Repair and Regeneration.pptx

REPAIR AND REGENERATION-
BASIC CONCEPTS
DEPARTMENT OF PERIODONTICS

CONTENTS
• Introduction
• Definition
• Cell and Tissue regeneration
• Repair by connective tissue
deposition
• Factors that influence tissue
repair
• Abnormalities in tissue
repair
• Wound strength
• Healing of epithelial injury
• Conclusion
• Reference
2

INTRODUCTION
• The survival of an organism depends on its ability to heal after
damage caused by toxic insults and inflammation.
• Healing is the body’s response to injury in an attempt to
restore normal structure and function.
• It involves two processes:
1. Repair
2. Regeneration
3
ROBBINS PATHOLOGIC BASICS OF DISEASE – 9TH EDITION

Melcher 1969
• Regeneration
 Biologic process by which the architecture and function of lost
tissue is completely renewed.
• Repair
Continuity of disrupted tissue is restored by new tissues which
do not replicate the structure and function of the lost tissues
4
DEFINITION
Repair or regeneration following periodontal therapy? Sigmund Stahl, J Clin Periodontol, 1979

5
Regeneration
• Reproduction or reconstitution of a lost or injured part
Periodontal regeneration
• Restoration of lost periodontium
Repair
• Healing of a wound by tissue that does not fully restore the
architecture or the function of the part.
- Glossary of Periodontal Terms (2001)
DEFINITION

Regeneration
• Some tissues are able to replace the damaged components and
essentially return to a normal state; this process is called
regeneration
Repair
• Sometimes called healing, refers to the restoration of tissue
6

• Regeneration: When healing takes place by proliferation of
parenchymal cells and usually results in complete restoration
of the original tissues.
• Repair: When healing takes place by proliferation of
connective tissue resulting in fibrosis and scarring.
7
HARSH MOHAN TEXTBOOK OF PATHOLOGY – 7TH EDITION

8
MECHANISM OF TISSUE REPAIR

CELLAND TISSUE REGENERATION
• The regeneration of injured cells and tissues involve:
 Cell proliferation
 Growth factors
 Development of mature cells from stem cells
9

CELL PROLIFERATION
• Cell proliferation is fundamental to development, maintenance
of steady-state tissue homeostasis, and replacement of dead or
damaged cells.
• Sequence of events that result in cell division is called cell
cycle.
10

• The ability of tissues to repair themselves is determined by
their intrinsic proliferative capacity.
• Based on intrinsic proliferative capacity cells are divided into:
11
LABILE
CELLS
STABLE CELLS
PERMANENT
CELLS

CELL CYCLE
13
G1 phase
S Phase
G2 Phase
M Phase

• G1 phase cell grows in size and synthesize mRNA and
protein that are required for DNA synthesis.
• S Phase DNA is replicated; occurring between G1 and G2
phase
• G2 Phase Rapid cell growth and protein synthesis during
which the cell prepares itself for mitosis
• M Phase Involves nuclear division and cytokinesis, where
two identical daughter cells are produced.
14

15
CELL CYCLE

EXTRACELLULAR MATRIX
• Extracellular matrix is assembled from components
synthesized and deposited outside the cell surface that provide
structural and functional integrity.
• Major extracellular matrix involved in wound healing process
are
– Structural proteins
– Glycosaminoglycans
– Matricellular proteins
– Multi-domain adhesive glycoproteins
16
Schultz and Wysocki, Interactions between extracellular matrix and growth factors in wound healing, Wound Rep
Reg (2009) 17 153–162

Structural proteins
• Contribute to the structural integrity during repair
• They involve
– Collagen
– Elastin
– Fibrin
17
Reg (2009) 17 153–162

Glycosaminoglycans
• Glycosaminoglycans are proteins that are deposited around
other ECM proteins such as collagen and elastin.
• They are hydrophilic substances which absorb upto 1000 times
their volume in water to form a gel-like material called the
ground substance.
• They include
– Hyaluronan
– Proteoglycans such as versican, syndecan, glypicans,
perlecans
18
Reg (2009) 17 153–162

Matricellular proteins
• Matricellular proteins are associated with ECM but do not
perform a structural role within tissues.
• They act temporarily and spatially to provide signals that
trigger specific cell activities.
• They are a group of proteins including
Galectin Tenascins
Thrombospondins Osteopontin
Secreted protein acidic Vitronectin
and rich in cysteine (SPARC)
19
Reg (2009) 17 153–162

