REVIEW REVIEW

Organogenesis 6:4, 225-233; October/November/December 2010; 2010 Landes Bioscience

Tissue repair
The hidden drama
Kristine P. Krafts
Department of Pathology; University of Minnesota School of Medicine; Duluth Campus; Duluth, MN USA

Key words: tissue repair, regeneration, scarring, wound healing, growth factors, extracellular matrix, stem cells

Abbreviations: cAMP, 3', 5' cyclic adenosine monophosphate; CDK, cyclin-dependent protein; DNA, deoxyribonucleic acid;
ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblast growth factor; GAG, glycosaminoglycan; KGF,
keratinocyte growth factor; MAD, mothers against decapentaplegic; MMP, matrix metalloproteinase; IL, interleukin; JAK, janus
kinase; OPN, osteopontin; PDGF, platelet-derived growth factor; RB, retinoblastoma; STAT, signal transducer and activator of
transcription; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinases; TNF, tumor necrosis factor; VEGF,
vascular endothelial growth factor

processes: regeneration and replacement. Regeneration refers to a

As living beings that encounter every kind of traumatic event
type of healing in which new growth completely restores portions
from paper cut to myocardial infarction, we must possess
ways to heal damaged tissues. While some animals are able
of damaged tissue to their normal state. Replacement refers to a
to regrow complete body parts following injury (such as type of healing in which severely damaged or non-regenerable
the earthworm who grows a new head following bisection), tissues are repaired by the laying down of connective tissue, a
humans are sadly incapable of such feats. Our means of process commonly referred to as scarring. While a few types of
recovery following tissue damage consists largely of repair tissue injury (such as minor paper cuts) can sometimes be healed
rather than pure regeneration. Thousands of times in our lives, in such a way that no permanent damage remains, most of our
a meticulously scripted but unseen wound healing drama tissue repair consists of both regeneration and replacement.
plays, with cells serving as actors, extracellular matrix as the Tissue repair may restore some of the original structures of the
setting and growth factors as the means of communication. damaged tissue (such as epithelial layers), but may also result in
This article briefly reviews the cells involved in tissue repair, structural abnormalities that impair organ function (such as the
their signaling and proliferation mechanisms and the function
scar formed in the healing of a myocardial infarction).
of the extracellular matrix, then presents the actors and script
for the three acts of the tissue repair drama.
Whether the healing of a wound proceeds down the regenera-
tion or the replacement pathway (or both) depends, in part, on
the type of tissue in which it occurs. Certain tissues of the body
are more capable of cellular proliferation (and hence regenera-
Proem tion) than others. In this regard, there are three types of tissues:
continuously dividing tissues, quiescent tissues and nondividing
All the worlds a stage, tissues. Continuously dividing tissues (also known as labile tis-
And all the men and women merely players; sues) are comprised of cells that are constantly proliferating in
They have their exits and their entrances order to replace dead or sloughed-off cells. Examples of such tis-
And one man in his time plays many parts, sues include epithelia (such as skin, gastrointestinal epithelium
His acts being seven ages and salivary gland tissue) and hematopoietic tissues. These tis-
sues contain pools of stem cells, which have enormous prolifera-
Shakespeare tive and self-renewing ability, and which give rise to more than
As You Like It one type of cell. Replicating asymmetrically, each stem cell gives
Act 2, scene 7, 139143 rise to one daughter cell that differentiates and matures and
another daughter cell that remains undifferentiated and capable
The term repair, when used in the context of the healing of of beginning another self-renewing cycle.
damaged tissue, is defined as the restoration of tissue architec- Some tissues, known as quiescent tissues (or stable tissues) are
ture and function after an injury. It encompasses two separate composed of cells that normally exist in a non-dividing state but
may enter the cell cycle in response to certain stimuli, such as
Correspondence to: Kristine P. Krafts; Email: [email protected] cell injury. Tissues falling into this category include parenchymal
Submitted: 05/01/10; Accepted: 06/01/10 cells of the liver, kidney and pancreas, mesenchymal cells such as
Previously published online: fibroblasts and smooth muscle cells, endothelial cells and lym-
www.landesbioscience.com/journals/organogenesis/article/12555 phocytes. It should be noted that the liver, unlike other quiescent
DOI: 10.4161/org6.4.12555
tissues, has a relatively robust proliferative capacity. When a lobe

