Tissue Repair, Regeneration, and Stem Cells

Tissue Renewal, Regeneration,
and Repair

• Injury to cells and tissues sets in motion a
series of events that contain the damage and
initiate the healing process. This process can
be broadly separated into regeneration and
repair.

• Regeneration results in the complete
restitution of lost or damaged tissue;
• repair may restore some original structures
but can cause structural derangements. In
healthy tissues, healing, in the form of
regeneration or repair, occurs after any insult
that causes tissue destruction, and is
essential for the survival of the organism.

• Regeneration refers to the proliferation of cells
and tissues to replace lost structures, such as the
growth of an amputated limb in amphibians.
• In mammals, whole organs and complex tissues
rarely regenerate after injury, and the term is
usually applied to processes such as liver growth
after partial resection or necrosis, but these
processes consist of compensatory growth
rather than true regeneration.

• Tissues with high proliferative capacity, such
as the hematopoietic system and the
epithelia of the skin and gastrointestinal (GI)
tract, renew themselves continuously and
can regenerate after injury, as long as the
stem cells of these tissues are not destroyed.

• Repair most often consists of a combination
of regeneration and scar formation by the
deposition of collagen. The relative
contribution of regeneration and scarring in
tissue repair depends on the ability of the
tissue to regenerate and the extent of the
injury

• For instance, a superficial skin wound heals
through the regeneration of the surface
epithelium. However, scar formation is the
predominant healing process that occurs
when the extracellular matrix (ECM)
framework is damaged by severe injury.

• Chronic inflammation that accompanies
persistent injury also stimulates scar
formation because of local production of
growth factors and cytokines that promote
fibroblast proliferation and collagen
synthesis.

• The term fibrosis is used to describe the
extensive deposition of collagen that occurs
under these situations.
• ECM components are essential for wound
healing, because they provide the framework
for cell migration, maintain the correct cell
polarity for the re-assembly of multilayer
structures, and participate in the formation
of new blood vessels (angiogenesis).

• Furthermore, cells in the ECM
(fibroblasts, macrophages, and other cell
types) produce growth factors, cytokines, and
chemokines that are critical for regeneration
and repair. Although repair is a healing
process, it may itself cause tissue
dysfunction, as, for instance, in the
development of atherosclerosis.

Control of Normal Cell Proliferation
and Tissue Growth
• In adult tissues the size of cell populations is
determined by the rates of cell
proliferation, differentiation, and death by
apoptosis and increased cell numbers may
result from either increased proliferation or
decreased cell death.[5] Apoptosis is a
physiologic process required for tissue
homeostasis, but it can also be induced by a
variety of pathologic stimuli

• Differentiated cells incapable of replication
are referred to as terminally differentiated
cells.
• The impact of differentiation depends on the
tissue under which it occurs: in some tissues
differentiated cells are not replaced, while in
others they die but are continuously replaced
by new cells generated from stem cells.

• Cell proliferation can be stimulated by physiologic and
pathologic conditions.
• E.g. The proliferation of endometrial cells under
estrogen stimulation during the menstrual cycle and
the thyroid-stimulating hormone–mediated
replication of cells of the thyroid that enlarges the
gland during pregnancy .
• Physiologic stimuli may become excessive, creating
pathologic conditions such as BPH resulting from
dihydrotestosterone stimulation and the
development of nodular goiters in the thyroid as a
consequence of increased serum levels of TSH.

• Cell proliferation is largely controlled by
signals from the microenvironment that
either stimulate or inhibit proliferation. An
excess of stimulators or a deficiency of
inhibitors leads to net growth and, in the
case of cancer, uncontrolled growth.

• The tissues of the body are divided into three
groups on the basis of the proliferative
activity of their cells: continuously dividing
(labile tissues), quiescent (stable tissues), and
nondividing (permanent tissues).

• In continuously dividing tissues cells proliferate
throughout life, replacing those that are destroyed.
These tissues include surface epithelia,the columnar
epithelium of the GI tract and uterus; the transitional
epithelium of the urinary tract, and cells of the bone
marrow and hematopoietic tissues.
• In most of these tissues mature cells are derived
from adult stem cells, which have a tremendous
capacity to proliferate and whose progeny may
differentiate into several kinds of cells.

• Quiescent tissues normally have a low level of
replication;
• cells from these tissues can undergo rapid
division in response to stimuli and are thus
capable of reconstituting the tissue of origin,e.g
parenchymal cells of liver, kidneys, and
pancreas; mesenchymal cells such as fibroblasts
and smooth muscle; vascular endothelial cells;
and lymphocytes and other leukocytes.

