TISSUE REPAIR ( pathalogy )

TISSUE REPAIR

Tissue Repair
The body’s ability to replace the injured or dead cell after inflammation is critical to the survival. The injurious agent set in motion not only the events that serve to contain the damage but also to prepare the non dead cells to replicate and replace the dead cells.

The repair of a tissue involves into 2 distinct processes:

a) Regeneration

b) Replacement with a connective tissue called fibroplasias or fibrosis which leaves a permanent scar.
The mechanism that controls both of them is similar and involves cell migration, proliferation. Differentiation and cell-matrix interaction.
The orderly regeneration of epith, tissue requires integrity of the basement membrane. If it is disrupted by injurious agent, orderly regeneration is not possible.
The basement membrane serves as extra cellular scaffold for accurate regeneration of the preexisting structure.
The size of the cell population is maintained by the rate of cell proliferation, differentiation and cell death by apoptosis.

Control of normal cell growth

Cell proliferation can be stimulated by injury, cell death and mechanical deformation. Cell replication is controlled by chemical factors in microenvironment stimulating or inhibiting cell proliferation. Growth is accomplished by shortening the cell cycle.


Cell cycle and proliferation potential 

Cells in our body can be divided into 3 groups on the basis of their proliferation capacity and relationship to cell cycle:

1. Continuously dividing cells (labile cells): follow the cell cycle from one mitosis to the next and proliferate throughout their life replacing cells continuously destroyed (epithelia and hematopiotic cells are examples)

2. Quiescent or stable cells: cells with low level of replication but can undergo rapid cell division in response to stimuli and able reconstituting the tissue of origin ( cells of the liver, kidneys, pancreas, mesechyma, fibroblasts, smooth muscle, endothelial) The cells are in Go of the cell cycle and stimulated go into G1.

3. Non dividing (permanent) cells: cells left the cell cycle and can not undergo mitosis in the postnatal life. In this group belong most nerve cells, skeletal and cardiac muscle cell.

Molecular events in cell growth 

They are complex and involve increasing array of intercellular pathways and molecules. Aberrations of such pathways may underlie the uncontrolled growth of cancer.
Some growth factors induce cell proliferation by affecting the genes involved in normal growth control pathways the so-called proto-oncogene. The expressions of these genes are tightly controlled during normal growth and regeneration. Alteration in these proto-oncogene can convert them into oncogenes and induce cancer. The normal and abnormal cellular proliferation follows similar pathways.
There are 3 ways in which intercellular signals are distributed depending on the distance of the target cells to secreting cells: autocrine, paracrine and endocrine

Cell surface receptors
Cell growth begins when the signal, usually, growth factor (GF) binds with the receptor protein which is on the surface, inside the cytoplasm or nucleus. The complex signal-receptor initiates specific cellular response. On ligand binding, they deliver signals to the nucleus, using variety of transduction pathways.


There are 3 major classes of surface receptors:

1) Receptors with intrinsic kinase activity: they have extra cellular domain for ligand binding, a single transmembrane region and cytosolic domain with tyrosine kinase activity e.g. EGF, FGF, PDGF

2) Receptors without intrinsic catalytic activity: is the same as first except for the cytosol domain which directly activates one or more cytosolic protein tyrosine kinase which in turn phosphorylate the receptor. They are called cytokine receptor super family.

3) Protein G linked receptors: they contain 7 transmembrane loops and are called seven-spanning receptors. They are usually for other cell function like inflammation.

Signal transduction system
Is the process by which extra cellular signals are detected and converted into intra cellular signals which in turn generate specific cellular response. The transduction is arranged as networks of sequential protein kinase. The most important that involve in cell growth regulation are: -

1) mitogen-activated protein kinase (MAP kinase) pathway,

2) 2)phosphoinositide-3-kinase pathway,

3) inositol-lipid pathway (IP3)

4) cyclic adenosine monophosphate (cAMP)

5) JAK/STAT pathway


Transcription factors (TF) 

The signal transduction system transfers information to the nucleus where specific change occurs in the regulation of gene expression which in turn is controlled by transcription factors. These factors have a vital role in cell growth.
The TF have two domains, one for DNA binding and the other for transcriptional regulation (regulatory domain).
The first specifically binds with a short-sequence motifs of DNA.
The second activates or inhibits the transcription. The TF are phospholrilated by specific proximal kinase which can change the TF’s location of affinity to the DNA which in turn alter gene expression.
Among the TF are proto-oncogenes in which their mutation is associated with tumors. The TF are also involved in the regulation of cell cycle itself.

Cell cycle and regulation of cell division

· The mechanism that controls the passage of the cell through specific phase of the cell cycle and orchestration of events leading to cell division are 2 molecular events:

1) Cascade of protein pathway involved by proteins called cyclins

2) A set of checkpoints that monitor the completion of the molecular events and if necessary delay progression to next phase.

