07 Wound Healing

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Periodontology 2000, Vol. 24, 2000, 127152 Copyright C Munksgaard 2000Printed in Denmark All rights reserved

PERIODONTOLOGY 2000ISSN 0906-6713

Cell biology ofgingival wound healingLARI HA KKINEN, VELI-JUKKAUITTO & HANNU LARJAVA

An overview

Wound healing is a critical process for the organism.

The attempt of this chapter is to critically summarize

the recent advancements in the wound-healing pro-

cess. Because of the excessive amount of infor-

mation (search with the key words wound healing

produced 2052 citations within the last 12 months),we will focus on two major events in wound healing,

namely re-epithelialization and granulation tissue

formation, leaving out many other key events such

as clot formation and angiogenesis. At the end, we

will also discuss the special features of wound heal-

ing in oral cavity. It is commonly stated that oral

wounds heal better than other wounds such as der-

mal wounds. We will explore evidence supporting

this assumption.

Most of the data presented in this chapter refer to

studies with gingival and dermal full-thicknesswounds and no special attempts have been made

to focus on the healing in tooth-gingiva interphase

because only a few studies are available to report

special molecular events in humans. In addition,

several previous articles have addressed periodontal

wound healing and regeneration in great detail (114,

276). It is reasonable to assume, however, that the

basic cell biological events of wound healing will fol-

low the same principles at the tooth-gingiva inter-

phase, at least at the supracrestal locations. We will

also try to make several back-and-forth citations be-

tween in vivo and in vitro studies. This is necessarybecause functional studies have been difficult to

perform with animal models of wound healing in a

manner that would allow meaningful conclusions to

be drawn. In vitro studies with cultured keratino-

cytes and fibroblasts are still the only way to explore

functions of various molecules. Recent molecular bi-

ology techniques have made it possible to eliminate

the expression of molecules of interest and investi-

gate how the function is changed consequently. In

vivo studies have dealt with immunolocalization of

127

extracellular molecules and their expression usingin

situ hybridization. In addition, transgenic animals

provide information how wound healing is altered in

the absence or following overexpression of a known

molecule (153). Many of these studies have provided

new critical information but others have resulted in

confusing results, such as showing no alterations

even though a molecule has been thought to be criti-cally important for wound healing. It is believed that

in these cases there are other related proteins that

can provide similar functions and compensate for

the absence of the studied molecule. Wound healing

has become well protected during evolution because

of its critical importance. Numerous molecules ap-

pear to overlap each others functions. During

wound healing, several molecules that are usually

present only during embryonal development are

found in the granulation tissue. Epithelial cells start

to express extracellular matrix receptors that are notnormally present in the resting epithelium. Fibro-

blastic cells with a special phenotype are found in

the healing granulation tissue. Where they come

from is still largery unknown but possible sources

will be discussed in this chapter. Expression of vari-

ous proteins by the resident cells is also influenced

by the new environment. For example, in fibroblasts

the expression of hundreds of genes is altered by the

exposure to serum alone. Proteolytic enzymes re-

lease growth factors from the wounded basement

membrane and connective tissue matrix. Matrix

degradation produces biologically active peptidesfrom the matrix proteins that could have specific

functions in tissue repair. Clarification of the com-

plex interplay between new matrix, growth factors,

matrix degradation products and cells during wound

healing will be a challenging task. This review fo-

cuses on re-epithelialization and granulation tissue

formation with the special emphasis on the inte-

grins, since cell adhesion serves as a foundation for

cell migration, matrix turnover and differentiation

during wound healing.


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Hakkinen et al.