www.landesbioscience.com Organogenesis 225

of the liver is resected for donation, for example, the remaining regenerated every 3 to 5 days. The bone marrow contains not
liver cells proliferate at such a rate that the liver reaches a size only hematopoietic stem cells, which give rise to all blood cell
similar to that prior to resection. While this process is commonly lineages, but also multipotent stromal stem cells, which travel to
described as regeneration, it is more accurately viewed as com- different regions in the body and generate chondrocytes, osteo-
pensatory growth, since the original lobe itself does not regrow. blasts, adipocytes, myoblasts and endothelial cells. These stromal
A few types of tissue are composed of cells that have left the cell stem cells participate in cell replenishment after tissue injury, but
cycle permanently, and are therefore unable to proliferate. These they do not seem to have a function in normal tissue homeostasis.
nondividing tissues (or permanent tissues) include cardiac and Beyond the stem cell, three other types of cells are critical to
skeletal muscle. Tissue repair in these tissues always leaves per- the process of tissue repair: fibroblasts, endothelial cells and mac-
manent evidence of injury, such as a scar.1 rophages. In most wounds, complete replacement of wounded
tissue to its original, unharmed state is impossible. The wound
Dramatis Personae must therefore be healed using externally obtained material to
reconnect the viable tissue margins. This process, discussed in
The cast of characters in the tissue repair drama is large and var- detail later, involves the laying down of acellular fibrous tissue
ied. Here we list the major actors, with a focus on those that may to replace the region of lost cells. The fibrous tissue is laid down
be less known to the reader. Central in the drama are the tissues by fibroblasts, which migrate to the injured area, proliferate and
own lost or damaged cells, which in most cases are terminally dif- secrete collagen under the influence of numerous growth factors
ferentiated and incapable of replication. In nondividing tissues, and cytokines. In the earliest stages of wound healing, fibroblasts
such as myocardial tissue, lost cells are simply never replaced. are few and far between, suspended together with tenuous new
In other tissues, however, replacement is possible. Continuously blood vessels in an edematous pink substance termed granulation
dividing tissues are particularly adept at self-renewal, undergoing tissue. Initially the new blood vessels are critical in the trans-
innumerable cycles of cell loss and replacement during a normal port of nutrients and cells to the new tissue, but after a time,
human lifespan. The regenerative capacity of these tissues lies they recede along with the fibroblasts, leaving a collagenous scar
not in their parenchymal cells (which are terminally differenti- that is remodeled and strengthened over time. Macrophages are
ated and thus unable to replicate), but in stem cells located deep essential directors of this drama, secreting growth factors that
within the tissue. entice and stimulate fibroblasts, endothelial precursor cells and
Stem cells are unique for two reasons: (1) they have the abil- (in skin wounds) keratinocytes. They also oversee the deposition
ity to self-renew and (2) they have the capacity to generate more and remodeling of extracellular matrix material.
than one cell type. Self-renewal occurs either by symmetric rep-
lication (in which a stem cell gives rise to two daughter stem The Script
cells, equally capable of self-renewal) or asymmetric replication
(in which one daughter cell remains a self-renewing stem cell The script of the tissue factor dramathe migration and prolif-
and the other daughter cell differentiates and matures). eration of cells, the laying down of extracellular matrix, and the
The capacity of a stem cell to give rise to multiple lineages of remodeling of collagen to form a durable scaris carried out by
cells is most striking in embryonic stem cells. These cells, which a process known as receptor-mediated signal transduction. Akin
are denoted as pluripotent, are capable of generating cells from to the words spoken between people, ligands such as growth fac-
any of the tissues of the body. Adult (or somatic) stem cells are tors and cytokines float between cells, carrying directives to per-
designated as multipotent, and give rise to a more restricted array form a certain action. Cells hear these words when the ligands
of cell types. As expected, somatic stem cells have been found in bind to cell-surface receptors, which bring the message into the
continuously dividing tissues, such as skin, gastrointestinal epi- cell, resulting in a new action, such as migration, proliferation or
thelial lining, cornea and hematopoietic tissue. However, they secretion of a substance. Herein we will discuss the words spoken
have also been discovered in certain quiescent tissues, such as between the actors, the way the actors hear these words and the
liver, pancreas and adipose tissue, in which they do not normally manner in which the message gets to the cell nucleus in order to
produce differentiated cells. Most surprising is the recent discov- effect change.
ery of stem cells residing in certain parts of the central nervous Growth factors and cytokines. Growth factors are specialized
system, an organ system whose tissues have long been thought to polypeptide molecules that bind to receptors on target cells and
be incapable of proliferating.2 deliver messages regarding migration, proliferation, differentia-
Most somatic stem cells are located in niches, micro-environ- tion, survival and secretion. The list of growth factors and their
ments within a tissue comprised of both stem cells and non-stem attendant functions is so long that it would tax even the most
cells. Neighboring non-stem cells signal the stem cell to divide capable memorizer. Herein we limit our discussion to the pri-
when necessary, a task that the stem cell generally performs very mary growth factors associated with each stage of tissue repair
slowly. In the skin, stem cells located in a niche within the hair (Table 1).
follicle bulge give rise to all the cells comprising the hair follicle One of the most critical growth factors in tissue repair is trans-
and contribute to the production of new surface epithelial cells forming growth factor beta (TGF-). This growth factor belongs
after wounding.3 Stem cells of the small intestine are located to a superfamily that also includes a number of other factors with
within monoclonal, stem cell-derived crypts that are completely wide-ranging functions, such as bone morphogenetic proteins,

