Tissue Healing Process

Dr.Djumadi Achmad, SpPA(K), SpF

Bagian Patologi, FK-Unhas

Healing Process
1. Regeneration - complete restitution of damaged
tissue
2. Repair - cause structural derangements

Regeneration refers to the proliferation of cells
and tissues to replace lost structures
Repair most often consists of a combination of
regeneration and scar formation
Healing after acute injury can occur by regeneration that restores
normal tissue structure or by repair with scar formation. Healing in
chronic injury involves scar formation and fibrosis.
Role of the extracellular matrix in regeneration and repair. Liver regeneration with
restoration of normal tissue after injury requires an intact cellular matrix. If the matrix is
damaged the injury is repaired by fibrous tissue deposition and scar formation.
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
Apoptosis is a physiologic process
required for tissue homeostasis, but it
can also be induced by a variety of
pathologic stimuli
Mechanisms regulating cell populations. Cell numbers can be altered
by increased or decreased rates of stem cell input, cell death due to
apoptosis, or changes in the rates of proliferation or differentiation.
TISSUE PROLIFERATIVE ACTIVITY
1. Labile cells continuously dividing :
hemopoietic cells, surface epithelia of the skin,
oral cavity, vagina, and cervix; mucosa of the
excretory ducts of the glands
2. Stable cells quiescent :
parenchymal cells of liver, kidneys, and pancreas
mesenchymal cells such as fibroblasts and smooth
muscle; endothelial cells; and lymphocytes and other
leukocytes
3. Permanent cells nondividing : neurons and
skeletal and cardiac muscle cells


STEM CELLS
Stem cells are characterized by their self-renewal
properties and by their capacity to generate
differentiated cell lineages
Stem cells need to be maintained by two
mechanisms :
1. Obligatory asymmetric replication : one cell retains its
self-renewing capacity and the other will differentiate
2. Stochastic differentiation : two self-renewing stem cells
or two cells that will differentiate
Stem cells may one day be used to repair
damaged human tissues, such as heart, brain,
liver, and skeletal muscle
Stem cell generation and differentiation. The zygote, formed by the union of sperm
and egg, divides to form blastocysts, and the inner cell mass of the blastocyst
generates the embryo. The cells of the inner cell mass, known as embryonic stem
(ES) cells, maintained in culture, can be induced to differentiate into cells of
multiple lineages. In the embryo, pluripotent stem cells divide, but the pool of these
cells is maintained. As pluripotent cells differentiate, they give rise to cells with
more restricted developmental capacity, and finally generate stem cells that are
committed to specific lineages.
STEM CELLS
In embryonic development, stem cells known as
embryonic stem cells or ES cells, are pluripotent,
that is, they can generate all tissues of the body
In adults, stem cells often referred to as adult stem
cells or somatic stem cells more restricted
capacity to generate different cell types
Somatic stem cells reside in special
microenvironments called niches, composed of
mesenchymal, endothelial, and other cell types
Niche cells transmit stimuli that regulate stem
cell self-renewal and the generation of progeny
cells
Stem cell niches in various tissues. A, Skin stem cells are located in the bulge area of the hair
follicle, in sebaceous glands, and in the lower layer of the epidermis. B, Small intestine stem cells
located near the base of a crypt, above Paneth cells. C, Liver stem (progenitor) cells, known as
oval cells, are located in the canals of Hering (thick arrow), structures that connect bile ductules
(thin arrow) with parenchymal hepatocytes (bile duct and Hering canals are stained for
cytokeratin 7). D, Corneal stem cells are located in the limbus region, between the conjunctiva
and the cornea.
STEM CELLS
Recent research has now demonstrated that
differentiated cells of rodents and humans can be
reprogrammed into pluripotent cells, similar to ES
cells, by the transduction of genes encoding ES
cell transcription factors.
These reprogrammed cells have been named
induced pluripotent stem cells (iPS cells). Their
discovery has opened open an exciting new era in
stem cell research and its applications
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, 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
Adult (Somatic) Stem Cells
Adult stem cells are present in tissues that
continuously divide (labile tissues) such as the
bone marrow, the skin, and the lining of the GI tract
It may also be present in organs such as liver,
pancreas, and adipose tissue (stabile tissues)
Somatic stem cells generate rapidly dividing cells
known as transit amplifying cells. These cells lose
the capacity of self-perpetuation, and give rise to
cells with restricted developmental potential known
as progenitor cells
Adult (Somatic) Stem Cells
A change in the differentiation of a cell from one
type to another is known as transdifferentiation
Hemopoietic stem cells (HSC) in culture have been
shown to transdifferentiate into other cell types,
such as hepatocytes, neurons, skeletal and cardiac
myocytes.
However, HSC transdifferentiation in vivo have
been difficult to reproduce
Stem Cells in Tissue Homeostasis
Stem cells in :
bone marrow
liver
brain
muscle
cornea
skin
Stem cells in the bone marrow
Hematopoietic Stem Cells (HSCs)
produces approximately 1.5 10
6
blood cells per second
can reconstitute the bone marrow after depletion caused by
disease or irradiation
widely used for the treatment of hematologic diseases

