Introduction Even as cells and tissues are injured, events that contain the damage and prepare the surviving cells to replicate are set into motion. Repair consists of the replacement of dead cells by viable cells. These new cells may be either derived from the parenchyma or from the connective tissue stroma of the injured tissue. During the evolutionary process, mammals lost the capacity to regenerate total structures, such as limbs, as many of the simpler aquatic and amphibious animals can. Indeed, the restorative capacity of humans is quite limited. Only some of their cells are capable of regeneration. ďƒ Repair of destroyed cells because usually involves some connective tissue proliferation with the formation of a fibrous scar. Although, the anatomic continuity of the tissue may be restored thereby, such impair is obviously imperfect, since it replaces functioning parenchymal cells with non-specialized connective tissue. Repair begins very early in the process of inflammation and involves two processes: I] Regeneration of the injured tissue. II] Replacement by connective tissue.
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I] Regeneration -
Some parenchymal cells are short-lived while others have a longer life span. In order to maintain the proper structure of tissues, these cells are under the constant regulatory control of their cell cycle. which is defined as the period between 2 successive cell
divisions and is divided into 4 unequal phases. (a)
M (mitosis) phase
(b)
(c)
G1 (gap 1) phase
S (synthesis phase)
the daughter cell enters G 1 phase after mitosis
synthesis of nuclear DNA takes place
(e)
(d) G2 (gap 2) phase after completion of nuclear DNA the cell enters G 2 phase
G0 (gap 0) phase is the quiescent or the resting phase of the cell after an M phase
Regeneration can be divided into: Movement of the surviving cells
Proliferation of the surviving cells
into the vacant space made
to replace the loss
available by loss due to wound / necrosis
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The cells of the body have been divided into 3 groups on the basis of their regenerative capacity and their relationship to the cell-cycle. (A)
Continuously dividing (labile cells)
ďƒ These cells continue to multiply throughout life under normal physiologic conditions. These include: -
Surface epithelial cells of epidermis.
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Alimentary tract.
-
Respiratory tract.
-
Urinary tract.
-
Vagina.
-
Cervix.
-
Uterine endometrium.
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Haemopoietic cells of bone marrow.
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Cells of lymph nodes and spleen.
(B)Quiescent (stable cells) -
These cells decrease or lose their ability to proliferate after adolescence but retain the capacity to multiply in response to stimuli throughout life. The growth capacity of stable cells is best exemplified by the
ability of the liver to regenerate after hepatectomy or following toxic / viral / chemical injury. These include:
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-
Parenchymal cells of organic like liver, pancreas, kidneys, adrenal and thyroid.
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Mesenchymal cells like smooth muscle cells
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Fibroblasts.
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Vascular endothelium
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Bone
-
Cartilage cells.
(C)Non-dividing (Permanent cells) -
Exited the cell cycle at some point in intra-uterine development and cannot undergo further mitotic division in post-natal life. These include: o Neurons of nervous system. o Skeletal muscle. o Cardiac muscle cells. i.e. why destruction of a neuron whether in central nervous
system / ganglia, represents a permanent loss. The perfection of parenchymal repair of an injury depends on more than the ability of the cells to regenerate. Preservation of the stromal architecture of the injured tissue is also necessary. Thus, the perfection of repair depends to a considerable extent on the survival of the basic framework of the
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tissue when this is lost, regeneration may restore mass but not complete function. Relationship of parenchymal cells to the cell cycle a. Labile cells which are continuously dividing cells remain in the cell cycle from one mitosis to the next. b. Stable cells are in the resting phase but can be stimulated to enter the cell cycle. c. Permanent cells leave the cell cycle and die after injury.
II] Repair -
Repair is the replacement of injured tissue by fibrous tissue. Two processes are involved. A. Granulation tissue formation. B. Contraction of wounds.
A. Granulation tissue formation Refers / derived from the pink, soft, granular gross appearance such as that seen beneath the scab of a skin wound.