• Nondividing tissues contain cells that have left
the cell cycle and cannot undergo mitotic
division in postnatal life,e.g. neurons and
skeletal and cardiac muscle cells. If neurons in
the central nervous system are destroyed, the
tissue is generally replaced by the proliferation
of the central nervous system–supportive
elements, the glial cells. However, recent results
demonstrate that limited neurogenesis from
stem cells may occur in adult brains

• . Although mature skeletal muscle cells do
not divide, skeletal muscle does have
regenerative capacity, through the
differentiation of the satellite cells that are
attached to the endomysial sheaths.
• Cardiac muscle has very limited, if
any, regenerative capacity, and a large injury
to the heart muscle, as may occur in
myocardial infarction, is followed by scar
formation.

STEM CELLS
• Stem cells are characterized by their self-
renewal properties and by their capacity to
generate differentiated cell lineages so, stem
cells need to be maintained during the life of the
organism.
• Such maintenance is achieved by two
mechanisms: (a) obligatory asymmetric
replication, in which with each stem cell
division, one of the daughter cells retains its
self-renewing capacity while the other enters a
differentiation pathway,

• (b) stochastic differentiation, in which a stem
cell population is maintained by the balance
between stem cell divisions that generate
either two self-renewing stem cells or two
cells that will differentiate

• In early stages of embryonic development, stem
cells, known as embryonic stem cells or ES
cells, are pluripotent, that is, they can generate
all tissues of the body.
• Pluripotent stem cells give rise to multipotent
stem cells, which have more restricted
developmental potential, and eventually
produce differentiated cells from the three
embryonic layers.
Transdifferentiation indicates a change in the
lineage commitment of a stem cell.

EMBRYONIC STEM CELLS
• ES cells have been used to study the specific
signals and differentiation steps required for the
development of many tissues.
• ES cells made possible the production of
knockout mice, an essential tool to study the
biology of particular genes and to develop
models of human disease. ES cells may in the
future be used to repopulate damaged organs.
• ES cells may be capable of differentiating into
insulin-producing pancreatic cells, nerve
cells, myocardial cells, or hepatocytes.

Reprogramming of Differentiated Cells:
Induced Pluripotent Stem Cells
• Differentiated cells of adult tissues can be
reprogrammed to become pluripotent by transferring
their nucleus to an enucleated oocyte
• may be used for therapeutic cloning in the treatment
of human diseases.
• In this technique the nucleus of a skin fibroblast from
a patient is introduced into an enucleated human
oocyte to generate ES cells, which are kept in
culture, and then induced to differentiate into various
cell types but is inaccurate.

• One of the main reasons for the inaccuracy is
the deficiency in histone methylation in
reprogrammed ES cells, which results in
improper gene expression.

Cell Cycle and the Regulation of Cell
Replication
• The replication of cells is stimulated by
growth factors or by signaling from ECM
components through integrins.
• To achieve DNA replication and division, the
cell goes through a tightly controlled
sequence of events known as the cell cycle.
The cell cycle consists of G1 (presynthetic), S
(DNA synthesis), G2 (premitotic), and M
(mitotic) phases

• . Quiescent cells that have not entered the
cell cycle are in the G0 state.
• the cell cycle has multiple controls and
redundancies, particularly during the
transition between the G1 and S phases.
These controls include activators and
inhibitors, as well as sensors that are
responsible for checkpoints.

Repair & Regeneration
• Repair begins early in inflammation
• Regeneration refers to growth of cells and
tissues to replace lost structure
– as liver and kidney growth after partial hepatectomy
and unilateral nephrectomy

• Two processes:
– Regeneration
– Fibrosis

Repair
• Regeneration of injured cells by cells of same
type as with regeneration of skin/oral mucosa
(requires basement membrane)
• Replacement by fibrous tissue
(fibroplasia, scar formation)
• Both require cell growth, differentiation, and
cell-matrix interaction

Tissue Regeneration
• Controlled by biochemical factors released in
response to cell injury, cell death, or
mechanical trauma
– Most important control: inducing resting cells to
enter cell cycle
– Balance of stimulatory or inhibitory factors
– Shorten cell cycle
– Decrease rate of cell loss

Healing
• Healing is usually a tissue response
– (1) to a wound (commonly in the skin)
– (2) to inflammatory processes in internal
organs
– (3) to cell necrosis in organs incapable of
regeneration

Repair & Regeneration
• Labile cells: continue to proliferate throughout life :
squamous, columnar, transitional epithelia;
hematopoitic and lymphoid tissues

• Stable cells: retain the capacity of proliferation but
they don’t replicate normally: parenchymal cells of
all glandular organs & mesenchymal cells

• Permanent cells: cannot reproduce themselves
after birth: neurons, cardiac muscle cells