Marrow Stromal Cells (MSCs)
have potentially therapeutic applications, because they can
generate chondrocytes, osteoblasts, adipocytes,
myoblasts, and endothelial cell precursors depending on
the tissue to which they migrate
Stem cells in the liver
The liver contains stem cells/progenitor cells in the
canals of Hering
Can give rise to precursor cells known as oval
cells, which can differentiate into hepatocytes and
biliary cells
Oval cell proliferation and differentiation are
prominent in the livers of patients recovering from
fulminant hepatic failure, hepatic tumors, and in
some cases of chronic hepatitis and advanced liver
cirrhosis
Stem cells in the brain
Neural stem cells (NSCs) : have been identified in
two areas of adult brains, the subventricular zone
(SVZ) and the dentate gyrus of the hippocampus
NSCs (also known as neural precursor cells),
capable of generating neurons, astrocytes, and
oligodendrocytes
NSCs transplantation, or the induction of
differentiation of endogenous NSCs, may be used
in treatment of stroke, neurodegenerative disorders
such as Parkinson and Alzheimer diseases, and
spinal cord injury
Stem cells in the skeletal and
cardiac muscles
Skeletal muscle myocytes do not divide, even after
injury
Regeneration of injured skeletal muscle occur by
replication of satellite cells (located beneath the
myocyte basal lamina)
The presence of stem cells in the heart continues
to be debated
It has been proposed that the heart may contain
progenitor-like cells with the capacity to generate
progeny after injury, but not during physiologic
aging
Stem cells in the cornea
Limbal stem cells (LSCs) are located at the junction
between the epithelium of the cornea and the
conjunctiva
LSC deficiency and corneal opacification can be
treated by limbal transplantation or LSC grafting
Stem cells in the skin
Stem cells are located in three different areas of
the epidermis:
Hair follicle bulge
Interfollicular areas of the surface epidermis
Sebaceous glands

They contribute to the regeneration of surface
epidermal cells in skin wounds but not during
normal homeostasis
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 G
1
(presynthetic), S
(DNA synthesis), G
2
(premitotic), and M (mitotic)
phases. Quiescent cells that have not entered the
cell cycle are in the G
0
state
The Cell Cycle
Regulated by proteins called cyclins and associated
enzymes called cyclin-dependent kinases (CDKs)
Activated cyclin-CDK complexes drive the cell cycle
by phosphorylating proteins that are critical for cell
cycle transitions. One such protein is the
retinoblastoma (RB) protein, which normally
prevents cell replication by forming a tight, inactive
complex with the transcription factor E2F
Phosphorylation of RB activates E2F and allows
it to stimulate transcription of genes whose products
drive cells through the cycle