Cyclins and cyclin dependent kinase (CDK)
The entry and progression of cells through cell cycle are controlled by the change in levels and activities cyclins termed A,B, E. They reach peak during specific phase of cell cycle and rapidly degraded as the cell enters the next phase. Cyclins to function complexes with CDK (E.G. CDK-1/cyclin B controls the transition from G2 to M) The cyclins are synthesized de novo while CDK are produced constitutively.
In addition to synthesis and degradation, the active CDK complexes are regulated by CDK inhibitors such as P12, P27, as well as other kinases and phophatases that counterbalance the activities of CDK complexes.

Checkpoints
They represent a second way of cell cycle regulation which ensures that critical transition occurs in the correct order and events are completed with fidelity. They sense problems in DNA replication, repair and chromosome segregation. When they are activated by damaged or under replicated DNA, signals are sent to cell cycle machinery that arrest the cell cycle. By delaying progression of cell cycle checkpoints provide more time for repair and reduce possibility of mutations.
Checkpoints arrest cell cycle either by promoting inhibitory pathways or by inhibiting activating pathways e.g. the tumor suppressing gene, P53, is activated in response to DNA damage and inhibits cell cycle by increasing the expression of the CDK inhibitor, P21.

Growth inhibitors
Their function is similar but inverse mechanism to growth stimulators: they have receptors and transduction system. They inhibit cell cycle progression through the CDK inhibitors. Good example of growth inhibition in vitro is contact inhibition of growth in confluent cell culture. In vivo, regeneration of liver after partial hepatoectomy.

Growth factors
Some of the GF act on a variety of cell types, whereas others have relatively specific targets. Beside cell growth, they have other effects which are also important to repair and healing wounds like: cell locomotion, contractility and differentiation.

The most important GF

1) EGF (epith, GF): first discovered by its ability to cause precocious tooth eruption. Binds receptor with tyrosine kinase activity. A mitogenic to various epith, fibroblast, hepatocytes.

2) PDGF (platelet derived GF): stored in α granules of the platelets, released in platelet activation. They cause migration and proliferation of fibroblasts, smooth muscle cells and monocytes and have proinflammatory properties.

3) FGF (fibroblast GF): are produced by a variety of cells; are of two types acid and basic. They stimulate angiogenesis, migration of cells (macrophages, endothelial, fibroblasts and epithelial to the damaged site), skeletal muscle development, lung maturation and hematopoiesis.

4) Vascular endothelial GF and placental GF: They promote asculogenesis in early development

5) TGF β: produced by different cells. They stimulate fibroblast chemo taxis, collagen and fibronectin production. They inhibit collagen degradation.

6) Cytokines: besides, inflammatory effects, they can be placed in the family of GF since many of them have growth promoting activities to variety of cells.


Extra cellular matrix (ECM)

Interaction cell/ matrix:
The ECM critically influence cell function. It forms significant proportion of the tissue volume and consist of macromolecules outside the cells. They sequester water to provide turgor to the soft tissue, minerals to provide rigidity to the skeletal tissue and provide reservoir for GF.

There 3 groups of macromolecules in the ECM:

1) Fibrose structural proteins

2) Adhesive glycoprotein like fibronectin and laminin

3) Gel of proteglycans and hyluronic acid.

These macromolecules assemble into 2 general organizations

1) Interstitial matrix and

2) Basement membrane



I. Interstitial matrix: is the matrix between the cells (epith, endoth, smooth muscle and connective) and consist of fibrillar (I, III, V) and non fibrillar collagen, elastin, fibronectin, proteoglycans, hylurunate and other components.

II. Basement membrane: is produced by epith, and mesenchymal cells and are closely associated with the cell surface. It consist of non fibrillar collagen mostly IV, laminin, heparan sulfate, proteoglycans and other glycoproteins.

Adhesive glycoproteins and integrins
These molecules link the ECM components one to another and ECM and the cells. Examples are fibronectin, laminin and integrins.
Fibronectin: Is multifunctional adhesive protein whose primary role is to attach cells to a variety of matrices. It’s large (450 kDa) glycoprotein of 2 chains held together by disulfides bonds associated with cell surface, BM, pericellular matrices. It is produced by fibroblasts, endoth, monocytes and other cells. It binds with ECM components via specific domain and binds cells via receptors that recognize the amino acids arginine-glycine-spartic acid ( RGD) The latter plays a key role in cell matrix adhesion,
Laminin: Is most abundant glycoprotein in the BM. It is large (820KD) hetero trimeric cross-shaped glycoprotein. It binds the cell surface through specific receptors and binds also with a domain to ECM components.