226 Organogenesis Volume 6 Issue 4

 Table 1. Major steps in wound healing and their associated growth factors
EGF FGF KGF PDGF TGF- TGF- TNF VEGF
Fibroblast migration X X X
Fibroblast proliferation X X X X X
Monocyte migration X X X X
Macrophage activation X
Epithelial migration X X X X
Epithelial proliferation X X X X
Angiogenesis X X X X X
Collagen synthesis X X
Collagenase synthesis X X X X X
Wound contraction X X
EGF, epidermal growth factor; FGF, fibroblast growth factor; KGF, keratinocyte growth factor; PDGF, platelet-derived growth factor; TGF, transforming
growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

activins, inhibins and Mllerian inhibiting substance.4 Made of new chemotherapeutic agents.11 Other growth factors include
by platelets, endothelial cells, lymphocytes and macrophages, epidermal growth factor (EGF) and hepatocyte growth factor
TGF- binds to cell-surface receptors that have serine/threonine (HGF). Both stimulate epithelial cell and hepatocyte prolifera-
kinase activity, triggering phosphorylation of cytoplasmic tran- tion and enhance epithelial cell migration. Once known as the
scription factors called Smads, so-named for the two drosophilia scatter factor, HGF also promotes cell scattering during embry-
proteins of which they are homologs: the mothers against deca- onic development.12
pentaplegic (MAD) and Caenorhabeitis elegans proteins.5 Smads In addition to growth factors, cytokines also carry impor-
then enter the nucleus and affect gene transcription. tant messages between cells during tissue repair. Small proteins
Although TGF- has many functions, its most important secreted by cells of the immune system, cytokines are perhaps
role in tissue repair is to promote fibrosis, a feat it accomplishes best known for their role as immunomodulators within both
by: (1) attracting fibroblasts and stimulating them to proliferate, the cellular and humoral arms of the adaptive immune system.
(2) triggering fibroblasts to secrete collagen and (3) inhibiting Three cytokines in particular are involved in tissue repair: tumor
extracellular matrix degradation by metalloproteinases. Not sur- necrosis factor (TNF) and interleukin-1 (IL-1) participate in the
prisingly, TGF- is a key factor in conditions involving patho- wound healing stage of tissue repair and TNF and interleukin-6
logic fibrosis, such as pulmonary fibrosis, systemic sclerosis and (IL-6) are involved in liver regeneration.13
Marfan syndrome.6 In addition to its fibrogenic effects, TGF- Receptor-mediated signaling. For humans, words must be
also inhibits epithelial cell growth, diminishes inflammation and seen or heard to have meaning. So it is with the messages between
promotes invasion and metastasis in tumors.7 cells in the tissue repair drama: the growth factor or cytokine
Another essential, multitasking growth factor is platelet- message must have a way to get into the target cell for action to
derived growth factor (PDGF), so named because it is stored in take place. This process is termed receptor-mediated signal trans-
platelet granules and released upon platelet activation. Made by duction and it involves binding of a ligand (a growth receptor or
a number of cells including macrophages, endothelial cells and cytokine) to a receptor molecule on a target cell. This binding
smooth muscle cells, PDGF is involved in nearly every aspect of triggers events that transduce the signal from the outside of the
tissue repair. It calls neutrophils, macrophages and fibroblasts to cell to the inside. The end result is a change in gene expression,
the wound area and subsequently stimulates and activates them. for example, transcription of genes that normally may be silent,
It also induces angiogenesis, triggers production of matrix metal- such as those that control cell cycle entry.
loproteinases (MMPs), fibronectin and hyaluronic acid and aids Receptor-mediated signaling often occurs between cells adja-
in wound contraction.8 cent to one another (paracrine signaling); for example, macro-
Fibroblast growth factor (FGF) calls virtually all of the major phages produce growth factors that bind to receptors on adjacent
players (macrophages, fibroblasts and endothelial cells) to the fibroblasts. However, signaling can also occur between cells
scene of the wound. It initiates the migration of epithelial cells located at some distance from each other (endocrine signal-
in from the margins of the wound, mediates wound contraction ing), as is often the case with cytokines and their distant targets.
and stimulates angiogenesis.9 Contrary to what one might infer Signaling may even occur within the same cell (autocrine signal-
from its name, however, FGF does not stimulate collagen synthe- ing), as when a tumor cell overproduces both a growth factor and
sis. The more appropriately-named vascular endothelial growth its receptor, which interact and lead to unbridled cell growth.
factor (VEGF) acts primarily on endothelial cells. It is a potent Whatever the mode of signaling may be, there are four main
inducer of blood vessel formation in tissue repair as well as in early types of receptors: those with intrinsic tyrosine kinase activ-
fetal development.10 Overexpressed in certain tumors, particu- ity, those without intrinsic tyrosine kinase activity, G protein-
larly renal cell carcinoma, VEGF is a target for the development coupled receptors and steroid hormone receptors. Receptors with