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Histologically, each granule corresponds to the proliferation of new small blood vessels which are slightly lifted on the surface by a thin covering of fibroblasts and young collagen. There are 3 components to granulation tissue formation. i)
PHASE OF ACUTE INFLAMMATION -
Following
trauma / injury, blood clots at the site of injury.
There is acute inflammatory response with exudation of plasma, neutrophils and some monocytes within 24 hours. ii)
PHASE OF CLEARANCE -
Combination of proteolytic enzymes liberated from neutrophils, autolytic enzymes from dead tissue cells, and phagocytic activity of macrophages clear off the necrotic tissue, debris and red blood cells.
iii)
PHASE OF INGROWTH OF GRANULATION TISSUE
Angiogenesis / Neovascularization
Formation of fibrous tissue
Formation of new blood vessels at Formation of fibrous tissue the site of injury takes place by - As the granulation tissue proliferation of endothelial cells natures, inflammatory cells from the margins of severed blood decrease in number, fibroblasts vessels. Initially, the proliferated lay down collagen and the endothelial cells are solid buds but capillaries become much less 6
within a few hours develop a prominent what emerges is an lumen and start carrying blood. avascular, relatively acellular scar The newly formed blood vessels with
inactive
are more leaky, accounting for the fibroblasts
tucked
spindle-shaped in
between
oedematous appearance of new collagen fibres. This is known as granulation tissue.
Cicartrisation.
Summarizing: -
Proteolytic – degeneration of the parent vessel basement membrane allowing formation of a capillary sprout.
-
Migration of the endothelial cells towards the angiogenic stimulus.
-
Proliferation
of
the
endothelial cells. -
Maturation endothelial
of cells
the with
organization. B. Contraction of wounds The wound starts contracting after 2-3 days and the process is completed by the 14 th day. During this time, the wound is reduced to approximately 80% of its original size. Contracted wound results
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in rapid healing since lesser surface area of the injured tissue has to be replaced. In order to explain the mechanism of wound healing, a number of factors have been proposed. These are: a)
Dehydration ďƒ as a result of fluid removal by the drying of the wound but this was not substantiated.
b)
Contraction of collagen ďƒ Was thought to be responsible for contraction but wound contraction proceeds at a stage when the collagen content of the granulation tissue is very small.
c)
Discovery of myofibroblasts appearing in active granulation tissue has resolved the controversy surrounding the mechanism of wound contraction. These cells have features intermediate between those of fibroblasts and smooth muscle cells. Their migration into the wound area and their active contraction decreases the size of the defect. With all this background on granulation tissue, we can now discuss the two forms of repair.
Healing by first intention
Healing by second intention
(primary union)
(secondary union) 8
The sequence of events are: 1.
Initial haemorrhage Within the first post-operative day, after the wound has been
approximated by surgical sutures, the line of incision promptly fills with blood clots which seals the wound against dehydration and infection. 2.
Acute inflammatory response This occurs within 24 hours by appearance of polymorphs from
the margins of the incision. By 3 rd day, polymorphs are replaced by macrophages. 3.
Epithelial changes The basal cells of epidermis from both the cut margins start
proliferating and migrating towards incisional space in the form of epithelial spurs. A well approximated wound is covered by a layer of epithelium in 48 hours. The migrated epidermal cells separate the underlying viable dermis from the overlying necrotic material and blood clot, forming a scab which is cast off. By 5th day a multilayered new epidermis is formed which is differentiated into superficial and deeper layer.
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4.
Organisation By 3rd day, fibroblasts also invade the wound area. By 5 th day,
the acute inflammatory response begins to subside and the neutrophils are replaced by macrophages which debride the wound margins of destroyed cells and bits and pieces of fibrin. In 4 weeks, the scar tissue with scanty cellular and vascular elements, new inflammatory cells and epithelialized surface is formed. I] Primary union This is defined as healing of a wound which has the following characteristics. -
Clean and uninfected.
-
Surgically incised.
-
Without much loss of cells and tissue, and
-
Edges of the wound are approximated by surgical sutures.