The Cell Cycle
Checkpoints : ensure that cells with damaged
DNA or chromosomes do not complete replication
The G
1
/S checkpoint monitors the integrity of DNA
before replication
The G
2
/M checkpoint checks DNA after replication
and monitors whether the cell can safely enter
mitosis
When cells sense DNA damage, checkpoint
activation delays the cell cycle and triggers DNA
repair
If DNA damage is too severe to be repaired, the
cells are eliminated by apoptosis
Growth Factors
The proliferation of many cell types is driven by
polypeptides known as growth factors
May have restricted or multiple cell targets
May also promote cell survival, locomotion,
contractility, differentiation, and 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, including genes that control cell cycle entry
and progression
Growth Factor Symbol Source Functions
Epidermal growth EGF Platelets, macrophages, saliva,
urine, milk, plasma
Mitogenic for keratinocytes
and fibroblasts; stimulates
keratinocyte migration and
granulation tissue formation
Transforming growth
factor
TGF- Macrophages, T lymphocytes,
keratinocytes, and many tissues
Similar to EGF; stimulates
replication of hepatocytes
and most epithelial cells
Heparin-binding EGF HB-EGF Macrophages, mesenchymal
cells
Keratinocyte replication
Hepatocyte growth
factor/scatter factor
HGF Mesenchymal cells Enhances proliferation of
hepatocytes, epithelial cells,
and endothelial cells;
increases cell motility,
keratinocyte replication
Vascular endothelial
cell growth factor
(isoforms A, B, C, D)
VEGF Many types of cells Increases vascular
permeability; mitogenic for
endothelial cells (see Table
3-3 ); angiogenesis
Growth Factor Symbol Source Functions
Platelet-derived growth
factor (isoforms A, B,
C, D)
PDGF 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, and HA;
stimulates angiogenesis
and wound contraction
Fibroblast growth factor
1 (acidic), 2 (basic),
and family
FGF Macrophages, mast cells, T
lymphocytes, endothelial cells,
fibroblasts
Chemotactic for fibroblasts;
mitogenic for fibroblasts and
keratinocytes; stimulates
keratinocyte migration,
angiogenesis, wound
contraction, and matrix
deposition
HA, hyaluronate; MMPs, matrix metalloproteinases; PMNs, polymorphonuclear leukocytes;
Growth Factor Symbol Source Functions
Transforming growth
factor (isoforms 1,
2, 3); other members
of the family are
BMPs and activin
TGF- Platelets, T lymphocytes,
macrophages, endothelial
cells, keratinocytes, smooth
muscle cells, fibroblasts
Chemotactic for PMNs,
macrophages,
lymphocytes, fibroblasts,
and smooth muscle cells;
stimulates TIMP
synthesis, angiogenesis,
and fibroplasia; inhibits
production of MMPs and
keratinocyte proliferation
Keratinocyte growth
factor (also called
FGF-7)
KGF Fibroblasts Stimulates keratinocyte
migration, proliferation,
and differentiation
Tumor necrosis factor TNF Macrophages, mast cells, T
lymphocytes
Activates macrophages;
regulates other cytokines;
multiple functions
BMP, bone morphogenetic proteins; MMPs, matrix metalloproteinases; PMNs,
polymorphonuclear leukocytes; TIMP, tissue inhibitor of MMP
Cytokines
Cytokines have important functions as mediators of
inflammation and immune responses
Some of these proteins can also be considered as
growth factors, because they have growth-
promoting activities for a variety of cells
Tumor necrosis factor (TNF) and IL-1 participate in
wound healing reactions
TNF and IL-6 are involved in the initiation of liver
regeneration
SIGNALING MECHANISMS IN CELL GROWTH
According to the source of the ligand and the
location of its receptors three general modes of
signaling :
Autocrine
Paracrine
Endocrine
Autocrine signaling
Cells respond to the signaling molecules that they
themselves secrete
Autocrine growth regulation 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
Paracrine stimulation is 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)
Endocrine signaling
Growth factors may also circulate and act at distant
sites, as is the case for HGF (Hepatocyte growth
factor) and several cytokines


Transcription Factors
Transcription factors that regulate cell proliferation
are :
Growth-promoting genes, such as c-MYC and c-JUN
Cell cycleinhibiting genes, such as p53
Growth factors induce the synthesis or activity of
transcription factors
Activation transcription factors :
Dimerization : c-FOS and c-JUN transcription factor
activator protein-1 (AP-1)
Phosphorylation : such as STAT
Release of inhibition : to permit migration into the nucleus,
as for NF-B
Release from membranes by proteolytic cleavage, as for
Notch receptors
Functions of Extracellular Matrix (ECM)
Mechanical support : for cell anchorage and cell
migration, and maintenance of cell polarity
Control of cell growth : via integrin
Maintenance of cell differentiation : The type of
ECM proteins can affect the degree of
differentiation of the cells in the tissue (also via
integrins)
Scaffolding for tissue renewal
Establishment of tissue microenvironments :
Basement membrane acts as a boundary between
epithelium and underlying connective tissue
Storage and presentation of regulatory molecules :
growth factors like FGF and HGF are secreted and
stored in the ECM in some tissues