Integrins

Integrins play key roles in re-epithelialization and

granulation tissue formation during wound healing

through their function in cell adhesion and sig-

naling. A brief overview is, therefore, presented de-

scribing the structure and function of integrins that

we hope will help the reader to better understandhow they participate in the regulation of wound

healing. Integrins are cell surfaceassociated dimeric

glycoproteins that function as cell-to-extracellular

matrix adhesion receptors (4, 16, 160). Through

binding to the extracellular matrix proteins integrins

mediate information transfer from the extracellular

matrix to the cell interior leading to alterations in

cell functions and ultimately in cell behaviour (100,

160). Integrins are known to play an important role

in regulating a wide range of cell functions during

growth, development, differentiation, and immune

response (4). Integrins are composed of a single aand a single b subunit that are non-covalently linked

to each other. At least 17 different a and 8 b subunits

are currently known. These subunits can variously

combine to form more than 23 different cell surface

receptors that have distinct ligand-binding speci-

ficities. Both a and b subunits are transmembrane

glycoproteins that cooperate in integrin binding to

ligands. Ligand binding causes clustering of the re-

ceptors, which leads to cytoskeletal organization and

signaling (Fig. 1) (160). Integrin-binding sites are tar-

gets for therapeutic applications aiming at blocking

integrin function. Domain crystal structures are

Fig. 1. Integrins and cell signalling. Integrins mediate cell

adhesion to extracellular matrix proteins (ECM) leading

to organization of cytoskeleton and signaling. Integrins

also collaborate with growth factors in regulation of cell

growth. Some integrins can also directly activate growth

factors such as transforming growth factor b1 (TGFb1).

Proteolytic enzymes are also found in cell adhesion sites

allowing cells to detach and subsequently migrate.

128

being used for designing new molecules that have

high affinity to the binding site but are irrelevant to

the original peptide sequence. Commercial products

are already available for cardiovascular applications

(146). Several other compounds are in clinical trials

and many other similar products will follow and may

be used for periodontal applications in the future.

The number of proteins known to associate withintegrins is rapidly increasing (96). Integrin associ-

ated protein, transmembrane-4 superfamily, growth

factor receptors and urokinase-type plasminogen ac-

tivator receptor appear to have a regulatory function

on integrins. Growth factor receptors accumulate in

the same structures as integrins and regulate inte-

grin functions (Fig. 1) (161). In the context of wound

healing, the synergy between integrin and growth

factor receptors is probably a key process in the

regulation of cell proliferation (100). Integrins avb1

and avb6 can also bind growth factors such as trans-

forming growth factor b1 through its latency-acti-vated peptide that contains an Arg-Gly-Asp (RGD)

peptide sequence (166, 167). It has recently been

shown that epithelial cells can bind and also activate

transforming growth factor b1 through avb6 integrin

(Fig. 1); this mechanism may play an important role

in the connective tissue bridge formation under-

neath the epithelium that has covered the wound. It

is evident that the epithelium has a much more ac-

tive role in the regulation of connective tissue forma-

tion than previously assumed.

Epithelial wound healing

Re-epithelialization

Wound healing is a complex phenomenon that in-

volves series of controlled events including the for-

mation of a provisional extracellular matrix that is

mainly composed of fibrin, fibronectin and vitronec-

tin and the migration of epithelial cells from the edg-

es of the wound (33, 108). After the epithelium has

been disrupted by tissue injury, re-epithelialization

must occur as rapidly as possible in order to re-es-tablish tissue integrity. Keratinocytes start moving

into the defect about 24 hours after the injury (277).

Epithelial cells from residual epithelial structures

dissolve their hemidesmosomal connections and de-

tach from basement membrane and move quickly

across the wound defect. Later, as the re-epithelializ-

ation proceeds, the proliferation of keratinocytes

that feed the advancing epithelial edge becomes

more important. It is still somewhat unclear which

cells in the epithelium first move into the wound.


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Cell biology of gingival wound healing