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The incised wound as well as sutures track on either side are filled with blood clot and there is inflammatory response from the margins. Spurs of epidermal cells migrate along the incised margin on either side as well as around the suture track. Formation of granulation tissue also begins from below. Removal of suture at around 7 th day results in scar tissue at the site of incision and suture track. Suture tacks Each suture track is a separate wound and incites the same phenomenon as in healing of the primary wound. When sutures are removed around 7 th day, much of the epithelialized suture tack is avulsed and the remaining epithelial tissue in the track is absorbed. However, sometimes the suture track gets infected (Stitch absecess), or the epithelial cells may persist in the track (implantation / epidermal cysts). II] Secondary union ďƒ This is defined as the healing of the wound having, the following characteristics:
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-
Open with a large tissue defect; at times infected.
-
Having extensive loss of cells and tissue and
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The wound is not approximated by surgical sutures but is left open.
-
The basic events in secondary union are similar to primary union but differ in having a larger defect which has to be bridged. Hence, healing takes place from the base upwards, as well as from the margins inwards. The healing by second intention is slow and results in a large at times, ugly, scar as compared to rapid healing and neat scar of primary union. The open wound is filled with blood clot and there is inflammatory response at the junction of viable tissue. Epithelial spurs from the margins of wound meet in the middle to cover the gap and separate the underlying viable tissue from necrotic tissue at the surface forming. After contraction of the wound, a scar smaller than the original wound is left.
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The sequence of events are: 1.
Initial haemorrhage As a result of injury, the wound space is filled with blood clot
and fibrin clot which dries. 2.
Inflammatory phase There is an initial acute inflammatory phase followed by
appearance of macrophages which clear off the debris as in primary union. 3.
Epithelial changes As in primary healing, the epidermal cells from both the margins
of wound proliferate and migrate into the wound in the form of epithelial spurs till they meet in the middle and re-epithelialise the gap completely. However, the proliferating epithelial cells do not cover the surface fully till granulation tissue from base has started filling the wound space. In this way, preexisting viable connective tissue is separated from necrotic material and clot on the surface, forming seal which is cast off.
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4.
Granulation tissue The main bulk of secondary healing is by granulations. The
newly-formed granulation tissue is deep red, granular and very fragile. With time, this scar on maturation becomes pale and white due to increase in collagen and decrease in vascularity. 5.
Wound contraction Due to the action of myofibroblasts present in granulation tissue,
the wound contracts to 1/3 rd – Ÿth of its original size. 6.
Presence of infection Bacterial contamination of an open wound delays the process of
healing due to release of bacterial toxins that provoke necrosis, suppuration and thrombosis. Surgical removal of dead and necrosed tissue helps in preventing the bacterial infection of open wounds. Complications of wound healing: -
Infection (due to entry of bacteria).
-
Implantation cyst.
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Pigmentation (healed wounds may at times have rust like colour due to staining with haemoglobin).
-
Deficient scar formation (due to inadequate G.T. formation).
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-
Incisional hermia (a weak scar may be site of bursting open of a wound) (Wound dehiscence). Two main aberrations occur in wound healing whether the
process of 1 st/2nd intention. a) The accumulation of excessive amounts of collagen may give rise to a protruding, tumorous scar known as a keloid thick interlacing bundles of collagen causing marked swelling at the site of the wound. b) The other deviation is the formation of excessive amounts of granulation tissue which protrudes above the level of the surrounding skin and in fact blocks re-epithelization. This has been called as ‘exuberant granulation / pround flesh’.
PATHOLOGIC ASPECTS OF REPAIR / Factors Influencing Repair In wound healing, normal cell growth and fibrosis may be altered by a variety of influences, frequently reducing the quality and adequacy of the reparative process. These factors may be either extrinsic / intrinsic.
