa.
• Guided tissue regeneration procedure and bone grafts is

recommended to prevent ingrowth of fibroblasts from
periosteum or submucosa into the bony defect and to
enhance periapical wound healing if periapical surgery is
necessary.
ENDODONTIC IMPLICATIONS (PATHOGENESIS
OF APICAL PERIODONTITIS AS EXPLAINED BY
FISH)
• Described the reaction of the periradicular tissues to
bacterial products, noxious products of tissue necrosis,
and antigenic agents from the root canal
• FISH in 1939 theorized that the zones of infection are

not an infection by themselves but the reaction of the
body to infection. Thus he concluded that the removal of
this nidus of infection will result in resolution of infection.
• Four well defined zones of reaction were found
during the experiment:
a. Zone of infection or necrosis (PMNLs)

b. Zone of contamination (Round cell
infiltrate –
lymphocytes)
c. Zone of irritation (Histiocytes and osteoclasts)
d. Zone of stimulation (Fibroblasts, capillary buds and
Osteoblasts)
FISH Zone
KRONFELD’S MOUNTAIN PASS
THEORY
Kronfeld’s mountain pass theory
PERIAPICAL WOUND HEALING
AFTER SURGICAL
ENDODONTIC THERAPY
• Faster than nonsurgical endodontic therapy.
• Surgical debridement is done.
• Goal of surgical endodontic therapy is to seal microbial

etiology within the root canal system by root- end filling
in most cases.
Periapical Index
(PAI)
HEALING OF ROOT FRACTURE
• Healing of root fracture depends upon:

a) Site of the fracture
b) Status of the pulp
• Two directions of wound healing response are expected:

a) At the pulpal side
b) At periodontal
ligament side
• If pulp is intact at the fracture site, the odontoblastic
progenitor cells will create a hard tissue bridge uniting
the fractures fragments.
• During initial stage of wound healing, traumatized tissue

stimulate an inflammatory response and trigger the
release of a series of osteoclastic activity ,subsequently
obliterating the fracture site.
Any of the following types of resorption may occur:
i. External surface resorption surrounding the

proximal fracture edges at the periodontal side of
fracture.
ii. Internal surface resorption surrounding the

fracture edges centrally at the pulpal side of the
iii. fracture.
Internal tunneling resorption burrows behind the pre-
dentin layer and along the root canal walls of the
coronal fragment.
The pattern of healing of root fracture are:
a) Healing with calcified tissue

b) Healing with interproximal calcified tissue
c) Healing with interposition bone and
of connective tissue
d) Interposition of granulation tissue
Healing With Calcified
Tissue
• The calcified tissue is formed at the fracture site.
• The innermost layer of repair maybe of dentin while the more

peripheral portion of the fracture is incompletely repaired with
cementum.
• Clinically the teeth appears normal.
• Radiographically, the fracture line is discernible,

but the fragments are in close contact.
Healing With Interproximal Calcified
Tissue
• Characterized by presence of connective tissue between the fragments.
• The fracture surfaces are covered by cementum along with connective tissue
fibres running parallel to the fracture surface or from one fragment to the
another.
• Clinically the teeth are firm or may be slightly
mobile.
• Radiographically, the fragments appear
separated by a narrow radiolucent line, and the
fractured edges appear rounded.
Interposition Of Granulation Tissue
• The fracture site is obliterated with granulation

tissue.
• The coronal portion is necrotic while the

apical portion is vital.
• Radiographically, widening of the fracture line
and/or a developing radiolucency corresponding
to the fracture line becomes apparent.
Healing with interposition of bone and
connective tissue
• This mode of healing is a sequelae of trauma prior
to
completed growth of the alveolar process
• Thus, the coronal fragment continues to erupt while the

apical fragment remains stationary.
• Interposition of bone and connective tissues is

seen along the fracture site.
• Clinically the teeth are firm and react normal to
pulp tests.
• Radiographically, the fragments are

seperated by a distinct bony ridge.
HEALING IN PATIENTS
ON CHEMOTHERAPY/
RADIATION THERAPY
• These patients have impaired healing responses.
• Pulp may become necrotic during radiation therapy.
• Symptomatic nonvital teeth should be

treated week endodontically
chemotherapy
1 whereasbefore
asymptomatic nonvital
initiating teeth may
radiation or
be delayed.
PERFORATION AND PROGNOSIS FOR
HEALING
• An endodontic perforation is an artificial opening in
the tooth or its root, created by the clinician during
entrycanal
the to system or by a biologic
pathologic resorption event such as or
communication between caries the root
that canal andresultsin
periodontal tissues. a
• A furcation perforation refers to a mid-curvature openingthe
into the periodontal ligament space and is a worst
possible outcome in root canal treatment.
• A post space perforation is defined as a
communication between the lateral root surface
and the surrounding periodontal structures due
to misdirection or an excessively large post
enlargement.
Timing Of Perforation Repair
• The main complication arising from perforation is the
potential for secondary inflammation of the periodontal
attachment with eventual infection and ultimately tooth
loss if untreatable.
• The timing of repair categorized into immediate or

delayed.
• Immediately closing the communication between the

periodontal tissues and the root canal system promotes
a superior healing potential.
Location Of Perforation
• Perforations can be categorized by location:
a) Subgingival
b) Midroot
c) Apical
• Once perforations exhibit the formation of
osseous defects, the prognosis is compromised
significantly.
• Any perforation located near the gingival sulcus
promoted inflammation and loss of epithelial
attachment, resulting in periodontal pocket
formation.
• Apical and midroot perforations without

communication to the oral cavity has a good
prognosis provided an immediate seal was
obtained .
Size Of Perforation
• Small perforations of the canal space promote a direct and immediate
restoration of the defect.
• It offers fewer chances for periodontal breakdown and epithelial

proliferation within the perforation site.
Vasculogenesi
s Angiogenesis
de novo blood vessel generation
•from
formation of newprogenitor
vascular blood vessels via extension or remodelling of
cells
existing blood vessels
Arteriogenesis
• maturation of blood vessels via increasing the lumen of vessels
John Hunter – first person to coin the term angiogenesis
Tied the artery supplying

one of the deer’s antler
Hunter expected deprived of

blood supply it would fall
off
But in a week or two the

antler warmed up
Thinking his procedure had failed he

investigated found the artery was
still blocked
Around the blockage he

observed a network of
new vessel growth
Formation of new blood vessels out of
pre-existing capillaries
Important and Continuous process

that occurs both in health and disease
When a tissue is formed, it is vital

that it has blood supply for its growth
and sustenance and for this purpose,
angiogenesis is important
Spindle shaped cells
Microvilli – 200 to 400nm
Supported by thin basement membrane

