The Cellular Biology of Wound Healing

The Cellular Biology of Wound Healing

M.C. REGAN and A. BARBUL
Abstract
Both in vitro and in vivo studies have demonstrated that the presence of both macrophages and T
lymphocytes at the wound site is essential in order for the normal healing process to occur. Both
macrophages and T lymphocytes possess the capacity to regulate essential steps in the process of
wound healing. The presence of macrophages is essential for the initiation and maintenance of wound
fibroblast activity. T cells do not appear to be required for the initiation of the healing process, and
healing can progress in the absence of T lymphocytes; however, the presence of an intact T cell
immune system is essential for a normal outcome, indicating that the T cells probably provide a
regulatory influence over macrophage-induced activities. Further research is still required into the
interaction of these immune cells, their secretory products, and other wound elements before our
understanding of the mechanism of wound healing is complete.
Introduction
The response of living tissue to injury forms the foundation of all surgical practice. Following tissue
disruption, whether operative or traumatic, the major priorities of any organism are cessation of
haemorrhage, prevention of infection and restoration of tissue integrity and function. In the
specialization which has followed our anatomical and physiological evolution, we have lost the facility
to regenerate organs and most tissues. Some lower forms of life, such as protozoa, can regenerate
any part of their unicellular structures, while lower vertebrates retain the ability to regenerate an
amputated limb or tail from totipotent cells; however, the mammalian system is only able to replace
certain tissues, such as an epidermal defect, and must therefore repair damage to other structures by
scar formation.
The process by which tissue repair takes place is termed wound healing and is comprised of a
continuous sequence of inflammation and repair, in which epithelial, endothelial, inflammatory cells,
platelets and fibroblasts briefly come together outside their normal domains, interact to restore a
semblance of their usual discipline and having done so resume their normal function.
The process of wound repair differs little from one kind of tissue to another and is generally
independent of the form of injury. Although the different elements of the wound healing process occur
in a continuous, integrated manner, it is convenient to divide the overall process into three overlapping
phases and several natural components for descriptive purposes (Fig. 1).
Inflammatory Phase (Day 0-5)
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The healing response is initiated at the moment of injury. Surgical or traumatic wounds disrupt the
tissue architecture and cause haemorrhage. Initially, blood fills the wound defect and exposure of this
blood to collagen in the wound leads to platelet degranulation and activation of Hageman factor [1].
This in turn sets into motion a number of biological amplification systems including the complement
kinin and clotting cascades and plasmin generation. These serve to amplify the original injury signal
and lead not only to clot formation, which unites the wound edges, but also to the accumulation of a
number of mitogens and chemoattractants at the site of wounding [2].
Production of both kinins and prostaglandins leads to vasodilatation and increased small vessel
permeability in the region of the wound [3]. This results in oedema in the area of the injury and is
responsible for the pain and swelling which occurs early after injury. Within 6 h, circulating immune
cells start to appear in the wound. Polymorphonuclear leucocytes (PMN) are the first blood leucocytes
to enter the wound site. They initially appear in the wound shortly after injury and subsequently their
numbers increase steadily, peaking at 24-48 h [4]. Their main function appears to be phagocytosis of
the bacteria which have been introduced into the wound during injury. The presence of PMN in the
wound following injury does not appear to be essential in order for normal wound healing to take place
[5, 6], with healing proceeding normally in their absence provided that bacterial contamination has not
occurred. In the absence of infection, PMN have a relatively short life span in the wound and their
numbers decrease rapidly after the third day [7].
The next cellular, immune element to enter the wound are macrophages. These cells are derived from
circulating monocytes by a combination of migration and chemotaxis. They first appear within 48-96 h
post-injury and reach a peak around the third day post-injury [4]. These macrophages have a much
longer life span than the PMN and persist in the wound until healing is complete. Their appearance is
followed somewhat later by T lymphocytes, which appear in significant numbers around the fifth day
post-injury, with peak numbers occurring about the seventh day after injury. In contrast to PMN, the
presence and activation of both macrophages and lymphocytes in the wound is critical to the progress
of the normal healing process [8, 9].
Macrophages just like neutrophils phagocytose and digest pathological organisms and tissue debris. In
addition, macrophages release a plethora of biologically active substances. Many of these substances
facilitate the recruitment of additional inflammatory cells and aid the macrophage in tissue
decontamination and debridement; in addition growth factors and other substances are also released
which are necessary for the initiation and propagation of granulation tissue formation. These
intercellular transmitters are known collectively as cytokines.