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Systemic factors: Systemic / Local 1. Hypoxia Healing is often imperfect in regions with a poor blood supply e.g., various ulcers in a lower limb made hypoxic by varicose veins, often fails to heal. In contrast to this, mild hypoxia helps healing by increasing the differentiation of fibrocytes, collagen formation and the cross-linkage of collagen-fibrils. 2. Scurvy Is a classic cause of defective wound healing though now rare. Scar that results is weak and in the granulation tissue, new capillaries are few and ill-formed. 3. Severe and prolonged protein deficiency Inhibits wound healing. Deficiency of sulfur containing amino acids like cystine and methionine is particularly important. 4. Glucocorticoids Of the adrenal gland interfere with wound healing when given in large doses.
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5. Age Wound healing is rapid in young and slow in aged and debilitated patients due to poor blood supply to the injured area. 6. Uncontrolled diabetic – are more prone to develop infections and hence delay in healing. 7. Local factors a) Movement of the injured part. b) Fixation of the injured tissue to an unyielding object such as bone, makes apposition of the wound edges difficult and hampers healing. c) Wounds made along a line of mechanical stress in the tissue tend to fall together and heal easily but wound across a line of stress tend to gape and heal more slowly. d) Infection of the wound. e) Any foreign bodies present. f) Type, size and location of injury determines whether healing takes place by resolution / organization. g) Exposure to u-v light facilitates healing.
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h) Exposure to ionizing radiation delays granulation tissue formation. MECHANISMS INVOLVED IN REPAIR 3 processes
Cell-cell interactions.
Cell-matrix interactions.
Stimulatory hormones / growth factors.
1. Cell-cell interactions As mentioned earlier, re-epithelialization of wound begins within 24 hours of injury and the gap is usually covered within 48 hours. During regeneration of liver, after partial hepatectomy, the liver cells burst into mitoses but cease to divide when normal liver substance is restored. What signals these cells to stop dividing? When certain normal cells are lightly seeded in Petri dishes to investigate the growth behaviour of cells, they proliferate migrate and eventually form confluent monolayers. At this point cells cease to divide – a process called contact inhibition. It is proposed that cells are inhibited from proliferation by interchange of signals or substances at contact point. 2. Cell-matrix interactions Much evidence has accumulated to indicate that the orderly movement and proliferation of the cells within a healing wound is 18
influenced not only by signals derived from other cells but also from the extra-cellular matrix, which is an organized complex of collagen, glycosaminoglycans, proteoglycans and glycoproteins. Of these much attention is focused on fibronectins ďƒ which are a family of adhesive high molecular weight glycoproteins. Experimental evidence suggests that migration of endothelial cells and their organization into capillaries is also facilitated by fibronectin. 3. Growth Factors Thus far, we have discussed the regeneration migration and organization of cells in the wound. But what triggers these cells to divide. Either loss of factors that
or release of growth
normally inhibit cell division
stimulating factors.
At one time, the concept that proliferation occurred because of reduced levels of growth inhibition substance called Chalones, was popular. But nowadays, the list of growth factors is ever increasing. E.g., -
Epidermal G.F. (E.G.F.).
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Nerve G.F. (N.G.F.).
-
Platelet derived G.F. (P.D.G.F.).
-
Fibroblast G.F. (F.G.F.).
All these are polypeptides with hormone like structure.
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HEALING IN SPECIALIZED TISSUES a) Internal surfaces – Endothelium The regeneration of the covering epithelium is very similar to that of the skin. b) Bone Repair of a bone injury is essentially another instance of connective tissue healing. It differs from soft tissue repair in so far as formation of the specialized calcified tissues of bone involves the activity of osteoblasts and osteoclasts. Repair of a fracture may be taken as a model for the process of bone healing. However, basic events in healing of any type # are similar and resemble healing of skin wound to some extent. Primary Union of fractures occurs occasionally in a few special situations when the ends of fractures are approximated because bony union takes place with formation of medullary callus. Secondary union is the more common process of # healing. It is described under 3 headings: -
Procallus formation.
-
Osseous callus formation.
-
Remodelling.
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1)
Procallus formation
a.
Haematoma Forms due to bleeding, filling the area surrounding the #.
b.