and adjacent collagen fibrils
Bound together by tight junctions of fascia

occludens
Weibel Palade bobies
Pinocytic vesicles
Pericytes :
 Periendothelial smooth muscle cells
 Mesenchymal origin
 Mechanical support & stability
 Contractile in nature
The vascular network formation consists of multiple
coordinated, sequential and interdependent steps
mediated by a wide range of angiogenic factors,
including growth factors, chemokines, angiogenic
enzymes, endothelial specific receptors and adhesion
molecules
1. Vasodilation (NO) and Increased
permeability
2. Separation of pericytes and
Breakdown of basement
membrane
3. Allows the endothelial cell to
escape from the parent
vessel walls
4. Proliferation of endothelial
cells just behind the leading
(‘tip’) of migrating cells
5. Remodelling into capillary
6. tubes Recruitment of
periendothelial cells
7. Suppression of endothelial
proliferation and migration and
deposition of basement membrane
In the process of angiogenesis
Tip cells – migrates
Stalk cells – proliferates and remains

attached to patent vessel
Patent BV are exposed to proangiogenic
stimuli
However….
Only a limited number of cells respond to it,
suggesting the existence of mechanisms
by which these behaviors can be reduced
in neighboring cells
Dll4 Notch receptors
VEGF
(tip cells) (stalk cells)
Highest VEGF Dampens the

conc. migratory ↓ VEGF2
behavior of stalk receptors
Tip cell cells
Promotes proper branching of new vessels & prevents

excessive
angiogenesis by decreasing the responsiveness to VEGF.
Key component for sprout formation

Mediators of angiogenesis
VEG
F
A B and PGF C and D
Embryogenic
After injury and
vessel lymphangiogenesis
in tumors
development
Tyrosine kinase
receptors
VEGFR-2- mediates
all cellular
responses
Hypoxia
Hypoxia-inducible factor 1 (Hif)
Release of VEGF-A
Under normoxemic
conditions HiF levels are
maintained low by
proteasome mediated
destruction regulated by
ubiquitin E3 ligase encoded
by the VHL tumor
suppressor gene
In short, it undergoes
protein degradation by
a protease enzyme
Angiogenesis
 ↑ Migration of endothelial cells
 ↑ Mitosis of endothelial cells
 ↑ Matrix metalloproteinase activity
 ↑ αvβ3 activity
 Creation of blood vessel lumen
Chemotactic for macrophages and granulocytes
Vasodilation (indirectly by NO release)
Platelets
Macrophage
s
Endothelium
Recruitment of Receptors
pericytes PDG Alpha and Beta
F
Induces Fibroblast,
endothelial cells,
smooth muscle cell
proliferation
Macrophages Chemotactic and
Mast cells mitogenic for
Endothelia fibroblasts and
l cells endothelial cells
FGF- Proliferation
1 and
differentiation
FGF-
FGF
of all cell types
2
3 isoforms TGF Beta 1, 2 & 3
Produced by platelets, ECs, lymphocytes &

macrophages
Suppresses Proliferation, migration, survival and

differentiation of ECs
Enhances Extracellular membrane synthesis by
production of collagen, fibronectin and
proteoglycans
Angiopoie
tins
Ang-1 Ang-2
Pericytes
Smooth muscle cells Endothelial cells
Fibroblasts
In contrast to normal endothelium, angiogenic
endothelium overexpresses specific members of integrin
family of ECM-binding proteins that mediate EC
adhesion, migration and survival
αvβ3, αvβ5, α5β1 mediates spreading and migration of ECs
In addition αvβ3 forms cell-surface complexes with matrix

metalloproteinases that cleave ECM proteins
Major contributor to angiogenesis
Zinc requiring proteases that cleave the

extracellular matrix proteins
This proteolysis allows the endothelial cells to

escape into the interstitial matrix (as seen in
sprouting angiogenesis)
STIMULATOR MECHANISM
VEGF Permeability, Migration and

Proliferation of endothelial cells.
FGF Promotes proliferation and
differentiation of endothelial cells,
smooth muscle cells and fibroblasts
Ang 1 and Ang 2 Stabilize vessels
PDGF Recruit smooth muscle cells
TGF β ↑ extracellular matrix production
Integrins Bind matrix macromolcules and

proteinases
MMP Cleaves ECM
INHIBITORS MECHANISMS
Thrombospondins Inhibit cell migration, cell

proliferation, cell adhesion and
survival of endothelial cells
Angiostatins Inhibit cell proliferation and

induce apoptosis of endothelial
cells
Endostatins Inhibit cell proliferation and
induce apoptosis of endothelial
cells
Semaphorins •Inhibit the VEGF and FGF

•Inhibit proliferation of EC
Platelet factor 4 Inhibits binding of FGF and VEGF
TIMP Inhibit MMP
Angiopoietin-2 Antagonises Angiopoietin-1

2 opposing capillary walls establish a zone of
contact
Endothelial cell junctions are reorganised and the

vessel bilayer is perforated to allow growth
factors and cells to penetrate into lumen
A core is formed between the 2 new vessels at the

zone of contact that is filled with pericytes and
myofibroblasts
Angiogenesis in health
The healthy body controls angiogenesis through a series
of on and off switches:
On switches– angiogenesis stimulating growth factors
Off switches- angiogenesis inhibiting growth factors
The normal healthy body maintains a perfect balance of

angiogenesis modulators.
Angiogenesis occurs in the healthy body for
healing wounds and for restoring blood
flow to tissues after injury or insult
Activation of coagulation pathway
Formation of blood clot
Release of cytokines, growth factors,

chemokines
Neutrophils (24 hrs)
Proteolytic enzymes that clear debris
Proliferation and migration of basal

epithelial cells(24-48hrs)
Macrophages (day 3)
Formation of granulation
tissue(angiogenesis and fibrogenesis)
In females, angiogenesis also occurs
during the monthly reproductive cycle

-to rebuilt the uterus lining,
-to mature the egg during ovulation
during pregnancy (to built the placenta)

