Chapter 3

TISSUE REPAIR
Sr. No. TOPIC Page No.
1 Cell cycle 2

2 Growth factors 3

3 Wound healing by repair 3

4 Cutaneous wound healing 3

5 Fracture healing 8

6 Important questions 10

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 CELL CYCLE

Definition
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:
1. G1 (presynthetic)
2. S (DNA synthesis)
3. G2 (premitotic)
4. M (mitotic) phases
Quiescent cells that have not entered the cell cycle are in the G0 state.

Gateway to the cell cycle: Cells can enter G1 either from G0 or after completing mitosis (as in
continuously replicating cells). This transition is regulated by proto-oncogenes and genes
required for ribosome synthesis and protein translation.

G1/S checkpoint: This point is regulated by proteins, cyclins and associated enzymes called
cyclin-dependent kinases (CDKs). The activity of cyclin-CDK complexes is tightly regulated by
CDK inhibitors. This checkpoint ensures that cells with damaged DNA or chromosomes do not
complete replication. Upon passing this point, normal cells become irreversibly committed to
DNA replication.

G2/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 mechanisms. If DNA damage is too severe to be repaired, the cells are eliminated by
apoptosis, or enter a nonreplicative state called senescence, through p53-dependent mechanisms.
Checkpoint defects that allow cells with DNA defects to divide produce mutations in daughter
cells that may lead to neoplasia.

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 GROWTH FACTORS

Definition
The proliferation of many cell types is driven by polypeptides known as growth factors. Some of
the growth factors involved in regeneration and repair are:
1. Epidermal growth factor α (EGF-α)
2. Transforming growth factor-α & -β (TGF-α & -β)
3. Platelet-derived growth factor (PDGF)
4. Fibroblast growth factor (FGF)
5. Vascular endothelial cell growth factor (VEGF)
6. Tumor necrosis factor (TNF)

WOUND HEALING BY REPAIR

Definition
Under normal circumstances, healing occurs by regeneration, whereby the normal tissue
components are reconstituted. But if tissue injury is severe or chronic, and results in damage of
parenchymal cells and stromal framework of the tissue, healing cannot be accomplished by
regeneration. Under these conditions, healing is accomplished by repair, where in there is
deposition of collagen and other ECM components, leading to scar formation of a scar.

CUTANEOUS WOUND HEALING

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Types of cutaneous wound healing

Healing by primary union or Healing by secondary union or

by first intension
Occurs in a clean, uninfected surgical
incision approximated by surgical sutures
Incision causes death of a limited number of
epithelial and connective tissue cells and
disruption of epithelial basement membrane
Healing results in a relatively thin scar
by second intention
Occurs in excisional wounds that create large
defects on the skin surface
Causes extensive loss of cells and tissue
Involves intense inflammatory reaction, the
formation of abundant granulation tissue, and
extensive collagen deposition, leading to the
formation of a substantial scar, which
generally contracts

Phases of cutaneous wound healing

Wound healing goes through three overlapping phases of inflammation, proliferation, and
maturation.

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Phase 1 (Inflammation):
1. Clot formation: Injury leads to activation of the coagulation system resulting into clot
formation on wound surface. VEGF increases vascular permeability and causes edema.
Dehydration over the surface causes the clot to dry and form a scab.

2. Chemotaxis: Clot not only serves to stop bleeding but also serves as a scaffold for
migrating cells. Neutrophils appear at the margins of incision within 24 hours.
Inflammatory cells secrete proteolytic enzymes that clean the debris and kill invading
bacteria.

Phase 2 (Proliferation):
1. Re-epithelization: In 24 to 48 hours, spurs of epithelial cells move from the wound edge
depositing basement membrane components as they move. They fuse in the midline
beneath the surface scab, producing a thin, continuous epithelial layer that closes the
wound. Subsequent epithelial cell proliferation thickens the epidermal layer. Mediators of
keratinocyte migration and proliferation are FGF-7 (keratinocyte growth factor), IL-6,
HGF, HB-EGF and chemokine receptor CXCR 3.