Cell Cycle
RESTING PHASE

G0
CYCLIN D / E
RESTRICTION POINT

G1

DNA SYNTHESIS
S

MITOSIS M
CYCLIN
A/B

G2

CYCLIN

GAP PHASE CDK
CYCLIN B

Intercellular Signaling
• 3 pathways
– Autocrine: cells have receptors for their own
secreted factors (liver regeneration)
– Paracrine: cells respond to secretion of nearby
cells (healing wounds)
– Endocrine: cells respond to factors (hormones)
produced by distant cells

Control of Normal Cell Proliferation
and Tissue Growth
In adult tissues the size of cell populations is determined
by
• rates of cell proliferation,
• differentiation
• death by apoptosis
• increased cell numbers may result from either
increased proliferation or decreased cell death.[5]
• Apoptosis is a physiologic process required for tissue
homeostasis, but it can also be induced by a variety of
pathologic stimuli

Cell proliferation can be stimulated by physiologic and
pathologic conditions.
• E.g. The proliferation of endometrial cells under
estrogen stimulation during the menstrual cycle
• thyroid-stimulating hormone–mediated replication of
cells of the thyroid that enlarges the gland during
pregnancy .
Physiologic stimuli may become excessive, creating
pathologic conditions such as
• BPH resulting from dihydrotestosterone stimulation
• development of nodular goiters in the thyroid as a
consequence of increased serum levels of TSH.

STEM CELLS
• Stem cells are characterized by their self-renewal
properties and by their capacity to generate differentiated
cell lineages so, stem cells need to be maintained during
the life of the organism.
• Such maintenance is achieved by two mechanisms:
• (a) obligatory asymmetric replication, in which with each
stem cell division, one of the daughter cells retains its self-
renewing capacity while the other enters a differentiation
pathway
• (b) stochastic differentiation, in which a stem cell
population is maintained by the balance between stem
cell divisions that generate either two self-renewing stem
cells or two cells that will differentiate

Stem Cell Progeny
• Capacity for unlimited division
• Whose daughters have a choice
Stem
Stem Stem

Stem DiffeDifferentiate

rentiate

Stem
1 3 2
Stem

Platelets

RBCs

Myeloid
Progenitor Neutrophils

Macrophages

Pluripotent
Stem Cell B lymphocytes
Lymphocyte
Progenitor
T lymphocytes

EMBRYONIC STEM CELLS
• ES cells have been used to study the specific
signals and differentiation steps required for
the development of many tissues.

• ES cells may be capable of differentiating into
insulin-producing pancreatic cells, nerve
cells, myocardial cells, or hepatocytes.

Imagination is better than knowledge -
Einstein

Cellular Signalling Pathways

 Vital for cell cycle progression, growth, differentiation
& death.
 Growth Factors – The key stone
 A delicate balance between activating and inhibitory
signals needs to be maintained normally
 Alteration in this balance - Dysregulated cellular
proliferation & survival of abnormal cells.

Growth Factors & Cell Cycle

Gene Transcription

Receptors
+ S

Priming

G0 G1
Cell Cycle G2

M

Growth factors increase synthesis &
decrease degradation of
macromolecules

• The proliferation of many cell types is driven
by polypeptides known as growth factors.
• can have restricted or multiple cell targets,
• promote cell survival
• locomotion
• contractility
• differentiation
• angiogenesis.

• All growth factors function as ligands that
bind to specific receptors, which deliver
signals to the target cells.
• These signals stimulate the transcription of
genes that may be silent in resting cells,
• Genes that control cell cycle entry and
progression.

Growth Factors and Cytokines
Involved in Regeneration and Healing

• EPIDERMAL GROWTH α
•
platelets, macrophages, saliva, urine, milk,
plasma.
• It is mitogenic for keratinocytes and
fibroblasts;
• stimulates keratinocyte migration and
granulation tissue formation.

Transforming growth factor α
 macrophages
 T lymphocytes
 keratinocytes
 many tissues
 stimulates replication of hepatocytes and
most epithelial cells

Heparin-binding EGF
• macrophages
• mesenchymal cells:
• Keratinocyte replication
• HEPATOCYTE GROWTH FACTOR/SCATTER
FACTOR
• mesenchymal cells
• Enhances proliferation of
hepatocytes, epithelial cells, and endothelial
cells;
• increases cell motility,
• keratinocyte replication.

Vascular endothelial cell growth
factor
• from many types of cells
• Increases vascular permeability;
• mitogenic for endothelial cells,
• angiogenesis

Platelet-derived growth factor
• platelets,
• macrophages
• endothelial cells
• keratinocytes
• smooth muscle cells
• Chemotactic for PMNs, macrophages, fibroblasts, and smooth
muscle cells;
• activates PMNs, macrophages, and fibroblasts;
• mitogenic for fibroblasts, endothelial cells, and smooth muscle
cells;
• stimulates production of MMPs,
• fibronectin stimulates angiogenesis and wound contraction

Fibroblast growth factor
 macrophages
 mast cells
 T lymphocytes
 endothelial cells
 fibroblasts
 Chemotactic for fibroblasts
 mitogenic for fibroblasts and keratinocytes
 stimulates keratinocyte migration, angiogenesis,
 wound contraction
 matrix deposition.