www.landesbioscience.com Organogenesis 227

intrinsic tyrosine kinase activity are common targets for growth translation. To move into the S phase and begin the process of
factors. When a growth factor binds to this type of receptor, the replication, cells must pass a checkpoint at the end of G1. Several
receptor dimerizes, causing phosphorylation of tyrosine residues such checkpoints built into the cycle operate as quality-control
and activation of the receptor. The activated receptor in turn monitors, validating that the cell has accurately completed
phosphorylates other molecules, inducing them to carry the mes- one phase of the cell cycle before allowing the cell to progress
sage (or signal) into the nucleus, resulting in a change of gene to the next phase. The G1 checkpoint assesses the integrity of
expression. Examples of actions mediated by this type of receptor DNA before allowing cells to pass through to S phase. It also
include: production of growth factors, production of growth fac- serves as a decision point, determining whether the cell should
tor receptors, production of proteins that control entry of the cell divide immediately, divide at a later point or enter a resting stage.
into the cell cycle and activation of cell proliferation and survival Another checkpoint at the end of G2 evaluates DNA after repli-
(through inhibition of apoptosis).14 cation to see if the cell can safely enter mitosis. A separate meta-
Receptors lacking intrinsic tyrosine kinase activity use a simi- phase checkpoint monitors the alignment of chromosomes at the
lar phosphorylation-activation system to transmit messages into mitotic plate and when alignment is achieved, allows chromo-
the cell; however, since they lack phosphorylating capability, they somes to separate into their sister chromatids. After splitting into
must recruit a separate kinase system, called the JAK-STAT path- two daughter cells, the cell again enters G1.
way, to complete the job. The receptor transmits the signal from Because the correct functioning of the cell cycle is crucial to
the ligand (often a cytokine) on the surface of the cell to a Janus its integrity, there are multiple controls built into the system,
kinase (JAK) protein inside the cell. Once activated, the JAK including activators, inhibitors and sensors that control check-
protein in turn phosphorylates and activates cytoplasmic tran- points in the cycle. Proteins called cyclins, together with asso-
scription factors called STATs (signal transducer and activator ciated cyclin-dependent protein kinases (CDKs), drive the cell
of transcription). As their name implies, STATs transduce the cycle by phosphorylating proteins that are essential for cell cycle
signal from the JAK protein to the nucleus of the cell, where they transitions.17 One such protein is the retinoblastoma (RB) pro-
activate the transcription of certain genes.15 tein, which in its normal, unphosphorylated state stalls the cell at
The largest family of signal transduction cell membrane recep- the G1/S transition by binding and inactivating the transcription
tors is comprised of G protein-coupled receptors. Many different factor E2F.18 When RB is phosphorylated by cyclins, it releases
types of ligands bind to this type of receptor, such as chemokines, (and activates) E2F, allowing transcription of genes necessary for
vasopressin, serotonin, histamine, epinephrine, norepinephrine, cell cycle progression. Cyclin proteins are themselves regulated
calcitonin, glucagon, parathyroid hormone, corticotropin and by CDK inhibitors.
rhodopsin.16 When the receptor receives a signal from a ligand, its
seven transmembrane alpha helices change conformation, allow- The Stage
ing it to interact with a G protein. Once activated, the G protein
transmits the signal to a second messenger molecule, such as 3', The stage upon which the drama unfolds is a special type of tis-
5' cyclic adenosine monophosphate (cAMP), which carries the sue known as extracellular matrix (ECM). ECM exists in two
signal to the nucleus of the cell. Such cAMP-mediated pathways forms: interstitial matrix (a gelatinous, amorphous intercellular
are involved in vision and olfactory sensing. Steroid hormone material) and basement membrane (a thin, highly-organized,
receptors are unique in that they are located inside the cell rather plate-like layer upon which epithelial cells rest). Although it
than on the cell membrane. Their ligands, which include steroid may appear inert and static, ECM has a long list of responsibili-
hormones, thyroid hormone, vitamin D and retinoids, must dif- ties and functions. Beyond its obvious physical characteristics
fuse through the cell membrane in order to bind to the receptor, imparting resilience to soft tissues and firmness to boneit also
which binds to DNA and alters gene expression. stores and presents growth factors, acts as a scaffold to which
The cell cycle. One of the main actions in the tissue repair migrating cells can adhere and establish polarity and facilitates
script is cell proliferation. In order to heal after injurywhether cell growth both in physiologic and tissue repair settings. ECM
by regeneration or scarringcells must enter and progress is the ever-changing background for regeneration and wound
through the cell cycle, a tightly-regulated process that consists of healing, but it also accompanies other processes such as mor-
two main activities: DNA replication and mitosis. Continuously phogenesis, chronic fibrotic processes and tumor invasion/
proliferating cells are always moving through the cell cycle, metastasis.
whereas quiescent cells must be called into the cell cycle by The many constituents of the ECM can be grouped into three
growth factors or cytokines (via receptor-mediated signal trans- types of molecules, according to their main functions: structural
duction) or by ECM components (via integrins). molecules (such as collagen and elastin), adhesive molecules
The cycle consists of four consecutive phases: the G1 (presyn- (such as integrins, fibronectin and laminin) and resilient com-
thetic) phase, the S (DNA synthesis) phase, the G2 (premitotic) ponents (such as proteoglycans and hyaluronan). The interstitial
phase and the M (mitotic) phase. Cells may begin their journey matrix is composed primarily of collagen, elastin, fibronectin,
through the cycle from G1 or G0 (a resting phase outside the cell proteoglycans and hyaluronan, whereas the basement membrane
cycle). In order to move from G0 to G1, cells must activate numer- is composed largely of nonfibrillar collagen, laminin and pro-
ous previously-silent genes including proto-oncogenes, genes teoglycans.1 A short description of each major ECM component
required for ribosome synthesis and genes required for protein follows.