2. Granulation tissue and angiogenesis:

Granulation tissue: The term, granulation tissue is derived from pink, soft, granular
appearance on the surface of wounds. It is a specialized type of tissue characterized by
presence of new small blood vessels (angiogenesis/neovascularization) and proliferation
of fibroblasts, which occurs in the first 24 to 72 hours of the repair process. It is the
hallmark of tissue repair. By 5 to 7 days, granulation tissue fills the wound area and
neovascularization is maximal.

Angiogenesis:
Processes: Angiogenesis can occur by two processes:

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a) Angiogenesis from preexisting vessels: Involves the following steps:
i) Vasodilation in response to NO and VEGF
ii) 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
iii) Migration of endothelial cells towards angiogenic stimulus
iv) Proliferation of endothelial cells just behind the migrating cells
v) Maturation of endothelial cells and remodeling into capillary tubes
vi) Recruitment of periendothelial cells (pericytes and vascular smooth muscle cells)
to form the mature vessel.

b) Angiogenesis from Endothelial Precursor Cells (EPCs): EPCs can be recruited from
the bone marrow into tissues to initiate angiogenesis, especially in ischemic organs,
cutaneous wounds, and tumors. These cells express markers of hematopoietic stem
cells, VEGFR-2 and vascular endothelial–cadherin (VE-cadherin).

Growth factors and receptors involved in angiogenesis:

a) Vascular Endothelial Growth Factor (VEGF): is the most important growth factor for
angiogenesis.
• Secreted by: many mesenchymal and stromal cells
• Receptor for VEGF: VEGFR-2, expressed by endothelial cells, their precursors,
other cells and many tumor cells.
• Action:
i) In angiogenesis from preexisting vessels: stimulates survival of endothelial
cells, their proliferation and motility, initiating sprouting of new capillaries.
ii) In angiogenesis from EPCs: enhances proliferation and differentiation of
endothelial cells at site of angiogenesis.

b) Fibroblast Growth Factor (FGF)-2: stimulates endothelial cell proliferation,

differentiation and migration.

Notch pathway: The pathway promotes proper branching of new blood vessels. There
are five Notch ligands [Jagged 1 and 2, and Delta-like ligand (DLL)- 1, 3, and 4] and four
receptors (Notch 1–4).

During angiogenesis the leading cell, known as a tip cell, undergoes proliferation and
migration, while stalk cells maintain their connection with the existing vessel. VEGF
induces DLL-4 in tip cells, while Notch-1 and -4 in stalk cells.

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 Stabilization of newly formed fragile vessels: Stabilization requires recruitment of
pericytes, smooth muscle cells and the deposition of ECM.

3. Provisional matrix: Neutrophils are largely replaced by macrophages by 48 to 96 hours.

They clear debris, fibrin, and foreign material and promote angiogenesis and ECM
deposition. Macrophages, to lesser extent other inflammatory cells and platelets secrete
chemokines and growth factors- TNF, PDGF, TGF- β, and FGF. This leads to migration
of fibroblasts to the site of injury and their subsequent proliferation.

Collagen fibers at first are vertically oriented and do not bridge the gap. At first the
provisional matrix contains fibrin, plasma fibronectin, and type III collagen. Later the
collagen is oriented horizontally, bridges the gap and is replaced by type I collagen.

Phase 3 (Maturation):
1. Scar Formation: The leukocytic infiltrate, edema, and increased vascularity largely
disappear during the second week. Granulation tissue is converted into a pale, avascular
scar, composed of spindle-shaped fibroblasts, dense collagen, fragments of elastic tissue,
and other ECM components. The dermal appendages that have been destroyed in the line
of the incision are permanently lost. By the end of the first month, the scar is made up of
acellular connective tissue devoid of inflammatory infiltrate, covered by intact epidermis.

2. Wound contraction: Wound contraction is generally seen in healing by secondary

intention, where contraction helps to reduce the wound surface area. Wound contraction
is achieved proliferation of myofibroblasts that express smooth muscle actin and
vimentin, responsible for contraction. They also produce large amounts of ECM
components such as type I collagen, tenascin-C, SPARC, and extra-domain fibronectin.