Transforming growth factor β
• platelets,
• T lymphocytes,
• macrophages,
• endothelial cells,
• keratinocytes,
• smooth muscle cells
• fibroblasts
• Chemotactic for
PMNs, macrophages, lymphocytes, fibroblasts, and
smooth muscle cells;
• stimulates angiogenesis, and fibroplasia;
• inhibits production of MMPs and keratinocyte
proliferation.

Keratinocyte growth factor
• Fibroblasts
• Stimulates keratinocyte
migration, proliferation, and differentiation
• TUMOR NECROTIC FACTOR
• Macrophages,
• mast cells,
• T lymphocytes
• Activates macrophages;
• regulates other cytokines; multiple functions.

SIGNALING MECHANISMS IN CELL
GROWTH
• receptor-mediated signal transduction is
involved, which is activated by the binding of
ligands such as growth factors, and cytokines
to specific receptors.
• Different classes of receptor molecules and
pathways initiate a cascade of events by
which receptor activation leads to expression
of specific genes.

Three general modes of signaling
• Autocrine signaling: Cells respond to the
signaling molecules that they themselves
secrete, thus establishing an autocrine loop
• plays a role in liver regeneration and the
proliferation of antigen-stimulated lymphocytes.
• Tumors frequently overproduce growth factors
and their receptors, thus stimulating their own
proliferation through an autocrine loop.

Paracrine signaling
• : One cell type produces the ligand, which
then acts on adjacent target cells that express
the appropriate receptor.
• common in connective tissue repair of
healing wounds, in which a factor produced
by one cell type (e.g., a macrophage) has a
growth effect on adjacent cells (e.g., a
fibroblast).
• It is also necessary for hepatocyte replication

Endocrine signaling
• : Hormones synthesized by cells of endocrine
organs act on target cells distant from their
site of synthesis, being usually carried by the
blood.
• Growth factors may also circulate and act at
distant sites, as is the case for HGF.

Receptors and Signal Transduction
Pathways
• The binding of a ligand to its receptor triggers
a series of events by which extracellular
signals are transduced into the cell resulting in
changes in gene expression.
• Receptors with intrinsic tyrosine kinase
activity. The ligands for receptors with
tyrosine kinase activity include
• most growth factors such as EGF, TGF-
α, HGF, PDGF, VEGF, FGF, c-KIT ligand, and
insulin.

EGFR Structure

Extracellular
Domain

Transmembrane
Domain

TK Intracellular
Domain

EGFR Function in Normal Cell

ATP ATP
TK TK

+

Gene Transcription
Cell Cycle Progression

Cell Proliferation Antiapoptosis

Angiogenesis

Consequence of proliferation of
Mutation EGFR receptors

Normal Cell
Cancerous Cell
Up Regulation

Signaling from tyrosine kinase receptors. Binding of the growth factor (ligand) causes
receptor dimerization and autophosphorylation of tyrosine residues. Attachment of
adapter proteins (e.g., GRB2 and SOS) couples the receptor to inactive RAS. Cycling
of RAS between its inactive and active forms is regulated by GAP. Activated RAS
interacts with and activates RAF (also known as MAP kinase kinase kinase). This
kinase then phosphorylates a component of the MAP kinase signaling pathway, MEK
(also known as MAP kinase), Activated MAP kinase phosphorylates other cytoplasmic
proteins and nuclear transcription factors, generating cellular responses.

Receptors lacking intrinsic tyrosine
kinase activity that recruit kinases

• . Ligands for these receptors include
• many cytokines, such as IL-2, IL-3, and
other interleukins;
• interferons α, β, and γ;
• erythropoietin;
• granulocyte colony-stimulating factor;
• growth hormone
• prolactin.

G protein–coupled receptors.
• These receptors transmit signals into the cell
through trimeric GTP-binding proteins (G proteins).
• A large number of ligands signal through this type
of receptor, including
• chemokines,
• vasopressin,
• serotonin, histamine,
• epinephrine and norepinephrine,
• calcitonin,
• glucagon,
• parathyroid hormone,
• corticotropin, and rhodopsin.

Steroid hormone receptors
• . These receptors are generally located in the
nucleus.
• involved in a broad range of responses that
include
• adipogenesis ,
• inflammation
• atherosclerosis.

Transcription Factors

• Many of the signal transduction systems used
by growth factors transfer information to the
nucleus and modulate gene transcription
through the activity of transcription factors.
• Include products of several growth-promoting
genes, such as c-MYC and c-JUN, and of cell
cycle–inhibiting genes, such as p53.