Proliferative Phase (Day 3-14)
In the absence of significant infection or contamination the inflammatory phase is short, and after the
wound has been successfully cleared of devitalized and unwanted material it gives way to the
proliferative phase of healing. The proliferative phase is characterized by the formation of granulation
tissue in the wound. Granulation tissue consists of a combination of cellular elements, including
fibroblasts and inflammatory cells, along with new capillaries embedded in a loose extra cellular matrix
of collagen, fibronectin and hyaluronic acid. Fibroblasts first appear in significant numbers in the wound
on the third day post-injury and achieve peak numbers around the seventh day [4]. This rapid
expansion in the fibroblast population at the wound site occurs via a combination of proliferation and
migration [10]. Fibroblasts are derived from local mesenchymal cells, particularly those associated with
blood vessel adventitia [11], which are induced to proliferate and attracted into the wound by a
combination of cytokines produced initially by platelets and subsequently by macrophages and
lymphocytes (Table 1). Fibroblasts are the primary synthetic element in the repair process and are
responsible for production of the majority of structural proteins used during tissue reconstruction. In
particular, fibroblasts produce large quantities of collagen, a family of triple-chain glycoproteins, which
form the main constituent of the extracellular wound matrix and which are ultimately responsible for
imparting tensile strength to the scar. Collagen is first detected in the wound around the third day post-
injury [12, 13], and thereafter the levels increase rapidly for approximately 3 weeks. It then continues to
accumulate at a more gradual pace for up to 3 months post wounding [10]. The collagen is initially
deposited in a seemingly haphazard fashion and these individual collagen fibrils are subsequently
reorganized, by cross-linking, into regularly aligned bundles oriented along the lines of stress in the
healing wound. Fibroblasts are also responsible for the production of other matrix constituents
including fibronectin, hyaluronic acid and the glycosaminoglycans [14]. The process of fibroblast
proliferation and synthetic activity is known as fibroplasia.
Revascularization of the wound proceeds in parallel with fibroplasia. Capillary buds sprout from blood
vessels adjacent to the wound and extend into the wound space. On the second day post-injury,
endothelial cells from the side of the venule closest to the wound begin to migrate in response to
angiogenic stimuli. These capillary sprouts eventually branch at their tips and join to form capillary
loops, through which blood begins to flow. New sprouts then extend from these loops to form a
capillary plexus [15, 16]. The soluble factors responsible for angiogenesis remain incompletely defined.
It appears that angiogenesis occurs by a combination of proliferation and migration. Putative mediators
for endothelial cell growth and chemotaxis include cytokines produced by platelets, macrophages and
lymphocytes in the wound [17, 18], low oxygen tension [19], lactic acid [20] and biogenic amines [21].
Of the potential cytokine mediators of neovascularization basic fibroblast growth factor (bFGF), acidic
FGF (aFGF), transforming growth factors- and (TGF- and -) and epidermal growth factor (EGF)
have all been shown to be potent stimuli for new vessel formation [22-24]. FGF, in particular, has been
shown to be a potent inducer of in vivo neovascularization [25, 26].
While these events are proceeding deep in the wound, restoration of epithelial integrity is taking place
at the wound surface. Re-epithelialization of the wound begins within a couple of hours of the injury.
Epithelial cells, arising from either the wound margins or residual dermal epithelial appendages within
the wound bed, begin to migrate under the scab and over the underlying viable connective tissue. The
epidermis immediately adjacent to the wound edge begins thickening within 24 h after injury. Marginal
basal cells at the edge of the wound loose their firm attachment to the underlying dermis, enlarge and
begin to migrate across the surface of the provisional matrix filling the wound. Fixed basal cells in a
zone near the cut edge undergo a series of rapid mitotic divisions, and these cells appear to migrate by
moving over one another in a leapfrog fashion until the defect is covered [27, 28]. Once the defect is
bridged, the migrating epithelial cells loose their flattened appearance, become more columnar in
shape and increase in mitotic activity. Layering of the epithelium is re-established and the surface layer
eventually keratinized [29]. Reepithelialization is complete in less than 48 h in the case of
approximated incised wounds, but may take substantially longer in the case of larger wounds where
there is a significant tissue defect. If only the epithelium is damaged, such as occurs in split thickness
skin graft donor sites, then repair consists primarily of re-epithelization with minimal or absent
fibroplasia and granulation tissue formation. The stimuli for re-epithelization remain incompletely
determined, but it appears that the process is mediated by a combination of loss of contact inhibition,
exposure of constituents of the extracellular matrix, particularly fibronectin [30], and by cytokines
produced by immune mononuclear cells [31]. EGF, TGF-, bFGF, platelet-derived growth factor
(PDGF) and insulinlike growth factor- (IGF-) in particular, have been shown to promote
epithelialization [32].