3. Connective tissue remodeling: Degradation of collagen and other ECM proteins is

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 achieved by matrix metalloproteinases (MMPs). MMPs are produced by fibroblasts,
macrophages, neutrophils, synovial cells, and some epithelial cells.

Metalloproteinases (MMPs) Degrade

Collagenases (MMP-1, -2, and -3) Collagen types I, II, and III
Gelatinases (MMP-2 and -9) Amorphous collagen and fibronectin
Stromelysins (MMP-3, 10, and 11) Amorphous collagen, fibronectin,
proteoglycans and laminin
Membrane-bound MMPs (ADAMs) Membrane-bound precursor forms of TNF
and TGF-α

4. Recovery of tensile strength: results from excess collagen synthesis over collagen
degradation during the first 2 months of healing, and later times from structural
modifications of collagen fibers (cross-linking, increased fiber size).

Age after injury Return of tensile strength

End of 1st week, at the time of suture 10 %
removal
3rd month 70-80%

FRACTURE HEALING

Types of union

Primary union of fractures Secondary union of fractures

Less common
Occurs when the ends of fracture are
approximated, as in rigid-fixation using
compression plates
Healing occurs in two stages:
1. Primary bone formation, where
connective tissue or fibrocartilage
(callus) is not laid down prior to new
bone formation.
2. Haversian remodeling, where
reabsorption cavities are formed within
the bone laid. These cavities act like
“cutting cones”, where advancing
osteoclasts remove bone and trailing
osteoblasts lay down new bone in
longitudinal direction parallel to the
More common
Occurs when the gap between the fractured
ends is more and cast fixation, external
fixation, bridge plating or intramedullary
nailing is done.
Bone healing closely resembles endochondral
ossification, which involves cartilage template
being replaced by bone, like normal bone
formation. The three stages include procallus
formation, callus formation and bone
remodeling.

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 long axis of bone.

Process of fracture healing by secondary union

Fracture Hematoma Granulation tissue Procallus Callus Remodeling

1. Procallus formation:
a. Hematoma: forms due to bleeding from torn blood vessels, filling the area surrounding
the fracture. This forms a framework for subsequent granulation tissue formation.
b. Local inflammatory response: occurs at the site of injury. Neutrophils are followed by
macrophages. Macrophages clear fibrin and debris. Macrophages and osteoclasts remove
the necrotic bone.
c. Ingrowth of granulation tissue: begins with neovascularization and proliferation of
mesenchymal cells from periosteum and endosteum. A soft tissue callus is thus formed.
d. Callus composed of woven bone and cartilage: the cells of the inner layer of periosteum
have osteogenic potential and lay down collagen and osteoid matrix in the granulation
tissue. The osteoid undergoes calcification and is called woven bone callus. This stage is
called procallus formation.

2. Osseous callus formation: Procallus acts as scaffolding on which osseous callus composed
of lamellar bone is formed. Woven bone is removed by osteoclasts. In their place, newly-
formed blood vessels and osteoblasts invade. Haversian system is formed around the blood
vessels and osteoblasts lay down the osteoid forming lamellar bone.

3. Remodeling: occurs as a result of laying down of lamellar bone by osteoblasts and removal
by osteoclasts, until the bony ends are united and indistinguishable from normal bone.
External callus is removed, compact bone is formed in place of intermediate callus and bone
marrow cavity develops in internal callus.

Complications of fracture healing

1. Fibrous union: may result if the immobilization of fractured bone is not done.
2. Non-union may result if some soft tissue is interposed between the fractured ends.
3. Delayed union: occurs with infection, inadequate blood supply, poor nutrition, movement
and old age.

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 IMPORTANT QUESTIONS

SHORT NOTES
1. Types of fractures and healing of fractures
2. Discuss wound healing

SHORT ANSWERS

1. Give two growth factors involved in regeneration and repair.

Ans. 1. Epidermal growth factor α (EGF-α)
2. Transforming growth factor-α & -β (TGF-α & -β)
3. Platelet-derived growth factor (PDGF)
4. Fibroblast growth factor (FGF)
5. Vascular endothelial cell growth factor (VEGF)
6. Tumor necrosis factor (TNF)

The End

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