Integrins: Are the major family of cell surface receptors that mediate cellular attachment to the ECM. Other integrins mediate cell to cell interaction involved in leukocyte adhesion. They are also important in the platelet aggregation, developmental processes and wound healing. Some cells need adhesion to ECM by integrins to proliferate and lack of it induce apoptosis.

· Integrins are transmembrane glycoproteins with α (14 types) and β ( 8 types) chains forming 20 hetrodimer integrins. The extra cellular domain binds with ECM components by recognizing the RGD sequences.

· Integrins are important in the transduction from ECM and actin cytoskeleton organization. Also important in clustering of receptors and formation of focal adhesion.


Proteoglycans and hyaluronan 

Together with the matricellular proteins, adhesive glycoproteins and integrins constitute ECM molecules. They are composed of core proteins linked by glycosaminoglycans.
Some are integral membrane proteins like the syndecan family that regulate cell growth and differentiation. They have short intracytoplasma segment and long external domain. They bind with ECM components and some GF like FGF.
The hyaluronan is associated with cell receptors that regulate cell proliferation and migration. It binds large amount of water, hydrated viscous gel which gives CT turgor pressure and ability to resist compression. It also gives resilience as well as lubricating feature to many types of CT and cartilage joints.   

Repair by connective tissue (fibroses) 

The destruction of the parenchyma and stroma framework produced by chronic inflammation in which repair cannot be done solely with regeneration of parenchyma cells the replacement is done by fibrosis and scaring.

There are 4 components in the scar formation:
Angiogenesis
Migration and proliferation of fibroblasts
Deposition of ECM
Maturation and organization of the fibroses tissue.



Repair begins early in the inflammation.
If resolution does not occur, fibroblasts and endothelial cells begin to proliferate forming in 3 to 5 days a tissue known as granulation tissue (it is soft, pink and has granulated in appearance on the surface of the wound.)
Histologically, there are new small blood vessels, proliferating fibroblasts and leukocytes. The new vessels are leaky so they allow passage of proteins and RBC to the extra vascular space and hence the edema and the redness of the granulation tissue

Angiogenesis

- In the embryo the vasculogenesis occurs with the proliferation of endothelial cell precursors called angioblasts. Afterwards the preexisting vessels send out new capillary bed or sprouts to form a new vessel. 

Steps of the development of new vessels:
Proteolytic degradation of the BM of the parent vessel to allow formation of new capillary sprout.
Migration of the endothelial cells to the site.
Proliferation of the endothelial cells behind the leading front.
Maturation of endothelial cells ( inhibition of growth and remodeling into capillary tubes.
Recruitment of periendothelial cells ( pericytes for capillaries and smooth muscle cells for larger ones) to support the endothelial tubes providing maintenance and accessory cell function for the vessels.


- All the steps of the angiogenesis are controlled by interactions among GF, vascular cells and the ECM.

- The most important GF for the angiogenesis is VEGF and angioproteins. They are produced by many cells (mesnchymal and stromal) but their receptor which has tyrosine kinase activity are restricted to endothelial cells.

- The ECM controls the motility and the direction of the endothelial migration (the integrins control these activities.)

Fibroses

- It occurs within the granulation tissue framework of new blood vessels and loose ECM initially formed at the repair site. It involves into 2 processes:



1. Proliferation and migration of fibroblasts at the site of injury.

- The increased permeably of the newly formed vessels provide leakage of plasma proteins like fibrinogen and fibronectin that deposits in the loose ECM of the granulation tissue providing provisional stroma for fibroblasts ingrowths.

- The migration and proliferation of fibroblasts to the site of injury are triggered by the GF (TGF-β, PDGF, EGF, FGF, fibrogenic cytokines – IL-1, TNF-α) produced by the cells (platelets, activated endothelial and macrophage cells) Other cells in the granulation tissue are mast cells, lymphocytes, eosinophiles.

2- ECM DEPOSITION.

- As the repair proceeds the number of fibroblasts and endothelial cells decreases. Fibroblasts produce more ECM especially fibrillar collagen which increases the strength of the healing wound. The same GF which stimulate fibroblasts proliferation also stimulate the ECM production.

- The granulation tissue is converted into scar tissue with spindle fibroblasts, dense collagen, elastic tissue and other components of the ECM. As the scar matures, it transforms into pale avascular scar.



Scar tissue remodeling.

- Replacement of granulation tissue with scar involves transition in the composition of the ECM. The GF stimulating the collagen formation also modulates the synthesis and activation of metalloproteinases (MP) that degradate ECM. The net result of synthesis versus degradation results in remodeling of the connective tissue of the scar.

- The degradation of collagen and other ECM proteins is achieved by matrix MP Zinc ion dependent for their activities.