LIVER REGENERATION

• The human liver has a remarkable capacity to
regenerate, as demonstrated by its growth after
partial hepatectomy, which may be performed for
tumor resection or for living-donor hepatic
transplantation.
• resection of approximately 60% of the liver in living
donors results in the doubling of the liver remnant in
about one month. The portions of the liver that
remain after partial hepatectomy constitute an intact
“mini-liver” that rapidly expands and reaches the
mass of the original liver.

• Restoration of liver mass is achieved without the
regrowth of the lobes that were resected at the
operation. Instead, growth occurs by
enlargement of the lobes that remain after the
operation, a process known as compensatory
growth or compensatory hyperplasia.
• the end point of liver regeneration after partial
hepatectomy is the restitution of functional
mass rather than the reconstitution of the
original form.
•

• Almost all hepatocytes replicate during liver
regeneration after partial hepatectomy. Because
hepatocytes are quiescent cells, it takes them
several hours to enter the cell cycle, progress
through G1, and reach the S phase of DNA
replication.
• The wave of hepatocyte replication is
synchronized and is followed by synchronous
replication of nonparenchymal cells (Kupffer
cells, endothelial cells, and stellate cells).

• hepatocyte proliferation in the regenerating
liver is triggered by the combined actions of
cytokines and polypeptide growth factors.

• Quiescent hepatocytes become competent to
enter the cell cycle through a priming phase
that is mostly mediated by the cytokines TNF
and IL-6, and components of the complement
system.
• Priming signals activate several signal
transduction pathways to start cell
proliferation

• . Under the stimulation of HGF, TGFα, and
HB-EGF, primed hepatocytes enter the cell
cycle and undergo DNA replication .
Norepinephrine, serotonin, insulin, thyroid
and growth hormone, act as adjuvants for
liver regeneration, facilitating the entry of
hepatocytes into the cell cycle.

• Individual hepatocytes replicate once or
twice during regeneration and then return to
quiescence in a strictly regulated sequence of
events, but the mechanisms of growth
cessation have not been established.
• Growth inhibitors, such as TGF-β and
activins, may be involved in terminating
hepatocyte replication, but there is no clear
understanding of their mode of action,

• Intrahepatic stem or progenitor cells do not
play a role in the compensatory growth that
occurs after partial hepatectomy.
• Endothelial cells and other nonparenchymal
cells in the regenerating liver may originate
from bone marrow precursors

Extracellular Matrix and Cell-
Matrix Interactions

• Tissue repair and regeneration depend not
only on the activity of soluble factors, but
also on interactions between cells and the
components of the extracellular matrix
(ECM).
• The ECM regulates the
growth, proliferation, movement, and
differentiation of the cells living within it

• It is constantly remodeling, and its synthesis
and degradation accompanies regeneration,
wound healing, chronic fibrotic processes,
tumor invasion, and metastasis.
• The ECM sequesters water, providing turgor
to soft tissues, and minerals that give rigidity
to bone, but it does much more than just fill
the spaces around cells to maintain tissue
structure.

Functions include
• Mechanical support for cell anchorage and
cell migration, and maintenance of cell polarity
• Control of cell growth. ECM components can
regulate cell proliferation by signaling through
cellular receptors of the integrin family.
• Maintenance of cell differentiation. The
types of ECM proteins can affect the degree of
differentiation of the cells in the tissue, also
acting largely via cell surface integrins.

• Scaffolding for tissue renewal. The maintenance of
normal tissue structure requires a basement
membrane or stromal scaffold. The integrity of the
basement membrane or the stroma of the
parenchymal cells is critical for the organized
regeneration of tissues.
• Although labile and stable cells are capable of
regeneration, injury to these tissues results in
restitution of the normal structure only if the ECM is
not damaged. Disruption of these structures leads to
collagen deposition and scar formation

• Establishment of tissue
microenvironments. Basement membrane
acts as a boundary between epithelium and
underlying connective tissue and also forms
part of the filtration apparatus in the
kidney.

• Storage and presentation of regulatory
molecules. For example, growth factors like
FGF and HGF are secreted and stored in the
ECM in some tissues. This allows the rapid
deployment of growth factors after local
injury, or during regeneration.

COMPOSITION OF ECM
• fibrous structural proteins, such as collagens and
elastins that provide tensile strength and recoil;
• adhesive glycoproteins that connect the matrix
elements to one another and to cells;
• proteoglycans and hyaluronan that provide
resilience and lubrication.
• These molecules assemble to form two basic
forms of ECM: interstitial matrix and basement
membranes.

• The interstitial matrix is found in spaces
between epithelial, endothelial, and smooth
muscle cells, as well as in connective tissue. It
consists mostly of fibrillar and nonfibrillar
collagen, elastin, fibronectin, proteoglycans, and
hyaluronan.
• Basement membranes are closely associated
with cell surfaces, and consist of nonfibrillar
collagen (mostly type IV), laminin, heparin
sulfate, and proteoglycans.