Multi-domain adhesive glycoproteins
• These are biological glue that mediates interaction between
cells and other ECM proteins.
• They include
– Fibronectin
– Vitronectin
– Laminin
20
Reg (2009) 17 153–162

21
• High molecular weight glycoprotein
• Produced by endothelial cells, fibroblasts, epithelial cells
• Primary matrix in the organization of collagenous tissue during
the repair process
FIBRONECTIN
• Component of platelets
• Contain receptors which act as binding site for membrane bound
integrins and provide anchor to extracellular matrix
VITRONECTIN
• Most abundant glycoprotein in basement membrane
• Binds cells to basal lamina of epithelial and connective tissue
LAMININ
Caffesse, Quliiones. Polypeptide growth factors and attachment proteins in periodontal wound healing and regeneration .Periodontology
2000.1999 ; I:69-79

GROWTH FACTORS
• Major role of growth factors is to stimulate the activity of
genes that are required for cell growth and cell division.
• Source:
– Macrophages activated by tissue injury
– Stromal cells
– Epithelial cells
– Endothelial cells
– Platelets
– Mesenchymal cells
22

Role of growth factors in wound healing
• Promotes cell proliferation and metabolism through interaction
with specific cell membrane bound receptors.
• Induce migration of cells into the wound space.
• Serve as chemoattractants to recruit leucocytes and fibroblasts
into the wound space.
23

Some of the important growth factors involved in
cell proliferation
• Epidermal growth factor (EGF)
• Transforming growth factor-α (TGF-α)
• Transforming growth factor-β (TGF-β)
• Vascular endothelial growth factor (VEGF)
• Platelet derived growth factor (PDGF)
• Fibroblast growth factors (FGF)
• Keratinocyte growth factor (KGF)
24

Epidermal growth factor(EGF)
• Sources:
– Activated macrophages
– Salivary glands
– Keratinocytes
• Functions:
– Mitogenic for
keratinocytes and
fibroblasts
– Stimulates keratinocyte
migration
– Stimulates formation of
granulation tissue
25

Transforming growth factor-α(TGF-α)
• Sources:
– Activated macrophages
– Keratinocytes
• Functions:
– Stimulates proliferation of
hepatocytes and many
other epithelial cells
26

Transforming growth factor-β(TGF-β)
• Sources:
– Platelets
– T-lymphocytes
– Macrophages
– Keratinocytes
– Fibroblasts
– Smooth muscle cells
• Functions:
• Chemotactic for
leucocytes and fibroblasts
• Stimulates ECM protein
synthesis
• Suppresses acute
inflammation
27

Vascular endothelial growth factor(VEGF)
• Sources:
– Mesenchymal cells
• Functions:
– Stimulates proliferation of
endothelial cells
– Increases vascular
permeability
28

Platelet derived growth factor(PDGF)
• Sources:
– Platelets
– Macrophages
– Smooth muscle cells
– Keratinocytes
• Functions:
– Chemotactic for neutrophils,
macrophages, fibroblasts and
smooth muscle cells
– Stimulates ECM protein
synthesis
– Activates and stimulates
proliferation of fibroblasts,
endothelial cells and other
cells
29

Fibroblast growth factor(FGF)
• Sources:
– Macrophages
– Mast cells
• Functions:
– Chemotactic and
mitogenic for fibroblasts
– Stimulates angiogenesis
and ECM protein
synthesis
30

Keratinocyte growth factor(KGF)
• Sources:
– Fibroblasts
• Functions:
– Stimulates keratinocyte
migration, proliferation
and differentiation
31

Types of ECM - Growth factor interactions
TYPES EXAMPLES
Growth factor binding to ECM FGF-2 must be bound to heparan
sulfate chains of proteoglycan to act
as a mitogen
Integrin mediated interactions Integrins-α1 and α2 necessary for
VEGF- induced angiogenesis
Matrikine ligand presentation by ECM
components
Tenascin-C and laminin bind to
epidermal growth factor receptors
where they act to enhance fibroblast
migration
Growth factor regulation of ECM TGF-β controls ECM production and
degradation
32
Christopher P, Role of Growth Factors in Cutaneous Wound Healing: A Review; Critical Reviews in
Oral Biology and Medicine, 1993