Maturation Phase (Day 7 to I Year)
Almost as soon as the extracellular matrix is laid down, its reorganization begins. Initially, the
extracellular matrix is rich in fibronectin, which forms a provisional fibre network. This serves not only
as a substratum for migration and ingrowth of cells, but also as a template for collagen deposition by
fibroblasts [33]. There are also significant quantities of hyaluronic acid and large molecular weight
proteoglycans present, which contribute to the gel-like consistency of the extracellular matrix and aid
cellular infiltration. Collagen rapidly becomes the predominant constituent of the matrix. The initially
randomly distributed collagen fibres become cross-linked and aggregated into fibrillar bundles, which
gradually provide the healing tissue with increasing stiffness and tensile strength [34]. After a 5-day lag
period, which corresponds to early granulation tissue formation and a matrix largely composed of
fibronectin and hyaluronic acid, there is a rapid increase in wound breaking strength due to collagen
fibrogenesis. The subsequent rate of gain in wound tensile strength is slow, with the wound having
gained only 20% of its final strength after 3 weeks. The final strength of the wound remains less than
that of uninjured skin, with the maximum breaking strength of the scar reaching only 70% of that of the
intact skin [34].
This gradual gain in tensile strength is due not only to continuing collagen deposition, but also to
collagen remodelling, with formation of larger collagen bundles [35] and alteration of intermolecular
crosslinking [36]. Collagen remodelling during scar formation is dependent on both continued collagen
synthesis and collagen catabolism. The degradation of wound collagen is controlled by a variety of
collagenase enzymes, and the net increase in wound collagen is determined by the balance of these
opposing mechanisms. The high rate of collagen synthesis within the wound returns to normal tissue
levels by 6-12 months [37], while active remodelling of the scar continues for up to 1 year after injury
and indeed appears to continue at a very slow rate for life.
As remodelling progresses, there is a gradual reduction in the cellularity and vascularity of the
reparative tissue which results in the formation of a relatively avascular and acellular collagen scar.
Grossly this can be observed as a reduction in erythema associated with the earlier scar and some
reduction in the scar volume, resulting in a pale thin scar. This is normally a desirable feature of
healing; however, in some cases shrinkage of the scar may give rise to an undesirable reduction in
skin mobility resulting in contracture.
Wound contraction, i. e. inward movement of the wound edge, is a further important element in the
healing process and should be distinguished from contracture. Sharply incised wounds without
significant tissue loss, approximated early after injury, heal rapidly without the need for significant
reduction in the wound volume. Such wounds are described as having healed by primary intention.
Large wounds, however, particularly those associated with significant tissue loss, heal by secondary
intention, with granulation tissue gradually filling the defect and epithelization proceeding slowly from
the wound edges. Contraction of the wound edges can lead to a significant reduction in the quantity of
granulation tissue required to fill the wound defect and a reduction in the area requiring
reepithelization, with a consequent reduction in scar volume. Contraction is only undesirable where it
leads to unacceptable tissue distortion and an unsatisfactory cosmetic result. Although contraction
normally accounts for a larger part of overall wound closure in looseskinned animals, it still accounts
for a significant proportion of the healing process in man, particularly in areas where the skin is not
tightly bound down to underlying structures, such as on the back, neck and forearms. Initially following
injury, where the wound edges are not approximated, there is a slight retraction of the wound edges
due to the release of normal elastic tension in the skin, with a resultant increase in wound volume. The
wound area starts to decrease rapidly from the third day onwards. While this is due in part to
reepithelization, the main reason is an inward movement of the uninjured skin edges. Wound
contraction usually begins around the fifth day postwounding and is complete by 12-15 days after
wounding [38-40]. Fibroblasts within the wound appear to be responsible for providing the force for this
contractile activity [41]. It was initially felt that specialized fibroblasts called myofibroblasts provided the
motive force for wound contraction via a musclelike cell contraction [42-44]. More recent studies reveal
that wound contraction occurs as a result of an interaction between fibroblast locomotion and collagen
reorganization [41, 45]. The contraction is thought to be mediated via the attachment of collagen fibrils
to cell surface receptors [46], with the resulting tractional forces generated by cell motility bringing the
attached collagen fibrils closer together and eventually compacting them [47].