Local inflammatory response Occurs at the site of injury with exudation of fibrin, polymorphs
and macrophages. Fragments of necrosed bone are scavenged by macrophages and osteoclasts. c.
Ingrowth of granulation tissue Begins with neovascularization. A soft tissue callus is that
formed which joins the ends of # bone without much strength. d.
Callus composed of woven bone and cartilage The cells of the inner layer of periosteum have osteogenic
potential and lay down the collagen as well as the osteoid matrix in the granulation tissue. The osteoid undergoes calcification and is called woven bone callus. At times, callus is composed of woven bone as well as cartilage, temporarily immobilizing the bone ends. This stage is called provisional callus / procallus formation and is arbitrarily divided into: -
External
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-
Intermediate.
-
Internal procallus.
2)
Osseous Callus The procallus acts as scaffolding on which osseous callus
composed of lamellar bone is formed. 3)
Remodelling During the formation of lamellar bone, osteoblastic laying and
osteoclastic removal are taking place, remodeling the united bone ends, which after sometime, are indistinguishable from normal bone. The external callus is cleared away, compact bone (cortex) is formed in place of intermediate callus and the bone marrow cavity develops in internal callus. Haematoma formation and local inflammatory response at the # site. Ingrowth of granulation tissue with formation of soft tissue callus. Formation of procallus composed of woven bone and cartilage with its characteristic furiform appearance and having 3 arbitary components -
External.
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-
Intermediate.
-
Internal callus
Formation of osseous callus composed of lamellar bone following clearance of woven bone and cartilage. Remodelled bone end; the external callus cleared away intermediate callus converted into lamellar bone and internal callus developing bone marrow cavity. 4)
Nervous tissue
Central Nervous System
Peripheral Nervous System
Regeneration does not occur. In cases of acute damage, the initial functional loss often exceeds the loss of actual nerve tissue because of the reactive changes in the surrounding tissue. As these changes diminish, functional restoration commences. Peripheral Nervous System (PNS) 3 types of degenerative processes in the PNS are: a) Wallerian degeneration Occurs after transection of the axon which may be as a result of knife wounds, compression, ischaemia. Then, there is initially 23
accumulation of organelles in the proximal and distal end of transection sites. Subsequently, the axon and myelin sheath distal to the transection site undergoes disintegration upto the next node of Ranvier followed by phagocytosis. The process of regeneration occurs by sprouting of axons and proliferation of Schwann cells from the distal end.
b) Axonal degeneration Degeneration of the axon begins at the peripheral terminal and proceeds backwards towards the nerve cell body. Changes similar to those seen in Wallerian degeneration are present but regenerative reaction is limited / absent.
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c) Segmental demyelination Is demyelination of the segment between two consecutive nodes of Ranvier, leaving a denuded axon segment. The axon, however remains intact. Schwann cells proliferation also occurs. This results in re-myelination of the affected axon. Repeated episodes of demyelination and remyelination are associated with concentric proliferation of Schwann cells around axons producing ‘onion bulbs’ found in hypertrophic neuropathy.
Traumatic Neuroma Normally, the injured axon of a peripheral nerve regenerates at the rate of approximately 1mm/day. However, if the process of regeneration is hampered due to an interposed haematoma or fibrous scar, the axonal sprouts together with Schwann cells and fibroblasts form a peripheral mass called Traumatic / Stump Neuroma.
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d) Muscles Muscle fibres of all 3 types – Skeletal, Caridac and Visceral have only limited capacity to regenerate. -
When a mass of muscle tissue is damaged, repair by scarring occurs.
-
If the damage affects individual muscle fibres diffusely and with varying severity, then regeneration of the specialized fibres is possible.
Books referred -
Harsh Mohan
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Stanley (Basic Pathology)
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Boyd’s Pathology
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Govan, MacFarlane and Callendar
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Cottran, Robin and Williams
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REPAIR (IN GENERAL) CONTENTS
• Introduction • Process Involved in Repair
Regeneration Replacement
• Wound Healing • Pathologic Aspects of Repair • Mechanisms Involved in Repair • Healing in Specialized tissues
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