DEVELOPMENT OF MATURE CELLS FROM
STEM CELLS
• In adults, the most important stem cells for regeneration after
injury are tissue stem cells.
• These stem cells live in specialized niches, and it is believed
that injury triggers signals in these niches that activate
quiescent stem cells to proliferate and differentiate into mature
cells that repopulate the injured tissue
33

STEM CELLS
• Stem cells are the primitive cells having capacity for self
renewal and that they can be modulated into multilineage
differentiation.
• Types (based on differentiation)
34
Totipotent stem
cell
Pluripotent stem
cell
Multipotent stem
cell
Unipotent stem
cell
Oligopotent
stem cell

TOTIPOTENT STEM CELL
• Ability to differentiate into all cell types and an entire
functional organism
• Eg Zygote
PLURIPOTENT STEM CELL
• Ability to differentiate into all cell types but cannot form a
functional organism
• Eg Embryonic stem cells
35
Bhattacharyya et al. The voyage of stem cell toward terminal differentiation: a brief overview, Acta Biochim
Biophys Sin.2012;44(6):463

MULTIPOTENT STEM CELL
• Ability to differentiate into closely related family of cells
• Eg Hematopoietic stem cell
UNIPOTENT STEM CELL
• Ability to differentiate into single cell type
• Eg Muscle cells
OLIGOPOTENT STEM CELL
• Ability to differentiate into a few cell types
• Eg Lymphoid or myeloid stem cells
36
Bhattacharyya et al. The voyage of stem cell toward terminal differentiation: a brief overview, Acta Biochim
Biophys Sin.2012;44(6):463

37
STEM CELL LINEAGE

REPAIR BY CONNECTIVE TISSUE
DEPOSITION
• If repair cannot be accomplished by regeneration alone it
occurs by replacement of the injured cells with connective
tissue.
• Steps in Scar formation:
– Tissue injury
– Inflammatory responses
– Angiogenesis
– Deposition of connective tissue
– Remodelling of connective tissue 38

• Inflammation is one of the body’s first reaction to injury.
• Release of damaged cells and tissue debris occurs upon injury
• These expelled particles act as antigens to stimulate a non
specific immune response and cause proliferation of leukocytes
• Resulting infiltration of tissues by leukocytes, plasma proteins
and fluid causes redness, swelling and pain.
39
TISSUE INJURY

INFLAMMATORY RESPONSES
Acute inflammation has 3 major components:
• Dilation of small vessels leading to an increase in blood flow
• Increased permeability of microvasculature enabling plasma
proteins and leucocytes to leave the circulation
• Emigration of leucocytes from the microcirculation, their
accumulation in the focus of injury
40

ACUTE INFLAMMATORY REACTIONS HAVE ONE OF
THREE OUTCOMES
• Complete resolution
• Healing by connective tissue replacement (scarring, or
fibrosis)
• Progression of the response to chronic inflammation
41

ANGIOGENESIS
• Formation of new blood vessels which supply nutrients and
oxygen needed to support the repair process.
• Angiogenesis involves several signaling pathways, cell-cell
interactions, ECM proteins and tissue enzymes
• Angiopoetin 1 & 2 play a vital role in angiogenesis and
structural maturation of new vessels.
42

STEPS IN ANGIOGENESIS
Vasodilation and increased vascular permeability by VEGF
Separation of pericytes and breakdown of basement membrane to
allow formation of vessel sprout
Migration and proliferation
of endothelial cells
Remodelling into capillary tubes
Formation of mature vessels by periendothelial cells
43

44
ANGIOGENESIS

DEPOSITION OF CONNECTIVE TISSUE
• The laying down of connective tissue occurs in two steps:
– Migration and proliferation of fibroblasts into the site of
injury
– Deposition of extracellular matrix
• Transforming growth factor-β (TGF-β) is the most important
cytokine for the synthesis of connective tissue proteins
45

STEPS IN CONNECTIVE TISSUE DEPOSITION
Proliferating fibroblasts and new vessels decreases
Increased deposition of ECM
Fibroblasts begin collagen synthesis(3-5 days)
Progressive vascular regression which transforms highly vascular
granulation tissue into pale avascular scar
46