The regulation of wound contraction remains poorly defined. Information regarding the effects of
specific cytokines on contraction is limited and often conflicting. TGF- has been found to promote
contraction even in the absence of serum [48, 49]; PDGF has also been found to either increase
contraction [50] or have no effect [49], while both FGF and EGF have been found by different authors
to either have no effect or cause a moderate enhancement of contraction [48-50].
Scar Formation
As mentioned previously, the process of wound healing is essentially similar in all tissues and is
relatively independent of the mode of injury; however, slight variation in the relative contribution of the
different elements to the overall result may occur. The final product of the healing process is a scar.
This relatively avascular and acellular mass of collagen serves to restore tissue continuity, strength
and function. Delays in the healing process cause the prolonged presence of wounds, while
abnormalities of the healing process may lead to abnormal scar formation. Successful completion of
wound healing may not always yield the desired clinical result, particularly where the final cosmetic
appearance of the scar is of primary importance.
The Role of Macrophages
Macrophages appear to have a dual role at the wound site. Initially, they participate in the inflammatory
and debridement process, superceding the PMN as the major wound phagocyte, and later, they play a
regulatory role in the mediation of the fibroblastic phase of healing. It is this latter role which is crucial
to the success of the wound healing process. In the classic studies of Leibovich and Ross, a
combination of systemic hydrocortisone to induce systemic monocytopenia and local antimacrophage
serum for local elimination of tissue macrophages resulted in a significant impairment of wound
debridement and fibroplasia in guinea pigs [8]. Wound fibrin levels were elevated and clearance of
fibrin, neutrophils erythrocytes and other miscellaneous debris from the wound was delayed in treated
animals. In addition, there was both a delay in the appearance of fibroblasts in the wound and in the
subsequent rate of expansion of the wound fibroblast population [51]. Recently, we have shown that in
vivo macrophage depletion by parenteral administration of macrophagespecific monoclonal antibodies
results in a significant reduction in both wound breaking strength and wound collagen deposition (A.
Barbul, unpublished data).
Further evidence for macrophage involvement in the regulation of wound healing is provided by the
findings that intradermal injection of allogeneic macrophages increases both collagen synthesis and
wound breaking strength in 8day-old rat skin wounds [52], while injection of wound macrophages into
rabbit corneas induces angiogenesis and scar formation [53, 54].
Activated macrophages are capable of influencing many aspects of wound healing, including the
proliferative [51] and synthetic activities of fibroblasts [53] and induction of neovascularization [55, 56].
Macrophages mediate their effects on other wound elements via the release of monokines. These
intracellular transmitters are capable of regulating fibroblast (Table 1) and endothelial function.
Of the cytokines produced by activated macrophages, TGF- [57, 58], TNF- and interleukin-1 (IL-1)
[59] have been detected in significant quantities in the extracellular fluid at the site of healing. Topical
application of TGF- [60, 61] and PDGF [62] to healing wounds results in significantly enhanced
wound breaking strength. Application of TGF- at the time of wounding in rats with steroid-induced
monocytopenia results in a wound breaking strength not significantly different from non-steroid-treated
rats; this effect is not seen with topical PDGF application. Neither treatment resulted in an increase in
the number of macrophages in the wound. TGF- appeared to act directly on the fibroblasts, inducing
type 1 procollagen gene expression, while the effects of PDGF appear to be mediated indirectly,
possibly by attracting futher macrophages and inducing the release of other monokines. This
hypothesis is supported by observations in models of impaired healing. Following total body irradiation,
which induces monocytopenia with selective sparing of skin tissue, there is a significant decrease in
wound breaking strength. Topical treatment with TGF- [63], but not PDGF [64], results in significantly
increased wound strength. By contrast, following megavolt electron beam surface irradiation to the skin
surface, which impairs skin fibroblasts but spares the bone marrow, TGF- [64] had no effect on
subsequent healing, while PDGF [63] led to a 50 % increase in wound breaking strength, associated
with an increase in the number of macrophages and fibroblasts in the healing wound.
IL-1, another macrophage product, has been shown to inhibit collagen synthesis in subcutaneously
implanted sponges in rats [65]. While TNF- has been found to have no effect on wound collagen
deposition [66] when administered alone, it acted synergistically with PDGF and inhibited the effects of
TGF- on collagen deposition. However, administration of TGF- into polyvinyl alcohol (PVA) sponges
induces increased collagen deposition, an effect which is abrogated by simultaneous administration of
indomethacin. This indicates a possible indirect inflammatory mode of action for TNF-. On the other
hand, TNF- antibody administration, which blocks the effects of TNF-, also induces an increase in
collagen deposition [67] (Fig. 2). This data is consistent with a direct inhibitory effect of TNF- on
fibroblast synthetic activity; however, this effect is masked by its strong pro-inflammatory and
macrophage activating effects.