Extracellular Matrix (ECM)
The ECM is composed of three groups of
macromolecules:
fibrous structural proteins : collagens and elastins
that provide tensile strength
Adhesive glycoproteins : connect the matrix
elements to one another and to cells
Proteoglycans and hyaluronan : provide resilience
and lubrication
Two basic forms of ECM: interstitial matrix and
basement membranes
Figure 3-12 Main components of the extracellular matrix (ECM), including
collagens, proteoglycans, and adhesive glycoproteins. Both epithelial and
mesenchymal cells (e.g., fibroblasts) interact with ECM via integrins. Basement
membranes and interstitial ECM have different architecture and general
composition, although there is some overlap in their constituents
CELL ADHESION PROTEINS = cell adhesion
molecules (CAMs)
Integrins, Selectins, Fibronectin, Laminin,
Cadherins
Integrins bind to ECM proteins such fibronectin,
laminin, and osteopontin providing a connection
between cells - ECM, and cell-to-cell contact
Selectins : leukocyte-endothelial interactions
Fibronectin : binds to many molecules, such as
collagen, fibrin, proteoglycans, and cell surface
receptors
Laminin is the most abundant glycoprotein in the
basement membrane and has binding domains for
both ECM and cell surface receptors
Cadherins and integrins link the cell surface with
the cytoskeleton (actin and intermediate filaments)
Healing process
In most healing processes, a combination of repair
and regeneration occurs
Influenced by:
1. The proliferative capacity of the cells of the tissue;
2. The integrity of the extracellular matrix;
3. The resolution or chronicity of the injury and inflammation
Healing by Repair, Scar Formation and
Fibrosis
If tissue injury is severe or chronic, and results in
damage of both parenchymal cells and the stromal
framework of the tissue, healing can not be
accomplished by regeneration. Under these
conditions, the main healing process is repair by
deposition of collagen and other ECM components,
causing the formation of a scar
Healing by Repair, Scar Formation and
Fibrosis
Repair by connective tissue deposition includes the
following basic features:
Inflammation
Angiogenesis
Migration and proliferation of fibroblasts
Scar formation
Connective tissue remodeling
MECHANISMS OF ANGIOGENESIS
Blood vessel formation in adults, known as
angiogenesis or neovascularization, involves
the branching and extension of adjacent pre-
existing vessels, but it can also occur by
recruitment of endothelial progenitor cells
(EPCs) from the bone marrow
Angiogenesis from Preexisting Vessels
1. Vasodilation in response to nitric oxide, and
VEGF-induced increased permeability of the
preexisting vessel
2. Proteolytic degradation of the basement
membrane of the parent vessel by matrix
metalloproteinases (MMPs) and disruption of cell-
to-cell contact between endothelial cells by
plasminogen activator
3. Migration of endothelial cells toward the
angiogenic stimulus
4. Proliferation of endothelial cells
5. Maturation of endothelial cells form capillary
tubes
6. Recruitment of periendothelial cells (pericytes and
vascular smooth muscle cells) to form the mature
vessel
Angiogenesis from Endothelial Precursor
Cells (EPCs)
EPCs can be recruited from the bone marrow into
tissues to initiate angiogenesis
EPCs may contribute to the re-endothelization of
vascular implants and the neovascularization of
ischemic organs, cutaneous wounds, and tumors
Growth Factors and Receptors Involved in
Angiogenesis
VEGF (Vascular Endothelial GF) : secreted by many
mesenchymal and stromal cells
Receptors for VEGF : VEGFR-2, a tyrosine kinase
receptor
FGF-2 and its receptors
Notch ligands (Jagged 1 and 2, and Delta-like ligand
[Dll] 1, 3, and 4) and their corresponding receptors :
Notch 1- 4
Angiopoietins 1 and 2 (Ang1 and Ang2), PDGF, and
TGF- participate in the stabilization of the new
vessles
Interactions between Notch and VEGF during angiogenesis.
VEGF stimulates delta-like ligand 4 (Dll4)/Notch, which inhibits VEGFR
signaling.
ECM Proteins as Regulators of Angiogenesis
1. Integrins especially
v

3
, which is critical for the
formation and maintenance of newly formed blood
vessels
2. Matricellular proteins, including thrombospondin
1, SPARC, and tenascin C, which destabilize cell-
matrix interactions and therefore promote
angiogenesis
3. Proteinases, such as the plasminogen activators
and MMPs, which are important in tissue
remodeling during endothelial invasion
CUTANEOUS WOUND HEALING
Three phases: inflammation, proliferation, maturation
(overlap)
Inflammation : platelet aggregation and clot
formation
Proliferation : formation of granulation tissue,
proliferation and migration of connective tissue
cells, and re-epithelialization of the wound surface
Maturation : ECM deposition, tissue remodeling,
and wound contraction
Phases of cutaneous wound healing:
inflammation, proliferation, and maturation
Wound healing and scar formation. A, Healing of wound that caused little loss
of tissue: note the small amount of granulation tissue, and formation of a thin
scar with minimal contraction. B, Healing of large wound: note large amounts
of granulation tissue and scar tissue, and wound contraction.
healing by
primary union
healing by
secondary
union
Healing of skin ulcers. A, Pressure ulcer of the skin, commonly found in
diabetic patients. The histologic slides show: B, a skin ulcer with a large gap
between the edges of the lesion; C, a thin layer of epidermal re-epithelialization
and extensive granulation tissue formation in the dermis; and D, continuing re-
epithelialization of the epidermis and wound contraction
Growth Factors and Cytokines Affecting Cutaneous Wound Healing
Monocyte chemotaxis Chemokines, TNF, PDGF, FGF, TGF-
Fibroblast migration/replication PDGF, EGF, FGF, TGF-, TNF, IL-1
Keratinocyte replication HB-EGF, FGF-7, HGF
Angiogenesis VEGF, angiopoietins, FGF
Collagen synthesis TGF-, PDGF
Collagenase secretion PDGF, FGF, TNF; TGF- inhibits
HB-EGF, heparin-binding EGF; IL-1, interleukin 1; TNF, tumor necrosis
factor