There is some evidence that the suprabasal keratino-

cytes are the first migratory cells sliding over the

basal keratinocytes. Epithelial cell migration on the

exposed connective tissue matrix underneath the

fibrin-fibronectin clot has been commonly de-

scribed. In small gingival wounds (Fig. 2), however,

the keratinocytes cut their way directly through the

clot and may not interact with the connective tissuematrix at all (132). Migrating keratinocytes are highly

phagocytic, allowing them to penetrate through

tissue debris or the clot (277). Degradation of the fi-

brin clot appears to be critical for wound healing,

since wounds in animals that lack the plasminogen

gene do not re-epithelialize (199). It seems likely that

integrins play a role in the fibrin clot removal. Mi-

grating cells must be able to focalize the proteolysis

into the leading edge of epithelium (227). This could

be done by activation of proteolytic enzymes at spe-

cific sites at the cell membrane. It has been found

that urokinase type plasminogen activator receptoris able to associate with integrins (283). This is an

example how a cell is able to focalize fibrinolysis by

plasmin and promote subsequent migration by inte-

grins. Modulation of other matrix componets may

Fig. 2. Stages of healing of full thickness gingival wounds (about 3 mm deep and 2 mm wide). FC: fibrin clot; GT:

granulation tissue.

129

also be necessary. Migrating keratinocytes express

matrix metalloprotease-9 (type IV collagenase), ma-

trix metalloprotease-1 (interstitial collagenase) and

matrix metalloprotease-10 (stromelysin), which all

may be required if the cells encounter the exposed

matrix (207, 210, 266). Blocking matrix metalloprote-

ase activity prevents keratinocyte migration into the

wounds in cell culture (147). This proteolytic modu-lation of the matrix underneath migrating cells must

be well controlled, since overexpression of matrix

metalloproteases is a common finding in nonhealing

chronic wounds (187). In chronic inflammation, ma-

trix metalloproteases such as matrix metalloprotea-

se-9 produced by keratinocytes may bind to the cell

surface and into the extracellular matrix resulting in

a delayed clearance (147). Thus, regulation of matrix

metalloproteases and their inhibitors must be well

balanced for normal wound healing (266).

During wound healing, keratinocytes function to

rapidly cover the exposed connective tissue (236,277). This process depends upon a variety of interac-

tions between cells and the extracellular matrix. As

the basal keratinocytes at the wound margin are ex-

posed to the new provisional matrix, the phenotype


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Hakkinen et al.

of the cells is changed from stationary to migratory

(176). In this process, keratinocytes detach them-

selves from the basement membrane, migrate lat-

erally into the wound bed and finally regenerate the

basement membrane (124). In mucosa and in skin,

the migration seems to involve similar patterns, al-

though the source of migrating epithelial cells is dif-

ferent. In the mucosa, the basal layers of the woundedge epithelium serves as a major source of the kera-

tinocytes migrating into the wound area. In the skin,

however, epithelial cells arise not only from the

wound edge but also from hair follicles and sweat

glands. During wound healing, most of the compo-

nents of the basement membrane zone such as type

IV and VII collagens, laminin-1 and heparan sulfate

proteoglycan are missing underneath migrating

keratinocytes (132, 178). Laminin-5, however, ap-

pears to be always deposited against the wound bed

matrix by keratinocytes during migration (Fig. 3)

(132). The role of laminin-5 appears to be criticaleven for healing of the blister wounds where lamina

densa remains intact (110). It is also the first base-

ment membrane component found against the con-

nective tissue in experimental cyst formation that re-

sembles wound healing (110). Keratinocytes show

poor migration in chronic aphthous ulcers and also

fail to deposit a proper laminin-5 rich matrix (196).

Fig. 3. Keratinocyte integrins and extracellular matrix pro- (LM5: laminin-5; TN: tenascin-C; FN ED-A: fibronectin

teins during re-epithelialization. A. Histological represen- isoform EIIIA) while they migrate through wound pro-

tation of a 3-day-old wound. B. Schematic view of the visional matrix. FC: fibrin clot; CT: connective tissue; E:

same wound. Keratinocytes express a number of integrin epithelium. refers to the strong immunoreaction at

receptors (a2b1 to a6b4) and extracellular matrix proteins this stage of healing.

130

Laminin-5 can be proteolytically modified (see be-

low) to produce different molecules that function

either as a nucleator of the hemidesmosomes (stop-

ping signal) or as a promoter of migration. Laminin-

5 seems to be the nucleator of a6b4 integrin and

therefore the nucleator of basement membrane or-

ganization (110). Migrating keratinocytes produce

other extracellular matrix molecules that they coulduse to support their own migration. Fibronectin-EII-

IA but not EIIIB (see below) appears to be present

underneath the migrating keratinocytes in vivo (Fig.