228 Organogenesis Volume 6 Issue 4

 Collagen, the most common protein in the animal world, con- In a process akin to regeneration, some organs are able to grow
sists of three chains that combine to form a triple helix trimer. in size in response to resection. If a lobe of the liver is resected,
Although 27 types of collagen have been identified, less than half the remaining liver will grow larger in response. A wave of repli-
of these have major roles in the human body. Fibrillar collagens cation occurs as hepatocytes are pulled from their quiescent state
(types I, II, III, V and IX) are found in many types of hard and and enter the cell cycle. Following a second wave of replication
soft tissues. Type IV collagen, which is arranged in long sheets in nonparenchymal cells (such as Kupffer cells and endothelial
rather than fibrils, is a primary component of basement mem- cells), hepatocytes return to their quiescent state. This process,
branes. Collagen imparts strength to tissues, but the ability to though often termed regeneration, is not true regeneration, but
stretch and snap back into shape is provided by elastic fibers. compensatory growth. True regeneration of lost organs is, at this
Elastic fibers are composed of a central core of elastin surrounded time, a process relegated to certain animal species and science
by fibrillin, a glycoprotein that controls the availability of TGF- fiction movies, but perhaps in the not-so-distant future stem cells
within the ECM. will be used for this purpose.21
Adhesive molecules, such as integrins, fibronectin and lam- If an injury damages only parenchymal cells in a continu-
inin, provide connections between cells or between cells and ously-dividing or quiescent tissue, repair by regeneration is pos-
ECM components. Integrins are transmembrane glycoproteins sible. However, if an injury is so severe as to damage not only
with an extracellular domain that attaches to ECM compo- the parenchymal cells but also the underlying stromal framework
nents, such as laminin and fibronectin and an intracellular of the tissue (as occurs in most injuries) or if the injury occurs
domain that links to cytoskeletal complexes.19 They are opera- in non-dividing tissues, regeneration is impossible. In these
tive in the earliest stages of wound healing, helping leukocytes instances, the tissue is repaired by the deposition of fibrous tissue
adhere to vascular endothelium in preparation for their trans- in the defect created by the wound. Regeneration involves res-
migration through the vessel. Fibronectin helps stabilize the titution of tissue components; repair involves patching rather
initial blood clot filling the wound by binding to fibrin; it also than restoring. The amount of regeneration vs. repair that occurs
provides a framework for building the collagen matrix during depends on the proliferative capacity of the cells, the integrity
wound healing. Laminin provides a connection between cells of the stromal framework and the duration of the injury and
and the ECM; it also binds with type IV collagen to form the inflammatory response.1
basement membrane. Repair by connective tissue involves the influx of debris-
The resilient nature of the ECM is provided by proteoglycans removing inflammatory cells, formation of granulation tissue
and hyaluronan. Both of these substances are composed of gly- (a substance consisting of fibroblasts and delicate capillaries in a
cosaminoglycans (GAGs), long repeating polymers of disaccha- loose extracellular matrix) and conversion of said granulation tis-
rides. Proteoglycans, which consist of GAGs linked to a protein sue into fibrous tissue that is remodeled over time to form a scar.
core, have many different roles: regulation of ECM permeability, There are five major components in this process: inflammation,
mediation of inflammatory and immune responses and control of new vessel formation, fibroblast proliferation, collagen synthesis
cell growth. Hyaluronan, comprised of many repeats of a single and scar remodeling.
dissacharide, binds huge amounts of water and provides strength Though the general principles are the same no matter what
and turgor to connective tissue. type of wound is being healed, the extent of granulation tissue,
inflammation and scarring vary depending on the size and type of
The Drama wound. In the case of skin wounds, there are two types of wound
healing: first-intention healing and second-intention healing.22
Preface: regeneration vs. scarring. True regeneration, in which Wounds healing by first intention are relatively small, with lim-
new cells replace damaged or dead cells such that the tissue is ited epithelial and connective tissue damage, such as paper cuts,
restored to its original state, occurs infrequently in humans. well-approximated surgical incisions and superficial stab wounds.
Two conditions must be met for a tissue to undergo pure regen- Healing in this type of wound is generally rapid, since the area of
eration (without scarring): (1) the injured cells must be capable the defect is relatively small. Inflammation and granulation tissue
of proliferation (as is the case in continuously dividing and qui- are present but not abundant and scarring is minimal.
escent tissues) and (2) the underlying stromal framework must In contrast, wounds healing by second intention are larger
be intact. wounds with margins that are not easily approximated, such
Regeneration occurs in a physiologic manner in continuously as burns, infarctions, ulcers and large excisional skin wounds.
dividing tissues. Sloughed-off gastrointestinal epithelial cells, for This type of wound necessarily heals more slowly, as the defect
example, are replaced by new cells arising from stem cells in the to be repaired is larger. The abundance of fibrin, necrotic debris
intestinal crypts. Pathologic examples of pure regeneration, how- and exudate necessitates a more intense inflammatory response
ever, are few and far between. One example occurs when the liver and consequently the chance of inflammation-mediated injury
undergoes acute, toxic injury from acetaminophen overdose. In is greater than it is in first-intention healing. Granulation tissue
this type of injury, hepatocytes are destroyed, but the underlying is abundant and wound contraction and scarring is maximal.
stromal framework remains intact, allowing for the possibility of Despite these differences between first and second intention heal-
complete regeneration of the injured tissue.20 ing, the underlying drama is very similar. We will present the