REMODELLING OF CONNECTIVE TISSUE
• After its deposition, the connective tissue in the scar gets
modified and remodelled.
• The degradation of collagens and other ECM components is
accomplished by a family of Matrix
Metalloproteinases(MMPs).
• MMPs are activated by proteases to remodel the deposited
ECM and inhibited by Tissue inhibitors of
metalloproteinases(TIMP) produced by mesenchymal cells.
47

Common MMPs involved in remodelling of
connective tissue
MMP NAME FUNCTION
MMP-1 Collagenases Cleaves fibrillar collagen
MMP-2,
MMP-9
Gelatinases Degrades amorphous collagen and
fibronectin
MMP-3,
MMP-10,
MMP-11
Stromelysins Degrades proteoglycans, laminins,
fibronectin and amorphous collagen
MMP-7 Matrilysin Disrupted cell aggregation, increased
cell invasion
48

FACTORS THAT INFLUENCE TISSUE REPAIR
• Infection
• Diabetes
• Nutrition
• Glucocorticoids
• Mechanical factors
• Poor perfusion
• Foreign bodies
• Type, extent and location of
the injury
49

50
Infection prolongs inflammation and potentially increases the local
tissue injury
Diabetes is a metabolic disease that compromises tissue repair,
systemic causes of abnormal wound healing
Prolonged inflammatory reaction
Increase in no of neutrophils
Secrete pro inflammatory cytokines TNFα,
IL-1β
Disruption in wound healing
MMP’S synthesised and degrade proteins and
growth factors

51
Nutrition particularly vitamin C deficiency, inhibits collagen
synthesis and retards healing
Mechanical factors such as increased local pressure or torsion may
cause wounds to pull apart, or dehisce.
Glucocorticoids (steroids) have anti inflammatory effects, and their
administration may result in weakness of the scar due to inhibition of
TGFβ production and diminished fibrosis

52
Type, size and location of injury determines whether healing
takes place by resolution or organisation
Poor perfusion, due either to arteriosclerosis and diabetes or to
obstructed venous drainage also impairs healing
Foreign bodies including sutures interfere with healing and cause
intense inflammatory reaction and infection.

53
SIZE OF WOUND
• Small wound heal
faster than larger
wound
LOCATION OF
WOUND
• Wound in
increased
vascularised area
can heal faster
TYPE OF TISSUE
• Complete
restoration of
tissues to normal
(stable or labile
cells)
• Replacement of
damaged tissue by
granulation tissue
is organisation
(permanent cells)

ABNORMALITIES IN TISSUE REPAIR
1. Inadequate formation of granulation tissue
2. Excessive formation of the components of the repair process
3. Exuberant granulation
4. Wound contracture
54

1. Inadequate formation of granulation tissue
– Wound dehiscence: Rupture of the wound, occurs most
frequently due to increased pressure.
– Wound ulceration: Occurs as a result of inadequate
vascularization during healing.
55

2. Excessive formation of the components of the
repair process
KELOID HYPERTROPHIC SCAR
Accumulation of excessive
amounts of collagen which
extends beyond the wound
margin
Accumulation of excessive
amounts of collagen confined
within the borders of the
wound margin
More common in African-
American population
No racial predilection
56

3. EXUBERANT GRANULATION
• Excessive formation of granulation tissue that protrudes above
the level of surrounding skin and blocks re-epithelialization.
• Excessive granulation tissue must be removed by cautery or
surgical excision to permit restoration of continuity of
epithelium.
57

4. WOUND CONTRACTURE
• Wound contraction is an important part of the healing process.
• An exaggeration of this process gives rise to contracture and
results in deformities of the wound and the surrounding
tissues.
58

WOUND STRENGTH
• Carefully sutured wounds have approximately 70% of the
strength of normal skin.
• 1 week after removal of suture wound strength is
approximately 10% of that of the normal skin.
• After 4 weeks, wound strength rapidly increases due to
increased collagen production and decreased collagen
degradation.
• Wound strength reaches approximately 70% to 80% of normal
by 3 months but never improves beyond that point.
59

HEALING OF EPITHELIAL INJURY
• This is a process that involves both epithelial regeneration and
formation of connective tissue scar.
• Based on the nature and size of the wound the healing occurs
by:
– Primary union or Healing by first intention
– Secondary union or Healing by second intention
60