3) (Hakkinen et al., unpublished). In addition, kera-

tinocytes in culture deposit fibronectin-EIIIA while

they migrate on substrates coated with serum

fibronectin (Koivisto et al., unpublished). Tenascin-

C large and small variants are also found underneath

the migrating keratinocytes. As we will discuss else-

where in this chapter, tenascin-C may function as a

modulator of cell adhesion to other matrix compo-

nents such as fibronectin. It appears obvious, there-fore, that keratinocytes themselves are capable of

making extracellular matrix that they can use to sup-

port or modulate their own migration if the pro-

visional matrix is not permissive to migration. One

could question whether keratinocytes in healing

wounds migrate on the provisional matrix at all.

Some of the migration could be intraepithelial, that


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Table 1. Epithelial integrins and their ligands during wound healing

Integrin Ligand Ligand source

a2b1 Collagen Connective tissueTenascin (?) Keratinocytes, granulation tissue

a3b1, a6b4 Laminin-5 Keratinocytes

a5b1, avb1 Serum fibronectin SerumFibronectin EIII/A Keratinocytes

a9b1 Tenascin-C Keratinocytes, granulation tissueavb5 Vitronectin Serum, connective tissue

avb6 Fibronectin EIIIA, fibronectin EIIIB Keratinocytes, granulation tissueTransforming growth factor b1 Keratinocytes, macrophages, connective tissue cells

is, suprabasal keratinocytes sliding on basal cells at

the leading edge. After reaching the provisional ma-

trix, migrating cells could stop moving and become

basal stationary keratinocytes. Two models for epi-

thelial sheet migration in wounds have been pro-

posed (236). In the sliding model the cells at themargin are active and pull the cells behind them. In

a leap frog model, the migrating cells become non-

motile when they adhere to the provisional wound

matrix. Then the cells behind the leading edge crawl

over the newly attached keratinocytes. The exact

mechanism by which multilayered epithelium mi-

grates remains to be discovered, but it is possible

that all three mechanisms described above could

function depending on the defect and the state of

the healing. Embryonic re-epithelialization appears

to occur through a different mechanism. Epidermal

cells do not crawl by extending lamellopodia into the

wound as they do during, for example, gingival

wound healing (132). They rather use the actin

filament cables to pull wound edges together (154).

In small gingival wounds (23 mm distance be-

tween the wound edges), the formation of new base-

ment membrane starts when the migrating epithelial

sheets have reached each other and fused (132). The

nucleation of the basement membrane appears to

occur at multiple sites at the same time. It has been

reported that in epidermal wounds the deposition of

basement membrane starts from the wound marginin a zipper-like fashion (35). Whether there is a real

difference in the sequence of basement membrane

organization between gingival and epidermal heal-

ing has not been shown. The reorganization of the

basement membrane is complete at 4 weeks at

which time the localization of all basement mem-

brane components such as type IV and VII collagens,

laminin-1 and heparan sulfate proteoglycan appear

normal (Hakkinen et al., unpublished). Keratinocytes

have been shown to synthesize these main compo-

131

nents of the basement membrane zone (236). Sig-

nificant portion of the basement membrane compo-

nents are, however, synthesized by the wound

fibroblasts (65). It is obvious that the cross-talk be-

tween the wound keratinocytes and fibroblasts is

crucial during the reorganization of the basementmembrane zone.

Extracellular matrix interactions of keratinocytesduring re-epithelialization

As the epithelial cells undergo major environmental

changes during the wound healing, they have to ad-

just their cell surface receptors for the new demands.

During wound healing keratinocytes have to migrate

on the provisional matrix different from their

stationary matrix. This requires a change of express-

ion levels and distribution of old integrins and ex-

pression of some totally new integrins (Table 1, Fig.

3) (131). As discussed throughout this chapter,

fibronectin is a critical early component of the clot

and the forming granulation tissue. Initially, the

blood clot contains plasma fibronectin, which is

later replaced by cellular fibronectin produced by

keratinocytes, fibroblasts and macrophages (33).