- There other proteases called serine proteinases (neutrophil elastase, cathepsin G, kinins, plasmin) which are different from MP, but also degradate ECM.

MP consists of:-

1. Interstitial collagease (for collagen I, II, III)

2. Gelatinase (for type IV, fibronectin)

3. Stromelysins (for proteglycans, laminin, fibronectin and amorphous collagen) -membrane-bound matrix metalloproteinase (MBMM): cell surface associated protease.

- These enzymes are produced by many cells like macrophages, neutrophiles, fibroblasts, senovial cells and epith, The production is stimulated by PDGF, FGF and cytokine IL-1, TNF-α, phagocytosis and physical injury. They are inhibited by TGF-β and steroids.

- These enzyme are harmful but elaborated in latent form to be activated by chemicals like HClO and plasmin

- The MP is inhibited by tissue inhibitors of metalloproteinase (TIMP) which are produced by most mesenchymal cells.

- The dual effects of the MP and TIMP regulate the wound healing.

Wound healing involves:

- Induction of acute inflammation

- Regeneration of parenchymal cells

- Migration and proliferation of both parenchymal and CT cells.

- Synthesis and deposition of ECM

- Collagenation and acquisition of wound strength.


Wound healing by first intention.

- The least complicated wound healing occurs in clean non infected surgical incision, with margins approximated by surgical suture. this type of wound heals by first intension.

- The incision causes death of epithelial and CT cells and disruption of the BM.

- The incision space is filled by clot which when dehydrated on the surface forms scab that covers the wound.

- In 24-48h, neutrophiles invade the clot and basal epidermal cells proliferate and migrate along the cut margin of the dermis depositing BM components. From both margins hey confuse at the mid line beneath the surface scab.

- By he third day, neutrophiles largely replaced macrophages and granulation tissue invade the incision space. Collagen fibers at the margin of the incision are oriented vertically and not bridge the cut. The epth, proliferate and thicken the epidermal covering layer.



- Fifth day, incision is completely filled by granulation tissue. Collagen fibers become abundant and bridge the incision. Epidermis recovers its normal thickness and there surface layer keratinization.

- From the second week the accumulation of collagen and fibroblasts continue and granulation tissue and neutrophiles are largely disappeared. The vessels regress and remodeling process begins.

- By the end of the first month, the scar comprises cellular CT devoid of inflammatory infiltrate covered by intact epidermis.

- The dermal appendages destroyed in the line of incision are permanently lost.

- The tensile strength of the wound increases thereafter but it may take months to obtain its maximal strength

- Although this healing is efficient, the end products may not be functionally perfect. Dermal appendages are lost, and scar CT replaces the efficient meshwork of collagen in unwounded dermis.



Wound healing by second intention.

- When there is large defects to be filled, parenchymal regeneration can not completely reconstitute the original architecture. Abundant granulation tissue grows from the margin to fill the defect.

- In this healing there contraction of the wound to reduce the size of the scar tissue.

- Whether a wound heals a first or a second intension is determined by the nature of the wound, rather than by the healing process itself.





Wound strength

- When the suture are removed by end of the first week the wound has 10% of the tensile strength of the normal skin. Then the strength increases rapidly over the next 4 weeks. Then the rate of increase slows at the third month from the incision and reaches a plateau at about 70% to 80% which may remain for life.


Factors affecting wound healing

Systemic factors

- Nutrition (protein and vitamin C and others)

- Metabolic status (diabetes)

- Circulatory status (arteriosclerosis, venous abnormality)

- Hormones like glucocorticoids



Local factors

- Infection is the most important

- Mechanical factors e.g. early motion

- Foreign bodies

- Size, location and type of the wound (richly vascularized sites heals faster)

Pathologic aspect of the wound repair

1. Inadequate formation of granulation tissue or assembly or scar leads wound dehiscence and ulceration.

- Dehiscence or rupture is common in abdominal wound surgery and is due to increased pressure generated by vomiting, coughing or ileus.

- Ulceration occurs in the wounds of the lower extremities due to inadequate vascularization. It usually affects individuals with vascular abnormalities.

2. Excessive formation of repair tissue.

a) Excessive collagen formation with resultant raised tumor scar (keloid with predisposition in black race)

b) Excessive granulation tissue which protrudes the level of the surrounding skin blocks re-epithelialization. This is called exuberant granulation or proud flesh or granuloma pyogenicum.

c) Exuberant proliferation of fibroblasts or other CT cells that recur after excision known as desmoids or aggressive fibromatosis ( sometimes referred as low grade malignant tumor)



d) Contraction.

· Contraction is normal in the wound repair but when exaggerated is called contracture and brings deformation of the wound and the surrounding tissue.

· It is common in palms, soles and anterior thorax. It usually results from serious burns and compromise joint movements.