In addition, topical application of bFGF results in an increase in wound breaking strength, collagen
accumulation [68] and granulation tissue accumulation [69].
The Role of Lymphocytes
Evidence supporting a central role for T lymphocytes in the control of wound healing is provided by
studies which examine the in vivo effects of alternate forms of T cell manipulation on various
parameters of healing. Administration of agents known to enhance T lymphocyte function, such as
growth hormone [70], vitamin A [71] or arginine [72], leads to increases in wound breaking strength
and collagen deposition, while agents which suppress T lymphocyte function, such as steroids [73],
retinoic acid, citral and cyclosporin A [74], markedly impair wound healing. Modification of T
lymphocyte function by adult thymectomy, which prevents the induction of T suppressor cells, causes
an increase in wound maturation. This effect could be reversed by intraperitoneal placement of
autologous thymic grafts in millipore chambers in thymectomized rats [75]. Conversely, administration
of purified thymic hormones , thymulin (FTS), thymopoietin and thymosin fraction V (TF5) results in
impaired wound healing as assessed by wound breaking strength and wound collagen deposition [76].
These data suggest that the thymus exerts an inhibitory effect on normal wound healing, possibly by
enhancing T suppressor cell activation following injury.
Direct evidence for lymphocyte involvement in the control of wound healing is provided by studies
examining the effects of in vivo lymphocyte depletion of wound healing parameters. Global T cell
depletion causes marked diminution in wound breaking strength and in the hydroxyproline content of
subcutaneously implanted PVA sponges, used as an index of wound reparative collagen deposition [9,
77, 78]. Selective depletion of the T suppressor/cytotoxic lymphocyte subset causes marked
enhancement of wound healing at 2 and 4 weeks post wounding. These findings suggests a possible
role for the T suppressor/cytotoxic lymphocyte subset in the overall down-regulation of wound healing
activity. It might be expected that the T helper/effector cells would promote such activity. Selective
depletion of this subset, however, has no effect on either wound breaking strength or wound collagen
deposition [9]. Simultaneous depletion of T helper/effector and T suppressor/cytotoxic T cells leads to
significant increases in both wound breaking strength and collagen synthesis [77]. This suggests that
an incompletely characterized T cell population bearing the T cell marker, but neither the T helper nor
the T suppressor antigenic determinant, is responsible for the promotion of wound healing, since its
deletion impairs wound healing.
Further support for these findings is provided by work in the congenitally athymic nude mouse [79].
These animals have a profoundly impaired T cell dependent immune system and display significantly
enhanced wound breaking strength and collagen deposition in response to injury when compared to
normal thymus-bearing animals. Administration of the anti-T cell monoclonal antibody to these athymic
animals in order to deplete the small numbers of extra-thymically derived T cells present had no effect
on either wound healing parameter but confirmed the previously observed significant decreases in
wound breaking strength and hydroxyproline deposition when administered to normal thymus-bearing
control mice. T cell reconstitution of nude mice, by injection of syngeneic T lymphocytes, resulted in
significant decreases in wound breaking strength towards the levels observed in normal controls (Fig.
3).
Lymphocytes exert many of their effects via cytokines (Table 1). In vitro studies have shown that
lymphokines are capable of modulating many fibroblast functions, including migration, replication and
collagen synthesis. Some, such as TGF-, lymphotoxin or -interferon (IFN-) are well characterized,
while many other proteins which can modulate in vitro fibroblast activity have not been fully
characterized. Both inhibitory and stimulatory lymphokines have been described for many fibroblast
functions [80]; however, exactly how these various signals interact in vivo is presently unknown.
As mentioned previously, the presence of a number of potential regulators of healing has been
confirmed in vivo at the wound site. The presence of biologically active TGF- [57] and IL-6 has been
shown in early wounds, although neither IL-2, IL-3 or IL-4 could be detected [59]. A similar model failed
to detect either IFN- or TGF- at 10 days post-injury [81]. The effects of TGF- administration on
wound strength have already been mentioned [23]. Similar increases in both fresh and fixed wound
breaking strength, with an associated rise in collagen deposition, were seen following administration of
human recombinant IL-2 (60 000 U and 140 000 U/day) in rats [82] (Fig. 4). In contrast, the
administration of IFN- via subcutaneously implanted osmotic pumps in mice resulted in a decrease in
the thickness and collagen content of the capsule which formed around the device [83].
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The Cellular Biology of Wound Healing

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