Fibronectin can vary structurally in a tissue specific

manner by alternative splicing of three regions,

namely EIIIA, EIIIB and V(IIICS) (284). Expression of

the alternatively spliced isoforms of cellularfibronectin containing either the EIIIA or EIIIB mod-

ules, which are omitted from the plasma fibronectin,

are upregulated specifically during embryonic devel-

opment and upon wounding (Hakkinen et al., un-

published). As discussed above, EIIIA fibronectin is

expressed by migrating keratinocytes in wounds

(Hakkinen et al., unpublished). The EIIIB fibronectin

that is expressed in embryonal tissues but not nor-

mally in adult connective tissue is strongly upregu-

lated during granulation tissue formation (Hakkinen


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Hakkinen et al.

et al., unpublished, see below). Plasma fibronectin

and the isoforms containing the EIIIA or EIIIB mod-

ules may have different properties in cell adhesion

and migration during wound repair. Human wound

keratinocytes express two major receptors that are

able to bind to fibronectins, namelya5b1 and avb6

integrins (Table 1) (91, 132). The classical fibronectin

binding receptor is a5b1 integrin that binds to theRGD sequence of the fibronectin molecule (1, 229).

Integrin a5b1 is considered to be a specialized recep-

tor for fibronectin and it mediates fibronectin-matrix

assembly, cell adhesion and migration on fibronec-

tin (1). Another fibronectin RGD site binding recep-

tor in epithelial cells is the avb6 integrin (17). Inte-

grin avb6 is expressed exclusively in epithelial cells,

and it functions as a receptor for the extracellular

matrix proteins fibronectin (17, 270) and tenascin

(191). Neither of the main fibronectin receptors,

a5b1 or avb6 integrin, appears to be present in kera-

tinocytes residing in the resting epithelium, but theexpression of both of these integrins can be induced

by wounding or placing epithelial cells in cell culture

(Fig. 3). These two fibronectin receptors appear in

the wound keratinocytes at different times, a5b1

during early migration and avb6 during reorganiza-

tion of the basement membrane zone suggesting

that their function may be different (91, Hakkinen

et al., unpublished). As mentioned earlier, epithelial

integrin avb6 may play a central role in the reorgan-

ization of the basement membrane zone (Fig. 4) as it

is expressed by wound keratinocytes after epithelial

sheets have joined and because it is able to activate

transforming growth factor b (167). In culture, kera-

tinocytes can switch between avb6 and a5b1 inte-

grins in fibronectin binding if one receptor is un-

functional (118).

Laminin-5 is found in the basement membrane

of skin and other epithelial tissues and serves as a

component of anchoring filaments that span

through the basement membrane (19, 203). The

extracellular domain ofa6b4 integrin interacts with

laminin-5 (173, 203). This integrin is known to link

the basal keratinocytes to the underlying basementmembrane, and this link mainly relies on the inter-

action with laminin-5 in the anchoring filaments.

The functional importance of a6b4 integrin is sub-

stantiated by experiments in knockout mice that lack

either a6 or b4 integrin and cannot form hemides-

mosomes, which results in blistering of skin. Lamin-

in-5 is also recognized bya3b1 integrin (19). Trans-

genic mice lackinga3 integrin also develop localized

blistering of the skin (49). In mice and man, a3b1

integrin is occasionally found at the basal surface of

132

the basal keratinocytes, suggesting that a3b1 inte-

grin collaborates with a6b4 integrin in laminin-5

binding and they may be functionally interchange-

able in some circumstances. Integrin a6b4 is present

in migratory epithelial cells although hemidesmo-

somes are absent (132). One possible scenario could

be connected to the functions of laminin-5 during

wound healing. Intact laminin-5 appears to be a mo-tility factor for keratinocytes, but when proteo-

lytically processed it starts to function as the nu-

cleator of hemidesmosomes and therefore promotes

the formation of basement membrane structures

(78). This is a good example demonstrating how

complex the wound-healing process is, since simple

proteolytic cleavage can totally reverse the function

of a protein important for the cell migration process.