Angiogenesis in
disease
Chronic inflammatory
disorder of autoimmune
origin that affect many
tissues and organs
Principally attacks the joints

producing a
nonsuppurative
proliferative and
inflammatory synovitis
CD4 T cells IFN γ Macrophages
Diabetic Retinopathy
Retinopathy of Prematurity
Age Related Macular Degeneration
Neovascular Glaucoma
Leading cause of blindness in people aged
20-64yrs
There is astrocyte precursor meshwork
which exists from disc to periphery and
forms template for glial fibres of new
vessels
The precursors of astrocytes secrete VEGF

along with PDGF synthesized by retinal
ganglion cells causing neovessel growth
ROP occurs because the retina of a
preterm infant at birth is incompletely
vascularized and if the postnatal
environment does not match the in
utero environment, the vessels and
neural retina will not grow normally.
Angiogenesis is stimulated by
“physiological hypoxia” of the
developing retina, when the metabolic
demands of the maturing retina
outspace the oxygen supplied by
underlying choroid, resulting in
secreting of VEGF by avascular area
Preterm birth into room air
often causes increase in
oxygen saturation
Non physiological
HYPEROXIA
Downregulation of
HIF
Disruption of normal
vasculature
The attenuated vasculature cannot supply
enough oxygen to developing retina
Hypoxia
Stimulate aberrant vessel formation

(angiogenesis) at the junction between
vascularised and avascular retina
Retinal folds, macular dragging,

retinal detachment
Most common cause
of severe, irreversible
vision loss in older
adults resulting from
ageing and thinning
of macular tissues
Non-exudative (dry)
Exudative (wet)
Wet
ARMD
Accumulation of the
RPE membrane and
metabolic debris and
cells degenerate
hyaline material
and atrophy sets
(drusen)
Photoreceptors and
New vessels grow pigment
behind the macula epithelium sends
signal to
choriocapillaries
Breakdown of
Scarring of macula
brush’s membrane
Common diseases
Kaposi sarcoma Hemangioma

Pyogenic granuloma Hemangioendothelioma
Psoriasis Vascular malformations
Angiofibroma Diabetic ulcers
Angiosarcoma
Atopic dermatitis
Organ Disease Angiogenic
Mechanism
Ski Hair Loss Retarded hair growth
n by angigenesis
inhibitirs
Reproductive system Pre eclampsia EC dysfunction due to
↓ VEGF production
Menorrhagia Fragility of SMC due to
low Ang-1
Lung Neonatal Respiratiory Insufficient lung
distress maturation and
surfactant production
due ↓ HIF 2α and VEGF
production
Pulmonary Fibrosis Alveolar EC apoptosis
Nervous System Alzheimer’s Disease Vasoconstriction,
microvasculatar
degeneration and
cerebral angiopathy
due to EC toxicity by
amyloid β
Tumor angiogenesis
The growth of primary and metastatic tumors to
larger than few mm requires the recruitment of
blood vessels and vascular endothelial cells to
support their metabolic requirements
DUAL EFFECT on tumor growth
 Supplies needed nutrients & O2
 Newlyformed ECs stimulate the growth of adjacent

tumour cells by secreting growth factors –IGF, PDGF
Angiogenesis plays a critical role in human
physiology that ranges from reproduction
and fetal growth to wound healing and tissue
repair
Therapeutic angiogenic drugs that decrease

the angiogenesis process are useful for
treating diseases of increase angiogenesis
and vice versa
Monoclonal antibody to VEGF

Inhibits proliferation &
survival of ECs
 Vessel normalization
 Block tumor regrowth
 Inhibit recruitment of EPCs

Many more promising drugs related to
angiogenesis are currently undergoing
clinical trials for a variety of diseases, but it
remains to be seen if they will prove
effective
Interactions Between Cells
and Their Environment
Introduction
➢Cells don’t exist alone.
➢Cells interact with extracellular
material to form defined tissues.
➢These interactions are crucial to the
formation of epithelial tissue and
connective tissue, which are crucial for
various cellular activities.
Introduction (Cont.)
➢Cell migration, cell growth, cell
differentiation, 3-D organization of
tissues and organs that emerges during
embryonic development.
Overview of cell organization
into tissues
• Click to edit ster text styles
– Second leve
– Third level
• Fourth leve
– Fifth lev
7.1 The Extracellular Space
(1)
➢The glycocalyx (cell
coat) is formed from
carbohydrate
projections form the
plasma membrane.
➢Outer surface of
the plasma
membrane
7.1 The Extracellular Space
(cont.)
➢Gycocalyx
➢Mediate cell-cell and cell-
substratum interactions
➢Provide mechanical protection to
cells
➢Barrier to particles moving
toward plasma membrane
The Extracellular Space
(cont.)
➢The extracellular matrix (ECM) is
an organized network of proteins
and polysaccharides beyond the
plasma membrane.
➢“Glue” that holds cells together
➢It often plays a regulatory role in
determining shape and activities of
the cell.
Organization of the
ECM
(cont.)
➢ECM (continued)
➢The basement membrane (basal lamina) is
a continuous sheet that underlies
epithelial tissue and surrounds blood
vessels.
➢Helps maintain cells attached.
➢Serves as substratum for cell migration.
➢Forms a barrier to macromolecules.
The basement membrane
Extracellular
matrix
➢Gel-like “ground substance”
➢Primarily made of polysaccharides
➢Gylycosaminoglycans (GAGs)
➢proteoglycans
➢Fibrous proteins
➢Collagen, laminin, elastin,
fibronectin
(cont.)
➢Collagens – fibrous glycoproteins found only
in the ECM.
➢Collagen is the most abundant protein
in the human body.
➢Provide high tensile strength.
➢Each collagen is restricted to
particular locations in the body.
➢All collagens are a trimer of polypeptide
chains (α chains) and 3 polypeptide chains
are wound around each other.
The structure of collagen I
Major types of collagen
➢Type I collagen
➢The chief component of tendons,
ligaments, and bones.
➢Type II collagen
➢Represents more than 50% of the protein in
cartilage and is the major component of
the vitreous body of the eye.
➢It is also used to build the notochord of
vertebrate embryos.
➢Type III collagen
➢Strengthens the walls of hollow structures like
arteries, the intestine, and the uterus.
➢Type IV collagen
➢Forms the basal lamina of epithelia. (The basal
lamina is often called the basement membrane.)A
meshwork of Type IV collagens provides the filter
for the blood capillaries and the glomeruli of the
kidneys.
The Extracellular Space (cont.)
➢Collagens (continued)
➢Provide the
insoluble framework
that determines
mechanical
properties of the
matrix.
➢Abnormalities in
collagen formation
lead to serious
disorders.
(cont.)
➢Collagens type I, II, III are
fibrillar collagens
➢Assemble into rigid, cable-like
fibrils (assembles like fibers)
➢Example: tendon – collagens are
parallel to tendons thus parallel to
pulling actions
(cont.)
➢Abnormalities in
fibrillar collagens
formation can lead to
serious disorders
➢Mutation in in genes
encoding type I
collagen can produce
osteogenesis imperfecta
➢Extremely fragile bones,
➢Mutation in genes encoding
(cont.)
type II alter the properties of
cartilage tissue causing
dwarfism and skeletal
deformities
➢Mutations in other
collagens genes that are
related in collagen matrix
structure can lead to Ehler-
Danlos sydromes
(cont.)
➢Not all collagens
form fibrils.
➢Collagen type IV is
non-fibrillar, and is
restricted to the
basement membrane.
(cont.)
➢Mutations in type IV
collagen genes causes
Alport syndrome
➢A kidney disease
in which
glomerular
basement
membrane is
disrupted
(cont.)
➢Proteoglycans – protein-polysaccharide
complex, with a core protein attached to
glycosaminoglycans (GAGs).
➢GAGs
➢Have a repeating
disaccharide structure.
➢Negatively charged
(cont.)
➢Negatively charged GAGs attract lots of
cations, which in turn attract water
forming a porous, hydrated gel.
➢Function:
➢to be able to withstand
compressional forces through
hydration and swelling pressure
(turgor) to the tissue
• Click to edi Master
tex
– Second level
– Third level
• Fourth level
– Fifth level
Structure of a proteoglycan complex
Structure of a proteoglycan complex
➢Forms complement to collagen
molecule
➢Together, they give cartilage and other
extracellular matrices strength and
resistance to deformation
➢Example: ECM of bones
➢Collagen + Proteoglycans +
calcium sulfate ions = bones
➢Fibronectin (Fn)
➢Multiple binding domains
➢Complex proteins that binds to
multiple substrates
➢Helps cells attach to matrix
➢Fn has binding sites for other components
of the ECM.
➢RGD
Structure of fibronectin
(cont.)
➢Fibronectin (FN) is involved in many
cellular processes, including tissue repair,
embryogenesis, blood clotting, and cell
migration/adhesion.
➢Fibronectin sometimes serves as a general
cell adhesion molecule
➢ FN also can serve to organize cellular
➢Laminins – extracellular
glycoproteins consisting
of three polypeptide
chains linked by disulfide
bonds.
➢Help cell migration
during development.
➢Components of
basement membranes.
➢Dynamic Properties
➢The ECM can be stretched during tension.
➢ECM materials degraded by
matrix metalloproteinases
(MMPs).
➢MMPs possibly involved in tissue remodeling,
embryonic cell migration, wound healing ,
and formation of blood vessels.
➢Excessive MMPs causes arthritis, hepatitis,
atherosclerosis, tooth and gum disease and
tumor progression
7.2 Interactions of Cells
with Extracellular
Materials
➢Integrins – family of membrane
proteins composed of heterodimers
with α and ß subunits.
➢Have a major role in
integrating extracellular and
intracellular environments.
➢Another role is adhesion of cells to
their substratum or other cells.
Model of integrin activation
Interactions of Cells with
Extracellular Materials
(cont.)
➢Integrins (continued)
➢Linkage between
integrins and their
ligands
mediates
adhesion between
cells and their
environment.
➢Binding of proteins
to integrins is
facilitated by
(cont.)
➢Integrins (continued)
➢Cytoplasmic domains of integrins
contain binding sites for a variety of
cytoplasmic proteins.
➢Integrins make the
connection between the ECM
and the cytoskeleton.
Blood
➢Injury clotting
conformational
change in platelets’
integrin activation
inc. fibrinogen affinity
aggregation o platelets f
Synthetic RGD peptides