www.landesbioscience.com Organogenesis 229

 Figure 1. Skin ulcer showing early-stage granulation tissue with numer- Figure 2. Early granulation tissue with blood vessels in a loose matrix of
ous blood vessels and scattered fibroblasts in a loose extracellular collagen and fibrin.
matrix. Numerous neutrophils are present within the granulation tissue.

drama as it occurs in first intention wounds, noting second inten- scattered fibroblasts. Initially, the new vessels are leaky and the
tion differences where necessary. granulation tissue has an edematous, loose appearance (Fig. 1).
Act I: inflammation. Immediately after the wound is inflicted, As the vessels grow stronger and fibroblasts begin to lay down
the most urgent task is not to repair the damaged tissue, but to collagen, it takes on a more substantial appearance, reaching its
stop the flow of blood from the wound. Within seconds of the maximum vascularity by day 5.1
injury, exposed collagen activates coagulation in the region of the Macrophageslarge, multitasking cells derived from mono-
injury. Platelets form an initial plug, coagulation cascade factors cytesnow replace the neutrophils that were so numerous in the
interact to form fibrin (which seals and solidifies the plug) and a immediate aftermath of the wounding. As their name suggests,
blood clot soon appears at the skin surface. Not only does the clot a primary function of macrophages is the ingestion of unwanted
stop the bleeding, but it serves as a scaffold for incoming cells and materials: bacteria, cell remnants, debris, fibrin and foreign mate-
a repository of cytokines and growth factors.22,23 rial. But macrophages also serve as directors for many other parts
At the skin surface, all appears static and calm as the clot of the drama. They call in and stimulate fibroblasts and kera-
dries and forms a scab. Underneath the surface, however, a tinocytes (through release of PDGF, TGF-, TNF, IL-1 and
flurry of activity begins. An army of neutrophils rushes to the KGF), stimulate angiogenesis (by secretion of VEGF, FGF and
scene through dilated local blood vessels, called to the area by PDGF) and direct the laying down and remodeling of extracel-
IL-1, TNF, TGF- and other growth factors. Within 24 hours, lular matrix [through the use of TGF-, PDGF, tumor necrosis
the neutrophils appear at the edges of the wound incision, using factor (TNF), osteopontin (OPN), IL-1, collagenase and matrix
the fibrin scaffolding of the clot to invade the region of destruc- metalloproteinases (MMPs)].22
tion, their primary tasks being to break down debris and kill The formation of new blood vessels (angiogenesis) begins
bacteria. within the first few days following injury, aided by VEGF and
Epithelial cells, meanwhile, begin their arduous process of FGF.27 The process is unexpectedly aided by a small number of
repair by crawling from the basal layers of the epidermis into the bone-marrow-derived endothelial precursor cells which home
wounded region, depositing basement membrane components to the wound by mysterious mechanisms. Their contribution to
as they migrate. They are called to begin this process by EGF the overall angiogenesis effort, however, is minimal.28 Formation
and TGF-a, secreted by activated platelets and macrophages. In of the correct blood vessel structure is aided by integrins on the
addition, fibroblasts secrete KGF (keratinocyte growth factor) surface of endothelial cells; surrounding perithelial cells are
and IL-6, factors that enhance keratinocyte migration and pro- recruited with the help of angiopoietin.
liferation.24,25 Fusing in the midline underneath the inert scab, The end point toward which the entire drama is directed is
these epithelial cell pioneers form the first thin, continuous layer the formation of a scar, a strong collagen filler that bridges the
of epithelium upon which the rest of the new epithelium will be gap left by tissue destruction, restoring strength and integrity to
built.26 the tissue. This process begins with the entrance of the fibroblast
Act II: proliferation. Following the initial flurry of clotting between days one and three, called to action by many growth fac-
and neutrophil activity, the work of cell proliferation begins in tors and cytokines, including PDGF, EGF and TGF-. At first,
earnest. The characteristic feature of this act in the drama is the type III collagen secreted by fibroblasts is arrayed vertically
the formation of a substance called granulation tissue, a pink, at the margins of the incision. Later, the collagen combines with
soft material so-named for its irregular, grainy appearance. fibrin and plasma fibronectin (which deposition is promoted by
Consisting of new blood vessels and fibroblasts in an extracel- the vascular permeability inherent in angiogenesis); this provi-
lular matrix, granulation tissue is laid down over a period of a sional matrix provides a framework for further fibroblast and
few days, beginning with a few small, tenuous blood vessels and endothelial cell ingrowth (Fig. 2).29