Expression ofb1 integrins is strongly upregulated

in keratinocytes during wound healing (20, 73, 97,

108, 132). Both a2b1 and a3b1 integrins seem to be

equally expressed. Integrin a3b1 is known to be ableto bind both fibronectin and laminin-5, both of

which are present in the provisional matrix (19, 132)

although its role as a fibronection receptor in kera-

tinocytes is doubtful. Integrin a2b1 is known to be

able to bind many types of collagens (Table 1), such

as type I, III, V and VI that serve as a substrate for

migrating keratinocytes. Direct interactions of kera-

tinocytes with type IV collagen are rare but may oc-

cur, for example, during healing of blister wounds,

in which case keratinocytes migrate on the compo-

nents of lamina densa. Keratinocytes are more likely

to interact with fibrillar collagens, such as types I,

III and V collagens during healing of full-thickness

wounds. In this case, interaction of keratinocytes

with collagens is mediated via a2b1 integrin. Inte-

grins are also involved in the regulation of extracellu-

lar matrix degradation. Cells in the tissue adapt to

their changing environment and sense alterations in

the matrix through integrins on the cell surface. Rec-

ognition of an altered matrix by integrins lead

changes in gene expression. For example, epithelial

and connective tissue cells attach to collagen by

a2b1 integrin which process induces the expressionof collagenase (matrix metalloproteinase-1) that can

cleave the exposed collagen matrix and facilitate the

migration of wound keratinocytes (186, 187, 278).

Vitronectin is an abundant protein present in

blood plasma, extracellular matrices and fibrin clots.

Vitronectin is known to be an active cell adhesion

mediator as well as playing a role in cell migration

and invasion (190). The avb1, avb3, avIIb3 and avb5

integrins are all vitronectin receptors binding to the

RGD cell attachment sequence of the protein (24).


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Fig. 4. Hypothetical role of keratinocyte avb6 integrin in thelium that cover the granulation tissue (GT) express

the regulation of granulation tissue formation. A. Fibrin avb6 integrin. C. Schematic representation of the putativestaining of a 7-day-old gingival wound. Arrowheads mark role of avb6 integrin in wound healing. TGFb1: trans-

the wound area. B. Immunolocalization ofavb6 integrin forming growth factor b1.

in a 7-day-old wound. Basal keratinocytes of fused epi-

Migrating keratinocytes in porcine epidermis have

been reported to express the avb5 integrin (73). The

avb5 integrin is present in cultured keratinocytes

and has been shown to mediate keratinocyte mi-

gration on vitronectin (116). Vitronectin is present in

the provisional wound bed matrix and it is, therefore,reasonable to assume that an avb5-vitronectin inter-

action can exist during wound healing (Table 1) (36).

Tenascins are a family of large extracellular matrix

proteins whose expression is tightly regulated, mak-

ing this protein particularly interesting. The express-

ion of tenascin is closely associated with morpho-

genetic events, including embryonic induction and

migration, wound healing and tumorigenesis (28,

122, 275). Tenascin-C knockout mice develop nor-

mally (208), suggesting that there are other mol-

133

ecules that can replace its function and compensate

for its absence. Tenascin-C is found in developing

brain and in mesenchyme associated with epithelial-

mesenchymal interactions (28, 247). In adult tissues

tenascin-C is found in the connective tissue under-

neath epithelium (138), but its expression is stronglyupregulated during wound healing (31, 144, 275). As

mentioned above, the large and small tenascin-C

variants are found under migrating keratinocytes,

suggesting their active role in re-epithelialization.

Whether tenascins modulate keratinocyte adhesion

to other matrix components or support directly kera-

tinocyte adhesion remains to be shown. Several

keratinocyte integrins have been found to bind tena-

scin-C, namely a2b1, avb6 and a9b1 integrins (14,

191, 290, 291). Integrin a9b1 in actively migrating,


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Hakkinen et al.

and avb6 integrin in nonmigrating wound keratino-

cytes, colocalize in the same area as tenascins (Table

1) (Hakkinen et al., unpublished). These two epi-

thelial integrins may, therefore, have a different role

in cell signaling caused by tenascin-C variants. Up-

regulation of a9b1 and tenascin-C is also observed

during corneal wound healing (239).