PRIMARY UNION VS SECONDARY UNION
FEATURE PRIMARY UNION SECONDARY UNION
Cleanliness of wound Clean Unclean
Infection Generally uninfected May be infected
Margins Surgical clean Irregular
Sutures Used Not used
Granulation tissue Scanty granulation
tissue at the incised gap
and along suture tracks
Exuberant granulation
tissue to fill the gaps
Outcome Neat linear scar Contracted irregular
wound
Complications Infrequent, epidermal
inclusion cyst formation
Suppuration, may
require debridement
61

HEALING BY FIRST INTENTION
• When the injury involves only the epithelial layer, the
principle mechanism of repair is epithelial regeneration.
• Example: Clean, uninfected surgical incision approximated by
sutures.
• The repair consists of three connected processes:
– Inflammation
– Proliferation of epithelial and other cells
– Maturation of connective tissue scar
62

Sequence of events in healing by first intention
• Immediate
 Wounding activates the coagulation pathways and leads to
formation of clot.
 Clot formed stops bleeding and acts as a scaffold for
migrating cells activated by chemokines released by
damaged tissue.
 Dehydration of the external surface of the clot leads to
formation of a scab covering the wound
63

• Within 24 hours to 48 hours:
 Neutrophils migrate towards the clot
and release proteolytic enzymes that
clear the debris.
 Basal cells at the cut margins show
increased mitotic activity.
 Epithelial cells from cut margins
proliferate and migrate along the
dermis and meet in the middle beneath
the scab
 This yields a thin but continuous
epithelial layer covering the wound.
64

65

• By day 3:
 Neutrophils are largely replaced by macrophages.
 Granulation tissue invades the incision space.
 Collagen fibers are now evident at incision margins.
 Epithelial proliferation continues and forms a covering
approaching the normal thickness.
66

• By day 5:
 Neovascularization reaches its peak as granulation tissue
fills the incision space.
 Fibroblasts produce extracellular matrix proteins and
collagen fibrils which become abundant and begin to
bridge the incision.
 Epithelium recovers its normal thickness as differentiation
of surface cells yields a mature epithelial architecture.
67

• During 2nd week:
 Leucocyte infiltration, edema and increased vascularity are
substantially diminished.
 The process of blanching begins due to increased collagen
deposition and regression of vascular channels.
• By the end of first month:
 Scar comprises a cellular connective tissue largely devoid
of inflammatory cells and covered by an normal
epithelium.
68

Healing by Second intention
• When cell or tissue loss is extensive, such as in large wounds,
abscesses, ulceration healing occurs by secondary union or
healing by second intention.
• This involves both the combination of regeneration and
scarring.
• The inflammatory reaction is more intense and there is
development of abundant granulation tissue, formation of a
large scar and wound contraction by myofibroblasts.
69

Sequence of events in healing by second intention
Wounds have large tissue deficits which are filled by large fibrin
clots and there is more exudate and necrotic debris.
Neutrophils accumulate initially which are then replaced by
macrophages which clear the necrotic debris.
Epithelial cells from both the wound margins proliferate and
migrate until they meet each other in the middle.
70

Proliferation of fibroblasts and neovascularization forms a larger
granulation tissue.
With time, the scar on maturation becomes pale and white which
is due to increase in collagen and decrease in vascularity.
Wound contracts to one-third to one-fourth of its original size.
71

72

CONCLUSION
• The goal of regenerative medicine is to restore the cells,
tissues and structures that are lost or damaged after disease or
injury.
• The understanding of these mechanisms can facilitate highly
relevant therapeutic strategies in regenerative medicine.
92

REFERENCE
• Robbins and Cotran Pathologic Basis of Disease, 9th edition
• Textbook of pathology, Harsh Mohan, 7th edition
• Carranza’s clinical periodontology, 11th edition
• Sigmund Stahl. Repair or regeneration following periodontal
therapy?, J Clin Periodontol.1969
93

• Christopher P. Role of Growth Factors in Cutaneous Wound
Healing: A Review; Critical Reviews in Oral Biology and
Medicine.1993
• Schultz and Wysocki. Interactions between extracellular
matrix and growth factors in wound healing. Wound Rep Reg.
2009;17:153–162
• Bhattacharyya et al. The voyage of stem cell toward terminal
differentiation: a brief overview. Acta Biochim Biophys Sin.
2012;44(6):463
94
REFERENCE

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