230 Organogenesis Volume 6 Issue 4

 Figure 3. Skin ulcer showing later-stage granulation tissue with blood Figure 4. Early scar formation showing fibrillar collagen, plump fibro-
vessels and early collagen deposition. blasts and spindle-shaped fibrocytes.

By day 3 to day 5, ECM formation is well underway, with down by a family of enzymes known as tissue inhibitors of metal-
granulation tissue serving as a substrate (Fig. 3). Shards of elas- loproteinases (TIMPs). Produced by mesenchymal cells, TIMPs
tic tissue form within the wound site. Plump fibroblasts mature help reign in collagen (and other ECM component) breakdown
into thin, spindle-shaped fibrocytes, which become entrapped in so that scar formation can proceed.
the dense, fibrillar collagen they have secreted (Fig. 4). Collagen Ultimately, strength is achieved by increased collagen synthe-
deposition is a balance between synthesis and degradation. In sis, followed by post-synthetic modifications of collagen, includ-
these early stages, the balance tips toward collagen synthesis, ing cross-linking and increased fiber size. Early in wound healing,
aided by TGF-, which inhibits the degradation of collagen. The the collagen is relatively thin and oriented parallel to the skin;
process of ECM deposition ends within 23 weeks. over time, the initial collagen fibrils are resorbed and replaced
Meanwhile, the epithelium quietly continues to proliferate. with thicker fibrils aligned with stress lines.22 Wound strength
Mediated by the aforementioned KGF and IL-6, it increases in increases rapidly during the first month, then slows, reaching a
thickness, eventually regaining its full, stratified architecture and plateau of 7080% of the original tensile strength by the end of
keratinized surface some time between 1 and 3 weeks (or later in the third month. Although full-thickness epithelial regeneration
second-intention healing).27 is possible, any skin appendages lost during the initial injury will
Act III: maturation. During the second week following the not be reformed.
incision, leukocytes gradually abandon the wound area and ves- Given their easily-approximated margins, wounds healing by
sels recede (Fig. 5). Type I collagen synthesis increases, due to first intention generally have minimal scarring. Wounds healing
both an increased number of fibroblasts and an increased rate of by second intention, however, are larger, with more epithelial
synthesis per fibroblast.30 Where there was once granulation tis- destruction, necessitating more collagen deposition to close the
sue, there is now a preliminary scar, pale and firm, composed of wound. The process of wound contraction eases the fibroblasts
dense collagen, fibroblasts and shards of elastic tissue. In another burden a bit. Specialized contractile, smooth-muscle-like cells
two weeks, even the fibroblasts begin to disappear and eventu- called myofibroblasts appear within the wound and pull the mar-
ally an acellular scar bridges the region of the wound, covered gins of the wound toward each other. These cells may be derived
by intact, fully-developed epidermis. Dermal appendages in the from tissue fibroblasts under the influence of FGF and PDGF;
region of the wound, however, are not reformed. they may also develop from epithelial cells or from bone marrow
Over the next several weeks, the composition of the extracel- precursor cells called fibrocytes. So efficient are these myofibro-
lular matrix changes, remodeling the newly-formed scar such that blasts that large wounds may decrease in size by up to 80%.32
it attains maximal strength at an appropriate size. Although sev- When the drama ends badly. Several systemic factors may
eral serine protease enzymes (such as neutrophil elastase, cathep- adversely affect the performance of the tissue repair drama.
sin G, kinins and plasmin) operate in the remodeling process, Patients with diabetes mellitus tend to have less granulation tis-
the key mediators of remodeling are zinc-dependent proteases sue formation, less collagen deposition and defects in collagen
known as matrix metalloproteinases (MMPs).31 There are many maturation, leading to slow and ineffective wound maturation.33
different types of MMPs, including collagenases (which cleave While this predisposition has been attributed to the microangi-
fibrillar collagen), gelatinases (which act on amorphous collagen opathy inherent in diabetes, recent research indicates that other
and fibronectin) and stromelysins (which degrade a number of factors, such as collagen glycosylation and pericapillary albumin
ECM components including laminin, fibronectin, proteogly- deposition, may play a role.22,34 Poor nutrition also retards the
cans and amorphous collagen). MMPs are produced by many healing process. Vitamin C, in particular, is necessary for col-
different types of cells, including fibroblasts and macrophages; lagen synthesis. Deprivation of this vitamin results in failure of
their secretion is stimulated by PDGF and FGF and inhibited activation of proline and lysine hydroxylases and formation of
by TGF-. Once they are produced, MMPs are rapidly broken unhydroxylated procollagen peptides, resulting in an unstable,