-> inhibit blood clot
(cont.)
➢Focal adhesions are found at the
cell membrane where the
cytoskeleton interacts with proteins
of the extracellular matrix
➢Focal adhesions – scattered,
discrete sites for cell adhesion to
their substratum in vitro.
➢They may act as a type of
sensory structure.
➢The clustering of integrins at these sites
attracts a large complex of proteins and
initiates intracellular regulatory processes, by
which such events as cell migration and
anchorage-dependent differentiation are
controlled.
➢Focal adhesion kinase (FAK) is a protein
tyrosine kinase which is recruited at an
early stage to focal adhesions and which
mediates many of the downstream
Focal adhesions
Focal adhesions
(cont.)
➢Hemidesmosomes are cell-
substratum adhesion sites that
connect the extracellular matrix to the
keratin cytoskeleton
➢basal attachments of epithelial cells to
the basement membrane in vivo.
➢Contain a dense plaque with
filaments consisting of keratin.
form rivet-like links between cytoskeleton and
extracellular matrix components such as the basal
lamina that underlie epithelia
Hemidesmosomes
7.3 Interaction of Cells
with Other Cells
➢Cells have surface-
recognition sites that
maintain
organization
Interaction of Cells with Other
Cells (cont.)
➢Selectins – family
of integral
membrane
glycoproteins that
bind to sugars on
the surface of
cells.
Cells (cont.)
➢Selectins (continued)
➢Contain a small cytoplasmic domain, a
single membrane-spanning domain, and a
large extracellular segment.
➢Three types:
➢E-selectin – on endothelial cells.
➢P-selectin – on platelets and
endothelial cells.
Cells (cont.)
➢Immunoglobulin
superfamily (IgSF) –
most proteins are
involved in immune
functions.
➢Most IgSF
molecules mediate
interaction of
lymphocytes with
cells required or
TIGHT JUNCTIONS
●
located at the apical end of the
junctional complex between adjacent
epithelial cells
●
sites where integral proteins of two
adjacent membranes meet
●
block the diffusion of solutes and
water
●
“fences”
●
claudin – major structural component
 expressed in TAL
●
•
claudin – 16
claudin – 1
 prevents water loss
●
•blood-brain barrier
 prevents drugs from entering CNS
●
GAP JUNCTIONS
●
sites for intercellular communication
● plasma membranes come very close, but
no contact
●
composed of connexin subunit:
connexon
●
allow molecules less than 1000
daltons
●
relatively nonselective
●
channel closure is triggered by
phosphorylation of connexin
●
have a potential to integrate individual
cells into functional unit
●
allow cells to share metabolites
●
connexons
 differ in conductance, permeability,
and regulation
 promote or prevent communication
 mutation resulting to disorder might
cause defects
●
tunneling nanotubules
 conducting cell surface proteins
PLASMODESMATA
●
cytoplasmic channels that pass
through cell walls
●
desmotubule
●
sites of cell to cell communication
●
capable of dilation
CELL
WALLS
●
bacteria, fungi, plants
●
gives polyhedral shape
●
“skeleton”
●
source of signal
●
cellulose
 fibrous component of
● cell wall
protiens and pectin
 provide matrix
●
cellulose
 cellulose synthase
 organized into rod-like microfibrils
●
provide rigidity
●
resistance to tensile forces
 polymerized at cell surface
●
matrix
 synthesized in the cytoplasm
 three types of macromolecules
●
hemicelluloses
 bind to the surfaces of cellulose
microfibrils
●
pectins
 holds water
●
proteins
 expansins – cell growth
 elongation
CELL
WALLS
●
thin cell plate
●
provide suporrt
●
primary walls
●
secondary walls
●
lignin
 structural
support
 in xylem, move water through the
plant
Cell-Cell Interactions
• The cell surface
• The extracellular layer
• Cell-cell connections
– Cell-cell attachments
– Cell-cell gaps
• Cell-cell communication (long distance)
The Cell Surface
• Recall the structure of the plasma (cell) membrane:

– Phospholipid bilayer w/ cholesterol molecules interspersed
– Both integral proteins and peripheral proteins
• Many of which have carbohydrate groups covalently attached!
The Extracellular Layer
• Most organisms have an extracellular layer just
exterior to the plasma membrane:
– Provides an extra layer of protection / defense.
– Helps define cell shape.
– Helps attach one cell to a neighboring cell.
• Broad types of extracellular layers:

– Cell wall:
• Surrounds plant, fungi, bacteria, and algal cells.
– Extracellular matrix:
• Surrounds animal cells.
The Extracellular Layer
• Usually “fiber composites”:

– Cross-linked network of long filaments (fibers)
surrounded by a stiff ground substance.
– Protects cell from stretching (tension) and
The Plant Primary Cell Wall
• Fibrous components = Cellulose microfibrils.

• Ground substance = Pectins and other
gelatinous polysaccharides.
The Plant Cell Wall
• Primary cell wall:

– Defines shape of plant cell.
– Counteracts force of water entering the plant cell via osmosis: cell wall
exerts wall pressure.
• Secondary cell wall:
– Secreted by certain plant cells. (e.g. xylem cells, above)
– Secreted interior to the primary cell wall.
– Can provide tough structural support (contains lignin).
The Plant Cell Wall
• Secondary cell wall:

– Contains the durable polymer lignin.
– Found primarily in the xylem (water conducting)
tissue of plants with a true vascular system:
• e.g. Ferns, “evergreen plants,” and flowering plants.
– Adaptation that allows vascular plants to grow tall
and resist the force of gravity:
• Xylem system acts like an internal skeleton!
The
Extracellular
Matrix (ECM)
• Fiber composite secreted
by animal cells.
• Fibrous component:
– Cable-like collagen protein
• Ground substance:
– Rich in proteoglycan
complexes:
• Contain hundreds of
proteoglycan
molecules:
– Core protein with many
hydrophilic carbohydrate
chains attached.
The Extracellular Matrix (ECM)
• Provides structural support.

• More pliable (flexible) than the plant cell wall.
• Helps cells adhere to each other.
• The cell’s internal cytoskeleton is physically connected to the

ECM via protein-protein interactions.
• Specifically, actin filaments are linked to transmembrane proteins

called integrins, which are linked to proteins (e.g. fibronectins and
laminins) which are linked to collagen proteins.
– Cell-cell gaps
Cell-Cell Connections
• Unicellular organisms may secrete

polysaccharide-rich biofilms, connecting
them to each other and to the substrate.
– e.g. Dental plaque in your mouth!
Multicellularity Through
Cell-Cell Connections
• In multicellular organisms (e.g. plants and

animals), various types cell-cell attachments and
cell-cell gaps help to connect neighboring cells
within a given tissue.
Cell-Cell Attachments
• Middle lamella (plants)
– Joins neighboring cell walls.
• Tight junctions (animals)

• Desmosomes (animals)
Middle Lamella (Plants)
• Gelatinous polysaccharides (pectins) glue

together neighboring plant cell walls.
Tight
Junctions
(Animals)
• Specialized proteins from adjacent cell

membranes line up and bind to each
other, “stitching” the cells together.
Tight Junctions
• Can form a watertight seal between cells.

• Common in cells lining your skin, stomach,
intestines, and bladder.
Desmosomes
(Animals)
• Anchoring and membrane proteins binding to

each other and to intermediate filaments link the
cytoskeletons of adjacent cells.
Desmosomes
• Made of proteins that link the cytoskeletons of

adjacent cells.
• Common in epithelial and muscle cells.
Cadherins
• A major class of cell adhesion proteins.
• An important component of desmosomes.
• Different types of cells express different types of

cadherins on their plasma membranes.
– Selective adhesion: adjacent cells of the same
cell type often adhere to one another due to
interactions of their cell-type specific cadherins.
– Cell-cell gaps
Cell-Cell Gaps
• Create a direct connection between the
cytoplasm of adjacent cells.
• Allows neighboring cells to communicate directly

through membrane “holes” and channels.
• Two major types:

– Plasmodesmata (plants)
– Gap junctions (animals)
Plasmodesmata (Plants)
• Cell-cell gaps connecting adjacent plant cells.

• Lined with plasma membrane.
• Allows a plant cell to directly share cytoplasm with
neighboring plant cells.
Plasmodesmata (Plants)
• Function in movement of water:

– Speeds the movement of water from the root
exterior to the root interior (location of xylem).
• Function in movement of sugars:
– Speeds the movement of sugars between
adjacent phloem cells.
Plasmodesmata
(Plants)
• Water, sugars, and other molecules

can travel through plant tissues via the:
– Symplastic route:
• Traveling via the symplast (continuous network of shared
cytoplasm between plant cells connected by plasmodesmata)
– Apoplastic route:
• Traveling around plant cells (e.g. through porous cell walls and
the middle lamella) without actually entering the cytoplasm of
individual cells.
• Apoplast: Extracellular space around cells.
Gap Junctions (Animals)
• Each gap junction consists of many channels (made of

) that connect adjacent cells.
• Allow water, ions, and small molecules to move
between adjacent cells.
Gap Junctions (Animals)
• Extensively found within muscle tissue:

– Speeds conduction of electrical impulses throughout the heart,
coordinating heart muscle contraction (your heartbeat!).
• Also found (to a limited extent) within
tissue:
– Allow electrical impulses to directly flow from neuron to neuron.
Cell-Cell Connections:
Summary
junctions
junction
– Cell-cell gaps
Long Distance Communication
• Distant cells communicate with each other
via :
– Information carrying molecules that:
• Are secreted by a cell,
• in the body, and
• Act on target cells far from the original cell.
– concentrations of hormones can have a
large impact on target cells!
– Hormone function and structure vary widely.
• Lipid soluble (steroids) vs. non lipid soluble.
Hormone Signal Receptors
• Signal receptors are _ that change conformation

(shape) upon hormone binding.
• Each hormone binds to a specific type of signal receptor:

– Steroid receptors: Located in .
– Other hormone receptors: Located in cell .
• Toto a particular hormone, a cell must express the appropriate sig

receptor!
Steroid
Hormone
Receptors
• diffuses across plasma membrane and binds

to receptor in cytosol.
• Hormone-receptor complex can enter
and change gene activity.
Steroid Hormone: Estradiol
• Estradiol, for example:

– Is released by follicle cells in the of
females.
– Binds to within the of
various cell types, ultimately causing target cells to:
• Differentiate (mammary gland cells during puberty).
• Proliferate (endometrial cells lining the uterine wall).
Other Hormone Receptors
• Non-lipid soluble (non-steroid) hormones bind to

receptors on plasma .
• Signal :
– Conversion of an extracellular signal (hormone) to an
intracellular signal.
Signal
Transduction
Pathways
• Involve several steps.