Transforming growth factor b andre-epithelialization

In wounds, specific regulatory signals are required

for normal repair. Currently it is believed that trans-

forming growth factors-b have a central role in the

wound healing process. Transforming growth factors

b are a family of polypeptides that have multiple

regulatory actions in cell growth, differentiation, and

developmental processes (155, 234, 244). Three

highly homologous transforming growth factor-b

genes have been identified in mammals, represent-ing transforming growth factor b1, transforming

growth factor b2 and transforming growth factor b3

polypeptides. Human keratinocytes of intact skin ex-

press transforming growth factor b3 (216). This

growth factor seems to play an important role in epi-

dermal maintenance. In animal studies, only small

amounts of transforming growth factor b2 and trans-

forming growth factor b3 messenger RNA have been

found in keratinocytes of intact dermis (215). All

transforming growth factor b isoforms are found in

healing wounds of animals (111, 137). However,

Schmid et al. (216) did not find any detectable levels

of transforming growth factor b2 messenger RNA in

human wound keratinocytes. Wound fibroblasts and

macrophages are known to express both trans-

forming growth factor b1 and transforming growth

factor b2 in cells adjacent to the wound (175, 267).

Transforming growth factor b1 is the major isoform

in wound keratinocytes, and induction of trans-

forming growth factor b1 in migrating keratinocytes

is crucial for the successful re-epithelialization of

skin wounds. In vitro studies have shown that trans-

forming growth factor b3 inhibits the growth of pri-mary human keratinocytes, while transforming

growth factor b1 seems to stimulate keratinocyte

motility by switching the cells from the differentiat-

ing to regenerative phenotype (152) and by inducing

their production of fibronectin (172) and laminin-5

(110). In cultured human keratinocytes, trans-

forming growth factor b1 has also been shown to in-

crease the levels of messenger RNA for some inte-

grins, such as a5, av and b5, that may facilitate mi-

gration of wound keratinocytes (73, 292). In cultured

134

HaCaT keratinocytes, transforming growth factor b1

and 2 specifically stimulate the expression of avb6

integrin, and promote both haptotactic and epi-

thelial sheet migration (118). As we have discussed

earlier, the most important role for avb6 integrin

may be the activation of transforming growth factor

b1 that then promotes the formation of the connec-

tive tissue bridge formation under the joined woundepithelium. The first collagen fibrils are, indeed, laid

down under the epithelium in areas where the ex-

pression ofavb6 is strong and transforming growth

factor b1 is present in active form. Transforming

growth factor b1 may also stimulate the proliferation

of wound keratinocytes at the wound margins in-

directly by inducing the expression of other polypep-

tide growth factors, such as platelet-derived growth

factor (3, 22).

In addition to transforming growth factors b,

many other growth factors regulate wound healing.

This review will not try to list all possible factors in-volved in re-epithelialization and the readers are ad-

vised to review other sources for the roles of platelet-

derived growth factor, epidermal growth factor, kera-

tinocyte growth factor, hepatocyte growth factor and

others in wound healing.

Connective tissue repair

Activation of fibroblasts

The resolution of a tissue defect after wounding re-

quires not only re-epithelialization but also prolifer-

ation and migration of fibroblasts into the wound

bed where they participate in the formation of

granulation tissue and synthesize, deposit and reor-

ganize various extracellular matrix molecules.

Wound repair involves phenotypic change of fibro-

blasts from quiescent to proliferating cells, and sub-

sequently to migratory, and then to stationary matrix

producing and contractile cells. In normal connec-

tive tissue, fibroblasts reside in a quiescent state and

have a slow proliferation rate and metabolic activity.