COLLAGEN
• Collagen is the most common protein in the
animal world, providing the extracellular
framework for all multicellular organisms.
Without collagen, a human being would be
reduced to a clump of cells interconnected by
a few neurons.
• Currently, 27 different types of collagens
encoded by 41 genes dispersed on at least 14
chromosomes are known.

• Each collagen is composed of three chains
that form a trimer in the shape of a triple
helix. The polypeptide is characterized by a
repeating sequence in which glycine is in
every third position (Gly-X-Y, in which X and Y
can be any amino acid other than cysteine or
tryptophan).

• Types I, II, III and V, and XI are the fibrillar
collagens,these proteins are found in
extracellular fibrillar structures.
• Type IV collagens have long triple-helical
domains and form sheets instead of fibrils;
they are the main components of the
basement membrane, together with laminin.

• Type VII collagen forms the anchoring fibrils
between some epithelial and mesenchymal
structures, such as epidermis and dermis.
• Still other collagens are transmembrane and
may also help to anchor epidermal and
dermal structures.

FIBRILLAR COLLAGENS
• I in hard and soft tissues
• Osteogenesis imperfecta; Ehlers-Danlos
syndrome—arthrochalasias type I
• II in Cartilage, intervertebral disk, vitreous
• Achondrogenesis type II
• III Hollow organs, soft tissues
• Vascular Ehlers-Danlos syndrome

• V in Soft tissues, blood vessels
• Classical Ehlers-Danlos syndrome
• IX in Cartilage, vitreous

BASEMENT MEMBRANE COLLAGENS
• IV,in Basement membranes
• Alport syndrome

• Procollagen is secreted from the cell and
cleaved by proteases to form the basic unit of
the fibrils.
• Vitamin C is required for the hydroxylation of
procollagen, a requirement that explains the
inadequate wound healing in scurvy.

ELASTIN, FIBRILLIN, AND ELASTIC
FIBERS
• Tissues such as blood
vessels, skin, uterus, and lung require
elasticity for their function.
• Proteins of the collagen family provide
tensile strength, but the ability of these
tissues to expand and recoil depends on the
elastic fibers. These fibers can stretch and
then return to their original size after release
of the tension.

• Morphologically, elastic fibers consist of a
central core made of elastin, surrounded by a
peripheral network of microfibrils.
• Substantial amounts of elastin are found in
the walls of large blood vessels, such as the
aorta, and in the uterus, skin, and ligaments.
The peripheral microfibrillar network that
surrounds the core consists largely of
fibrillin,a glycoprotein.

• The microfibrils serve as support for
deposition of elastin and the assembly of
elastic fibers. They also influence the
availability of active TGFβ in the ECM.
• inherited defects in fibrillin result in
formation of abnormal elastic fibers in
Marfan syndrome, manifested by changes in
the cardiovascular system (aortic dissection)
and the skeleton.

CELL ADHESION PROTEINS

• Most adhesion proteins, also called CAMs
(cell adhesion molecules), can be classified
into four main families:
• immunoglobulin family CAMs,
• cadherins
• integrins
• selectins.

• These proteins function as transmembrane
receptors but are sometimes stored in the
cytoplasm.
• As receptors, CAMs can bind to similar or
different molecules in other cells, providing
for interaction between the same cells
(homotypic interaction) or different cell types
(heterotypic interaction).

• other secreted adhesion molecules
• (1) SPARC also known as
osteonectin, contributes to tissue remodeling
in response to injury and functions as an
angiogenesis inhibitor;
• (2) the thrombospondins, a family of large
multifunctional proteins, some of which also
inhibit angiogenesis;
• .

• (3) osteopontin is a glycoprotein that
regulates calcification and is a mediator of
leukocyte migration involved in
inflammation, vascular remodeling, and
fibrosis in various organs
• (4) the tenascin family, which consist of large
multimeric proteins involved in cell adhesion

GLYCOSAMINOGLYCANS AND
PROTEOGLYCANS
• GAGs make up the third type of component
in the ECM, besides the fibrous structural
proteins and cell adhesion proteins.
• four structurally distinct families of GAGs:
heparan sulfate, chondroitin/dermatan
sulfate, keratan sulfate, and hyaluronan.

• The first three of these families are
synthesized and assembled in the Golgi
apparatus and rough endoplasmic reticulum.
• By contrast, HA is produced at the plasma
membrane by enzymes called hyaluronan
synthases and is not linked to a protein
backbone.

• HA is a polysaccharide of the GAG family
found in the ECM of many tissues and is
abundant in heart valves, skin and skeletal
tissues, synovial fluid, the vitreous of the
eye, and the umbilical cord.
• It binds a large amount of water, forming a
viscous hydrated gel that gives connective
tissue the ability to resist compression forces.