• Message is as it changes
Non-Steroid
Hormone:
Epinephrine
• Epinephrine is a non-steroid hormone:

– Produced and released by the
glands in response to short-term
stress.
– Binds to epinephrine
embedded in the cell membranes of liver cells:
• Triggers a signal transduction cascade that
ultimately activates phosphorylase:
Signal Transduction Pathways
• G-protein cascades:
– Binding of hormone to receptor activates a
inside the cell, which then in turn activates
other proteins inside the cell.
– e.g. Epinephrine binding to epinephrine receptor on
liver cell membranes.
• Enzyme-linked receptor cascades:

– Binding of hormone to receptor triggers a cascade of
phosphorylation events inside cell.
• Usually, the hormone-bound receptor is the first target to be
phosphorylated. (Autophosphorylation)
– e.g. binding to insulin receptor on liver cell
membranes.
G-Protein Cascades
• G-protein initially in “ ”
conformation.
G-Protein Cascades
• Receptor changes and

activates.
G-Protein Cascades
• Activated G-protein binds to and activates an

.
• Enzyme catalyzes formation of a
messenger.
Second
Messengers
• intracellular signaling molecules.

• May open ion _ or activate protein kinases.
• Protein :
– Enzymes that activate/inactivate other proteins by adding
phosphate groups to them (phosphorylation).
Epinephrine Action
• 1.) Epinephrine binds to
and activates the
epinephrine
on liver cell membranes.
• 2.) Receptor activates an
intracellular :
– G-protein activates an
enzyme, adenylyl cyclase.
• 3.) Adenylyl cyclase
catalyzes the formation of a
second messenger, cyclic
AMP ( ).
Epinephrine Action
• 4.) cAMP activates the
enzyme protein
A.
• 5.) Protein kinase A
activates phosphorylase
kinase.
• 6.) Phosphorylase kinase
activates phosphorylase.
• 7.) Activated
phosphorylase catalyzes
the cleavage of
into
monomers!
Enzyme-linked Receptors
• Hormone binding to receptor results in
autophosphorylation and
of receptor.
• Activated receptors then induce

phosphorylation of many other
in the cell: a phosphorylation cascade.
• Cascade causes of signal.
• Best understood subgroup:

– Receptor tyrosine kinases (RTKs)
Signal amplification!
Insulin Action
• Insulin is a non-steroid hormone:

– Released by the in
response to elevated blood glucose levels.
– Binds to insulin on the
cell membrane of cells:
• Enzyme-linked receptors that initiate a
“phosphorylation” cascade within the liver cell.
Insulin Action
• 1.) Insulin binds to insulin
receptor on liver cells.
• 2.) Insulin
becomes phosphorylated.
• 3.) protein
becomes activated.
• 4.) Ras activates an
called
MAPKKK.
• 5.) MAPKKK activates
another enzyme: MAPKK.
Insulin Action
• 6.) MAPKK activates
another enzyme: MAPK.
• 7.) MAPK activates a
transcription factor, which
enters the .
• 8.) Transcription factor
increases the the
expression of
involved in
glycogen synthesis.
• 9.) Liver synthesizes
more
from
glucose monomers.
Signal
Transduction
Pathways
• Convert an
extracellular signal to
an intracellular signal.
• Original message is
as it changes
form.
• May ultimately lead to
the activation of:
– Intracellular
– factors
Signal Deactivation
• How are cell signals turned off?
– Hormone away from

receptor.
– G-proteins turn back “ ”-
deactivate.
– Second messengers are degraded.
– Phosphatases remove
groups from proteins.
Signal Transduction Pathways
• As a biologist, you
will encounter
signal transduction
pathways often,
especially when
studying:
– The system
– The system
– The nervous
system
LIFE AT THE END OF
CHROMOSOMES
INTRODUCTION
• A telomere is a region of repetitive

nucleotide sequences at each end of a
chromosome.
• Blackburn, Carol Greider and Jack Szostak
were awarded the 2009 Nobel prize in
physiology and medicine for the discovery of
telomere and telomerase.
INTRODUCTI
ON
• Unique structures at the end of chromosomes are
necessary for chromosomal integrity and overall
genomic stability called as telomeres which
protect our genetic data, make it possible for cells
to divide, and hold some secrets to how we grow
old and get cancer.
• An entire chromosome has about 150 million base
pairs. Each time a cell divides, an average person
loses 30 to 200 base pairs from the ends of that
cell's telomeres
STRUCTURE OF
TELOMERE
STRUCTURE…..
• Telomeres are comprised of repeat sequences
and bound by multiple telomeric interacting
proteins. In mammalian cells, telomere DNA
contains double-stranded tandem repeats of
TTAGGG followed by terminal 3′ G-rich single-
stranded overhangs. Telomere DNA is thought
to adopt the T-loop structure, where the
telomere end folds back on itself and the 3′ G
strand overhang invades into the double-
stranded DNA (the so-called D-loop).
Telomere : a multi protein complex
• Mammalian telomeres have a SIX PROTEIN complex
called “SHELTERIN”.
• TRF1 and TRF2 bind to the TTAGGG sequences in the double strand
telomeric DNA.
• POT1 binds to the sequences in single strand form
• TIN2 and TPP1 proteins keep TRF1, TRF2 and POT1 together.
• This six protein complex, SHELTERIN prevents the activation of the DNA
damage response.
• SHELTERIN is required for the recruitment of telomerase.