www.landesbioscience.com Organogenesis 231

 Figure 6. Keloid showing dense, haphazardly-arranged collagen
bundles.

Figure 7. Proud flesh with exuberant granulation tissue.

or pressure at the wound site. However, the single most impor-

tant cause of delayed healing of wounds is infection.1 If any beta-
hemolytic Streptococcus organisms are present or if there are
over 105 organisms of any bacterial species per gram of tissue, the
wound will not heal.35 More common in second-intention heal-
ing, infection prolongs the inflammatory phase of wound heal-
ing, impeding epithelialization, collagen deposition and wound
contraction. Bacterial endotoxins stimulate collagenase secretion,
Figure 5. Wide excision of skin. (A) Previous punch biopsy site (center) leading to degradation of not only the forming scar, but of sur-
in late stages of repair. (B) Dense, acellular, normal dermal collagen rounding normal tissue.22
bundles (bottom) abutting new, still-cellular fibrous tissue of the repair
On the other hand, sometimes acts in the drama are played so
process (top). (C) Blood vessel in process of involution.
well, so exuberantly, that the end result is an abnormally-healed
wound. Occasionally, the balance of collagen formation and deg-
poorly cross-linked collagen helix. Conditions in which blood radation tips in favor of formation, leading to hypertrophic scars
flow is compromisedsuch as cardiac insufficiency or arterio- or keloids. Hypertrophic scars are raised scars that remain con-
sclerosisdelay the delivery of necessary cells and factors to the fined to the region of the wound. They generally occur within 4
scene of the wound. Certain drugs may also slow wound healing; weeks of injury (often a severe traumatic or thermal injury to the
glucocorticoids, for example, inhibit inflammation and collagen dermis) and may regress over time.22,36 An aberrant autocrine loop
formation. in which myofibroblasts produce excessive TGF- and, hence,
Local factors can also greatly affect the quality of wound heal- collagen, appears to contribute to their formation.36 Keloids are
ing. The size of the wound is important (small injuries heal faster scars that have overgrown the boundaries of the initial incision,
than large ones) as is the location (wounds in richly-vascularized presenting as nodules or masses of fibrous tissue (Fig. 6). While
areas of the body, such as the face, heal relatively quickly). The keloids generally appear within one year of the inciting injury,
presence of foreign bodies may prolong healing, as may motion some may begin growing years later.22 For unknown reasons,

232 Organogenesis Volume 6 Issue 4

keloids occur much more frequently in patients with darkly- Conclusion
pigmented skin; certain people seem to have a predisposition
towards their formation. Wound healing is a carefully-scripted drama performed count-
Formation of granulation tissue may likewise occur in an less times throughout a human lifespan. Though occasionally
excessive fashion. In a lesion known as proud flesh, the process the drama does not go as planned, in most cases damage is con-
of wound healing is interrupted by masses of granulation tissue tained, dead cells and tissue are removed and a scar restores
protruding above the skin surface, preventing re-epithelialization integrity to the injured tissuea triumphant performance
and appropriate scar formation (Fig. 7). indeed.

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