Tissue Healing Tissue Healing ProcessProcess

Dr.Djumadi Achmad, SpPA(K), SpFBagian Patologi, FK-Unhas

Healing ProcessHealing Process1. 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 Control of Normal Cell Proliferation and Tissue Growthand Tissue GrowthIn 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 ACTIVITYTISSUE PROLIFERATIVE ACTIVITY

1.1. Labile cells Labile cells continuously dividing : hemopoietic cells, surface epithelia of the skin, oral cavity, vagina, and cervix; mucosa of the excretory ducts of the glands

2.2. Stable cells 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

3.3. Permanent cells Permanent cells nondividing : neurons and skeletal and cardiac muscle cells

STEM CELLSSTEM 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 : ◦ Obligatory asymmetric replication : one cell

retains its self-renewing capacity and the other will differentiate

◦ 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 CELLSSTEM 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 CELLSSTEM 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 CellsEmbryonic 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 CellsAdult (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 CellsAdult (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 Stem Cells in Tissue HomeostasisHomeostasisStem cells in :

bone marrow liver brain muscle cornea skin

Stem cells in the bone marrowStem cells in the bone marrow

Hematopoietic Stem Cells (HSCs) Hematopoietic Stem Cells (HSCs) produces approximately 1.5 × 106 blood cells per

secondcan reconstitute the bone marrow after depletion

caused by disease or irradiationwidely used for the treatment of hematologic

diseases

Marrow Stromal CellsMarrow Stromal Cells ( (MSCsMSCs))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 liverStem cells in the liverThe liver contains stem cells/progenitor cells

in the canals of HeringCan 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 brainStem cells in the brain Neural stem cells (NSCs) 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 sStem cells in the skeletal and keletal and cardiac musclescardiac musclesSkeletal muscle myocytes do not divide,

even after injuryRegeneration of injured skeletal muscle

occur by replication of satellite cellssatellite 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 corneaStem cells in the corneaLimbal 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 skinStem cells in the skinStem 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 Cell Cycle and the Regulation of Cell Replicationof Cell ReplicationThe 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 cyclecell 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 The Cell CycleCell 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 The Cell CycleCell Cycle

CCheckpointsheckpoints : ensure that cells with damaged DNA or chromosomes do not complete replication

The GG11/S checkpoint /S checkpoint monitors the integrity of DNA before replication

The GG22/M checkpoint /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 DNA repair

If DNA damage is too severe to be repaired, the cells are eliminated by apoptosisapoptosis

Growth FactorsGrowth Factors

The proliferation of many cell types is driven by polypeptides known as growth factorsgrowth factors

May have restricted or multiple cell targetsMay also promote cell survival, locomotion,

contractility, differentiation, and angiogenesisAll 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 FunctionsEpidermal growth α

EGF Platelets, macrophages, saliva, urine, milk, plasma

Mitogenic for keratinocytes and fibroblasts; stimulates keratinocyte migration and granulation tissue formation

Transforming growth factor α

TGF-α 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 FunctionsPlatelet-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 FunctionsTransforming growth factor β (isoforms 1, 2, 3); other members of the family are BMPs and activin

TGF-β 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

CytokinesCytokines

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 GROWTHSIGNALING 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 signalingAutocrine 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 signalingParacrine 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 signalingEndocrine signaling

Growth factors may also circulate and act at distant sites, as is the case for HGF (Hepatocyte growth factor) and several cytokines

Transcription FactorsTranscription FactorsTranscription factors that regulate cell

proliferation are :◦ Growth-promoting genes, such as c-c-MYCMYC and c-c-JUNJUN◦ Cell cycle–inhibiting genes, such as p53p53

Growth factors induce the synthesis or activity of transcription factors

Activation transcription factors : ◦ Dimerization : c-FOSc-FOS and c-JUN 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-κBNF-κB◦ Release from membranes by proteolytic cleavage, as

for Notch receptors

Functions of Functions of Extracellular MatrixExtracellular Matrix (ECM)(ECM) Mechanical supportMechanical support : for cell anchorage and cell

migration, and maintenance of cell polarity Control of cell growthControl of cell growth : via integrin Maintenance of cell differentiationMaintenance 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 renewalScaffolding for tissue renewal Establishment of tissue microenvironmentsEstablishment of tissue microenvironments :

Basement membrane acts as a boundary between epithelium and underlying connective tissue

Storage and presentation of regulatory moleculesStorage and presentation of regulatory molecules : growth factors like FGF and HGF are secreted and stored in the ECM in some tissues

Extracellular MatrixExtracellular Matrix (ECM) (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 cellsProteoglycans and hyaluronan : provide resilience and lubricationTwo 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 PROTEINS = cell adhesion molecules (CAMs)

Integrins, Selectins, Fibronectin, Laminin, CadherinsIntegrins bind to ECM proteins such fibronectin, laminin, and osteopontin providing a connection between cells - ECM, and cell-to-cell contactSelectins : leukocyte-endothelial interactionsFibronectin : binds to many molecules, such as collagen, fibrin, proteoglycans, and cell surface receptorsLaminin is the most abundant glycoprotein in the basement membrane and has binding domains for both ECM and cell surface receptorsCadherins and integrins link the cell surface with the cytoskeleton (actin and intermediate filaments)

HealingHealing process 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 Healing by Repair, Scar Formation and Fibrosisand 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 Healing by Repair, Scar Formation and Fibrosisand FibrosisRepair by connective tissue deposition includes the following basic features: InflammationAngiogenesisMigration and proliferation of fibroblastsScar formationConnective tissue remodeling

MECHANISMS OF ANGIOGENESISMECHANISMS 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 Angiogenesis from Preexisting VesselsVessels1. Vasodilation in response to nitric oxidenitric oxide, and VEGFVEGF-

induced increased permeability of the preexisting vessel

2. Proteolytic degradation of the basement membrane of the parent vessel by matrix metalloproteinases matrix metalloproteinases (MMPs) and disruption of cell-to-cell contact between endothelial cells by plasminogen activatorplasminogen activator

3. Migration of endothelial cells toward the angiogenic stimulus

4. Proliferation of endothelial cells5. Maturation of endothelial cells form capillary tubes6. Recruitment of periendothelial cells (pericytes and

vascular smooth muscle cells) to form the mature vessel

Angiogenesis from Endothelial Precursor Angiogenesis from Endothelial Precursor Cells (EPCs)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 Growth Factors and Receptors Involved in Angiogenesisin 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 receptorsNotch 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-β 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 ECM Proteins as Regulators of AngiogenesisAngiogenesis

1. Integrins especially αvβ3, 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 HEALINGCUTANEOUS 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 surfaceMaturation : 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 Growth Factors and Cytokines Affecting Cutaneous Cutaneous Wound Wound HealingHealing

Monocyte chemotaxis Chemokines, TNF, PDGF, FGF, TGF-β

Fibroblast migration/replication PDGF, EGF, FGF, TGF-β, TNF, IL-1

Keratinocyte replication HB-EGF, FGF-7, HGF

Angiogenesis VEGF, angiopoietins, FGF

Collagen synthesis TGF-β, PDGF

Collagenase secretion PDGF, FGF, TNF; TGF-β inhibits

HB-EGF, heparin-binding EGF; IL-1, interleukin 1; TNF, tumor necrosis factor