STRUCTURE……
FUNCTIONS
• They protect the chromosomes.
• They separate one chromosome from another
in the DNA sequence.
• Without telomeres, the ends of the
chromosomes would be "repaired", leading to
chromosome fusion and massive genomic
instability.
FUNCTION CONT….
• Telomeres are also thought to be the "clock"

that regulates how many times an individual
cell can divide. Telemetric sequences shorten
each time the DNA replicates.
FUNCTIONS CONT…
Telomere function
Replication Capping
(RED)
A) The telomeres are seen as the
bright red signals on the
chromosome ends.
B) Fusions between the

chromosomes are indicated by
the arrows, no telometric DNA is
observed at these points.
Telomeres and aging
• It has been proposed that telomere shortening
may be a molecular clock mechanism that counts
the number of times a cell has divided and when
telomeres are short, cellular senescence (growth
arrest) occurs.
• It is believed that shortened telomeres in mitotic
(dividing) cells may be responsible for some of
the changes we associate with normal aging.
Cells normally can divide only about 50 to 70
times, with telomeres getting progressively
shorter until the cells become senescent, die or
sustain genetic damage that can cause cancer.
Example:
In human blood cells, the length of telomeres

ranges from 8,000 base pairs at birth to 3,000
base pairs as people age and as low as 1,500 in
elderly people.
Telomeres do not shorten with age in
tissues such as heart muscle in which cells
do not continually divide.
People who are older have
chromosomes that have replicated
more times.
Number of cell division Vs Number of
nucleotides lost in normal cell :
No. of nucleotides
16000
14000
12000
10000 No. of cell
8000 divisions
6000
4000
2000
0
Why do telomeres get shorter each
time a cell divides?
• While replicating DNA, the eukaryotic DNA
replicating enzymes, cannot replicate the
sequences present at the end of
chromosomes. Hence these sequences and
the information they carry may get lost.
• They cap the end sequences and
themselves get lost in the process of DNA
replication.
• In 1972, James Watson called this as End-
replication problem.
Since the DNA structure can be rebuilt on both
parent strands, two identical DNA helices are
produced, each containing one original parent
strand and one newly synthesized strand, called
a complementary strand.
Due to the nature of the mechanism via which
DNA is replicated, one strand of the DNA is left
with an incompletely replicated end. Without
specialized means of maintaining chromosomes,
this causes chromosome ends to shrink with
each successive cell division.
END Replication problem
WHAT
NEXT?
• Dr. Jerry Shay and his colleagues (The

University of Texas Southwestern Medical
Center at Dallas ) found that cellular
aging can be bypassed or put on hold by
the introduction of the catalytic
component of telomerase.
TELOMERASE
• Telomerase (TEE-LÓM-ER-ACE) is a
ribonucleoprotein enzyme complex (a cellular
reverse transcriptase) that has been referred
to as a cellular immortalizing enzyme.
• It stabilizes telomere length by adding
hexametric (TTAGGG) repeats onto the
telomeric ends of the chromosomes, thus
compensating for the erosion of telomeres
that occurs in its absence
HOW DOES TELOMERASE WORKS?
• Telomerase works by adding back telomeric
DNA to the ends of chromosomes, thus
compensating for the loss of telomeres that
normally occurs as cells divide.
• Most normal cells do not have this enzyme
and thus they lose telomeres with each
division.
• In humans, telomerase is active in germ cells,
in vitro immortalized cells, the vast majority of
cancer cells and, possibly, in some stem cells.
• High telomerase activity exists in germ cells,
stem cells, epidermal skin cells, follicular hair
cells, and cancer cells.
• Some cells are immortal because their
telomerase is switched on
• Examples of immortal cells: blood cells
and cancer cells
• Cancer cells do not age because they
produce telomerase, which keeps the
telomere intact.
Telomere and
CANCER
• Telomeres were first discovered in cancer cells
because, cancer cells are saturated with an
enzyme called telomerase.
• Telomerase is the key enzyme for human cells to
accquire immortality.
• As a cell begins to cancerous, it divides more
often and its telomere becomes very short. If its
telomeres get too short, the cell may die, whereas
normal cell is devoid of telomerase activity.
Telomere and
CANCER
• It can escape this fate by becoming cancerous cell
by activating telomerase (or) ALT pathway is
activated, resulting in abnormal
telomere lengthening & proliferative growth
• Telomerase is over expressed in many cancers cells.
• When cells lose the function of P53 pathway, they can
no longer arrest cells in G1 an important point in cell
cycle for repairing DNA damage response. Cells
without P53 are able to divide with deprotected
telomeres, which cause genomic instability a common
feature of malignant cells.
CONCLUSIO
N…
• Measuring telomerase may be a new way to detect
cancer.
• If scientists can learn how to stop telomerase, they might be
able to fight with cancer by making cancer cells age and die.
• Some of the drugs are showed positive results by inhibiting
telomerase and associated proteins and finding the way to
shortening of telomere which results in cell death/apoptosis.
• Most of anti-telomerase drugs are still in Clinical phases I and II.
Cell Adhesion and Cell
Migration
Outline:
• Overview of the kinds of adhesions that
cells make
• Anchoring junctions
– adherens junctions
– desmosomes
• The organization of adhesions at epithelia
• Tight junctions
• Migration
Four functional classes of cell junctions in animal tissues:
• Anchoring junctions
– Cell-cell and cell-matrix
• Transmit stresses through tethering to cytoskeleton
• Occluding junctions
– Seal gaps between cells to make an impermeable barrier
• Channel-forming junctions (gap junctions)
– Link cytoplasms of adjacent cells
• Signal-relaying junctions
– Synapses in nervous system, immunological
Figure 19-2 Molecular Biology of the Cell (© Garland Science 2008)

Anchoring junctions transmit stresses and are
tethered to the cytoskeletal elements:
• Connective tissue -
– Main stress-bearing component is the ECM
• Epithelial tissue
– Cytoskeletons transmit mechanical stresses

Anchoring junctions:
Table 19-2 Molecular Biology of the Cell (© Garland Science 2008)

Transmembrane adhesion proteins link the cytoskeleton to extracellular structures:
• Cell-cell adhesions usually mediated by cadherins

• Cell-matrix adhesions usually mediated by integrins
• Internal linkage to cytoskeleton is mediated by intracellular
anchor proteins

The cadherin superfamily includes hundreds of different proteins:
• Take their name from their

dependence on calcium
• Extracellular domain containing
multiple copies of the cadherin
motif
• Intracellular portions varied
• Adhesive and signaling functions

Cadherins mediate Ca2+-
dependent cell-cell adhesion in all
animals:
• Main adhesion molecules holding cells together in
early embryonic tissues

Cadherins mediate homophilic
adhesion:
• Cadherins of a specific subtype on one cell will bind
cadherins of the same type on another cell
Figure 19-9a Molecular Biology of the Cell (© Garland Science 2008)