• HA helps provide resilience and lubrication to
many types of connective tissue, notably for
the cartilage in joints.
• Its concentration increases in inflammatory
diseases such as rheumatoid
arthritis, scleroderma, psoriasis, and
osteoarthritis.

Repair by connective tissue
fibrosis

Overview

• Severe or persistent tissue injury with damage both
to parenchymal cells and to the stromal framework
leads to a situation in which repair cannot be
accomplished by parenchymal regeneration alone.

• Repair occurs by replacement of the non
regenerated parenchymal cells with connective
tissue.

 General components of this process are:

 Formation of new blood vessels (Angiogenesis)
 Migration and proliferation of fibroblast
 Deposition of ECM
 Maturation and reorganization of the fibrous
tissue (Remodeling)

Angiogenesis from endothelial
precursors

 Common precursor: hemangioblast that gives rise
to angioblast and hematopoietic stem cells.
 Angioblasts then proliferate and differentiate into
endothelial cells that form arteries,veins and
lymphatics.
 Epcs (angioblast like cells) in the bone marrow can
be recruited into tissues to initiate angiogenesis.
 What is VASCULOGENESIS?

Angiogenesis from preexisting vessel
• Vasodilation in response to nitric acid and VEGF.

• Increased permeability of pre-existing vessel.

• Proteolytic degradation of the parent vessel BM by
metalloproteinases and disruption of cell to cell
contact btw endothelial cells of the vessel by
plasminogen activator.

• Migration of endothelial cells from the original
capillary toward an angiogenic stimulus.

• Proliferation of the endothelial cells behind
the leading edge of migrating cells.

• Maturation of endothelial cells with inhibition
of growth and organization into capillary
tubes.

• Recruitment and proliferation of pericytes (for
capillaries) and smooth muscle cells (for larger
vessel) to support the endothelial tube.

Growth factors and receptors involved
in angiogenesis
VEGF secreted by mesenchymal cells and stromal
cells.

Angiopoietins(Ang1 and Ang2)
FGF-2

PDGF

TGF-B

Angiopoietins ,PDGF,TGF-B participate in the
stabilization process,

PDGF participates in the recruitmenmt of the
smooth muscle cells,

TGF-b stabilizes newly formed vessel by
enhancing the production Of ECM
production.

ECM proteins as REGULATORS OF
ANGIOGENESIS
• Integrins ,
• Thrombospondin 1 and SPARC, tenascin
C,destabilize cell matrix interactions and
promote angiogenesis.
• Proteinases , plasminogen activators and
matrix metalloproteinases : important role in
tissue remodeling during endothelial invasion.

Scar formation
• Emigration and proliferation of fibroblasts in
the site of injury
• Deposition of ECM
• Tissue remodeling

Fibroblast Migration and Proliferation
• Exudation and deposition of plasma
protein, fibrinogen and plasma fibronectin in
the ECM of granulation tissue provides a
provisional stroma for fibroblasts and
endothelial cell ingrowth.
• Migration of fibroblasts to the site of injury
and subsequent proliferation are triggered by
multiple growth factors TGF-B, PDGF, EGF, FGF
and the cytokines IL-1 and TNF

ECM Deposition and Scar Formation
• TGF-B appears to be the most important
because of the multitude of effects that favors
fibrous tissue deposition.

• It is produced by most of the cells in
granulation tissue and causes fibroblast
migration and proliferation, increased
synthesis of collagen and fibronectin and
decreased degradation of ECM by
metalloproteinases.

• Fibrillar collagens form a major portion of the
connective tissue in repair sites and
development of strength in healing wounds

• As the scar matures, vascular regression
continues, eventually transforming the richly
vascularized granulation tissue into a
pale, avascular scar

Tissue Remodeling
• The balance between ECM synthesis and
degradation results in remodeling of the CT
framework.
• Degradation of collagen and other ECM
proteinS is achieved by a family of matrix
metalloproteinases which are dependant on
zinc ions for their activity.

• MMPs include interstitial collagenases (MMP1, 2, 3) which
cleave the fibrillar collagen type 1, 2 and 3
• Gelatinases (MMP2 and 9) degrade amorphous collagens and
fibronectin.
• Stromelysin (MMP-3,10,11) act on proteoglycan
, laminin,fibronectin,amorphous collagen.
• MMPS are synthesized as propeptides that require proteolytic
cleavage for activation.
• Their secretion is induced by certain stimuli such as growth
factors,cytokines,phagocytosis,physical stress and is inhibited
by TGF-b and steroids

• Collagenases are synthesized in as a latent
precursor (procollagenase) that is activated by
chemicals such as free radicals produced in
oxidative burst of leukocytes and proteinases
• Once formed activated collagenases are
rapidly inhibited by a family of specific tissue
inhibitor of metalloproteinases (TIMP).

WOUND HEALING
• Cutaneous wound healing is divided into three
phases
• Inflammation (early and late)
• Granulation tissue formation and
reepithilialization
• Wound contraction ,ECM deposition and
remodeling.

• Wound healing is a fibroproliferative response
that is mediated through growth factors and
cytokines.
• Skin wounds are classically described to heal
by primary or secondary intention.

Healing by first intention (wounds with
opposed edges)
• Common example of wound repair is healing
of a clean, uninfected surgical wound
approximated by sutures. such healing is
called as primary union or healing by first
intention.

• Incision causes,
• Death of limited number of epithelial cells and
connective tissue.
• Disruption of epithelial BM.

Process
• Within 24 hours;
• Neutrophils appear at the margin of the
incision moving towards the fibrin clot.
• In 24 to 48 hours;
• Movement of the epithelial cells from wound
edges from dermis cut margins depositing BM
components as they move

• Day 3;
• Neutrophils are replaced by macrophages.
• Invasion of granulaton tissue invading the
incision space.
• Collagen fibers are present in the margin of
the incision but does not bridge the incision.
• Epithelial cell proliferation thickens the
epidermal layer

• Day5;
• Incisional space filled with granulation tissue.
• Neovascularization is maximal
• Abundant collagen fibers ,bridging the
incision.
• Mature epidermal architecture with surface
kertinization.

• Second week;
• Continued accumulation of collagen and proliferation
of fibroblast .
• Dissapperance of edema ,leukocyte infiltrate, and
increased vascularity.
• End of first month;
• Scar is made of cellular C.T inflammatory cell
infilterate , covered now by intact epidermis.
• Dermal appendages that have been destroyed in the
line of incision are permanently lost.

Healing by second intention (wound
with separate edges)
extensive loss of cells and tissue.
Large defects.
Abundant granulation tissue grows in from the
margin to complete the repair

Difference
• Larger tissue defect
• Larger fibrin clot
• More necrotic debris and exudate
• Inflammatory reaction is more intense.
• Abundant granulation tissue is formed

Wound contraction
• Formation of a network of actin containing
fibroblasts at the edge of the wound.
• Permanent wound contraction requires the
action of myofibroblasts.
• Contraction of these cells at the wound site
decreases the gap between the dermal edges
of the wound.

Wound strength
• When sutures are removed at the end of the
first week ,strength is approximately 10% that
of unwounded skin, but strength increases
rapidly over the next 4 wks.
• At the end of third month it the rate decreases
and the wound strength is about 70 to80% of
the unwounded skin.

Factors that influence wound healing
• Systemic factors;
• Nutrition (protein def,Zn, vit.C def) inhibit
collagen synthesis
• Metabolic status, diabetes mellitis ,delayed
wound healing, ( microangiopathy)
• Inadequate B.S (arteriosclerosis, varicose
veins)
• Hormones. ( glucocorticoids)

Local factors
• Infection (persistent tissue injury and
inflammation)
• Mechanical factors ,(mobility ,compression of
B.V)
• Foreign bodies, (sutures ,fragments of
bone, steel or glass)
Localization and size of wound (face, foot)

Complications
Inadequate formation of granulation tissue.

Wound dehiscence
• Rupture of a wound is most common after
abdominal surgery and is due to increased
abdominal pressure

• Vomiting, coughing , ileus

• Exuberant granulation is another deviation in
wound healing consisting of the formation of
excessive amounts of granulation
tissue, which protrudes above the level of
the surrounding skin and blocks re-
epithelialization called proud flesh.

• Ulceration of the wounds because of
inadequate vascularization during healing.
(peripheral vascular diseases)

• Non healing wounds are also found in areas of
denervation. (diabetic peripheral neuropathy)

Excessive formation of the
components of the repair
• Excessive amount of collagen lead to
hypertrophic scar.
• Is a raised scar
• Keloid ,it is a scar tissue which goes beyond
the boundaries of the original wound and
does not regress.

• Incisional scars or traumatic injuries may be
followed by exuberant proliferation of
fibroblasts and other connective tissue
elements that may recur after excision.
Called desmoids, or aggressive
fibromatoses, these lie in the interface
between benign proliferations and malignant
(though low-grade) tumors.

• Contracture
• Exaggeration in the process of contraction in
the wound is called contracture.
• Severe burns
• Palms,soles.thorax

• Healing wounds may also generate excessive
granulation tissue that protrudes above the
level of the surrounding skin and in fact
hinders re-epithelialization. This is called
exuberant granulation, or proud flesh, and
restoration of epithelial continuity requires
cautery or surgical resection of the
granulation tissue.

Tissue Repair, Regeneration, and Stem Cells

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