In the absence of calcium the structure becomes floppy:
• Series of compact domains (cadherin repeats) joined by

flexible hinges
Figure 19-9b Molecular Biology of the Cell (© Garland Science 2008)

The “Velcro” principle of adhesion:
• Low-affinity binding to ligand
• Strength comes from multiple bonds in parallel
• Allows for easy disassembly
Figure 19-9c Molecular Biology of the Cell (© Garland Science 2008)

Selective cell-cell adhesion enables dissociated
vertebrate cells to reassemble into organized tissues:
• Homophilic attachment allows for highly selective recognition

• Cells of similar type stick together and stay segregated from other cell types

Cadherins control the selective assortment of cells:
• Appearance and
disappearance of specific
cadherins
• A. is labeled for E-cadherin
• B. is labeled for N-cadherin
Figure 19-12a,b Molecular Biology of the Cell (© Garland Science 2008)

Selective dispersal and reassembly of cells to form
tissues in a vertebrate embryo:
• Cells from epithelial neural
tube alter their adhesive
properties
• Epithelial-mesenchymal
transition
• Migrate
– Chemotaxis
– Chemorepulsion
– Contact guidance
• Re-aggregate

Twist is a transcription factor that regulates
epithelial-mesenchymal transitions:
• Epithelial cells can dis-assemble, migrate away from
parent tissue as individual cells -- epithelial-
mesenchymal transition
• Part of normal development, e.g., neural crest
• Twist is essential for neural crest cell development in
embryogenesis
• Twist represses transcription of E-cadherin
• Twist contributes to metastasis in human breast cancers
Figure 19-12c Molecular Biology of the Cell (© Garland Science 2008)

Catenins link classical cadherins to the actin cytoskeleton:
• Intracellular domains
of the cadherins
provide anchorage for
cytoskeletal filaments
• Intracellular anchor
proteins assemble on
the tail of the cadherin
• Catenins
– , , p120-catenin

Adherens junctions coordinate the actin- based
motility of adjacent cells:
• Allow cells to coordinate the activities of their cytoskeletons
• Form a continuous adhesion belt around each of the interacting cells in a
sheet of epithelium
• Network can contract via myosin motor proteins
– Motile force for folding of epithelial sheets

Adherens junctions coordinate the actin- based
motility of adjacent cells:
• Oriented contraction of bundles of actin filaments running
along the adhesion belts causes narrowing of the cells at
the apex

Desmosome junctions give epithelia mechanical
strength:
• Structurally similar to adherens junctions
• Link to intermediate filaments
Figure 19-17a Molecular Biology of the Cell (© Garland Science 2008)

Molecular components of a desmosome:
Figure 19-17b Molecular Biology of the Cell (© Garland Science 2008)

Desmosomes, hemidesmosomes, and the
intermediate filament network:
• Form a structural framework of great tensile strength

Desmoplakin mutations:
• Clinical features include varying degrees of keratoderma,
blisters, nail dystrophy, wooly hair, cardiomyopathy
From Clinical and Experimental Dermatology, 30, 261-266 (2005)

Clinical importance of desmosomal junctions:
• Pemphigus
– Auto-antibodies against desmosomal cadherins
• Cells become “unglued” from each other
– Severe blistering of the skin
Pemphigus foliacious – antibodies against desmoglein 1

Cell-cell junctions send signals into the cell interior:
• Cross-talk between adhesion machinery and cell

signaling pathways allows cell to make or break
attachments as dictated by circumstances
– Analagous to cross-talk between integrin signaling and other
signaling pathways
• Contact inhibition
– In general, when cells are attached to other cells, proliferation is
inhibited
– When attachments are broken, proliferation is stimulated
• Physiologic example
– Repair a breach in the epithelium

Unit 2 - 18BTE416T Tissue Engineering For Regenerative Medicine

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Organotypic Models:

i. Growth of cell layer on top of filter (Fig. A).

Figure: Illustration of various organotypic culture methods

Rank and Wakefield classification

• Healing is the body’s response to injury in

• The goal of all surgical procedures should be

• Repair typically results in the formation of scar

• Healing by first intention (wounds with opposed

• Healing by secondary intention (wounds with

• The narrow incisional space immediately fills

• Neutrophils replaced by macrophages

•Collagen fibers are now

•Epithelial cell proliferation

• Continued of collagen and

• Dermal appendages that have been destroyed in the line

• Tensile strength of the wound increases thereafter, but it

• When there is more extensive loss of

 Much larger amounts of granulation tissue are formed

 Wound contraction occurs in large surface

CLEANLINESS CLEAN NOT CLEAN

INFECTION NOT INFECTED INFECTED

MARGINS SURGICALLY CLEAN IRREGULAR

SUTURES USED NOT USED

HEALING SMALL GRANULATION LARGE GRANULATION

OUT COME LINEAR SCAR IRREGULAR WOUND

COMPLICATION NOT FREQUENT FREQUENT

2. Stage of granulation tissue formation and organisation.

4. Stage of scar formation and resorption.

•For soft tissue wound healing:

2.Proliferative phase 3.Maturation phase

• This result in formation of a coagulum.

• Begin to enter the wound site within 6 hours of

• The number of PMNs increases steadily, peaking

• Reaches peak concentration by

• They have longer life than PMNs and remain in

 wound decontamination through phagocytosis

• Characterized by formation of granulation tissue in the wound.

• GTR procedures are based on control of

– This action is stimulated by combination of cytokines

– Fibroblasts produces collagen (first detected in the

– Fibroblasts produce type III collagen initially and as

– Myofibroblasts align themselves parallel wound

– These cells are eliminated by apoptosis after wound closure.

–Formation of new blood vessels at the site of injury

–The newly formed blood vessels are more leaky

• There is conversion of granulation tissue to fibrous

• Maturation of the epithelial layer quickly follows

PDGF- platelet-derived growth factor IL- interleukin

• The inflammatory and proliferative phases

• The maturation phase differs markedly from that

• Osteoclasts acts as an organizational unit to debride

• In this process osteoblasts produce matrix vesicles through

• Osteoblasts an organic matrix composed of

• Mineralization by mineral deposition directly

• Begins 10 to 12 days after root end resection.

• The exact sequence leading to the formation of

• Cementum covers the resected root end

• A sinus tract is a drainage duct for the suppuration

• It drains onto the epithelial tissue through either a

• The presence of a sinus tract in the oral cavity is usually

• If there is fibrosis of the sinus tract trajectory

• The healing pattern following pulpectomy is characterized by an initial

• Inflammatory cells accumulate close to the root filling