Upon wounding, fibroblasts become activated andchange their gene expression. This is supported by a

recent finding with quiescent fibroblasts that show a

rapid change in expression of hundreds of genes

when stimulated by serum. Interestingly, many of

the serum-activated genes are known to be involved

in the physiology of wound repair, including control

of cell cycle and proliferation, coagulation and

hemostasis, inflammation, angiogenesis, tissue re-

modeling, cytoskeletal reorganization and re-epi-

thelialization (107). This indicates that exposure of


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fibroblasts to serum that is present in the blood clot

in the wound initiates not only a rapid general

stimulus for cell proliferation but also a more speci-

fic gene expression that controls function of other

cells involved in inflammation, angiogenesis and re-

epithelialization. Therefore, it is likely that fibro-

blasts play a more important role in the physiology

of wound repair than has been previously realized(107).

In addition to serum stimulation, cell adhesion to

different matrix molecules can also alter gene ex-

pression and protein synthesis (4, 120, 121, 143). Cell

adhesion also modulates signaling initiated by

growth factors (109, 163). During tissue repair,

fibroblasts are exposed to a variety of extracellular

matrix molecules and soluble growth factors that can

exert multiple signals at the same time. Additionally,

cells experience tensional forces from the extracellu-

lar matrix during cell migration and reorganization

of the wound matrix, which can modulate signalinginside the cell (27, 205, 228). The function of extra-

cellular matrix proteins may be further modulated

by proteolytic modification (13, 143, 227), and by

conformational changes induced by tensional forces

(295). Therefore, the regulation of fibroblast gene ex-

pression upon wounding is likely to be very complex

and dependent on the balance between different ac-

tivated signaling pathways (41).

Origin of wound fibroblasts

The multiple functions of wound fibroblasts raise

the question of whether all of the tasks are per-

formed by a single cell type or multiple different

phenotypes are involved (Table 2). If the latter case

is correct, does the heterogeneity result from mi-

gration of phenotypically different cells into wound

or does it result from differentiation of the fibro-

blasts that have populated the wound? These ques-

tions are still largely unanswered. There is evidence,

however, that connective tissue fibroblasts are het-

erogeneous in several properties, including, respon-

siveness to growth factors and in the ability to pro-duce specific extracellular matrix proteins (93, 94,

Table 2. Origin of wound fibroblasts

Possible sources of wound fibroblasts Steps involved

Surrounding connective tissue Migration, differentiation

Pericytes Proliferation, migration

Bone marrow Systemic control, homing, differentiation

135

185, 188, 219, 225) suggesting that signals initiated

by wounding may stimulate certain subpopulations

to enter the wound space. On the other hand, there

is also evidence that some of the fibroblasts that

have migrated into wound actually change pheno-

type and differentiate into myofibroblasts (see

below).

Progenitor fibroblast populations have been iden-tified in wounds for decades using autoradiographic

labeling techniques. Based on these studies, the

main source of wound fibroblasts seems to be the

surrounding connective tissue. Typically, 2 or 3 days

after wounding, the label-retaining cells are located

at the subepithelial connective tissue underlying the

wound edge and at the perivascular location. It

seems, however, that only a subpopulation of the lo-

cal fibroblasts proliferate in response to tissue injury

(225), suggesting that certain fibroblast subsets are

more responsive to growth stimulation than others.

Local fibroblasts are not the only source of woundfibroblasts. After wounding, pericytes that are resi-

dents of the surroundings of the vascular endo-

thelium of capillaries and venules are also induced

to proliferate and migrate into the wound (79, 106,

158). Although fibroblasts have multiple functions in

wound repair, their hallmark characteristic is the

property to produce collagen and other structural

extracellular matrix proteins that replace the blood

clot. This property separates wound fibroblasts from

inflammatory and vascular endothelial cells that are

also involved in the formation of granulation tissue.

Accordingly, upon wounding, the pattern of gene ex-

pression in pericytes is reprogrammed from a con-

tractile to a migratory collagen-producing cell, sug-

gesting that these cells are able to function as ma-

trix-producing wound fibroblasts.

Could some of the wound fibroblasts also arise

from bone marrow stem cells? Interestingly, early

work by Cohnheim in 1867 suggested that some of

the wound fibroblasts may originate from bone mar-

row. This was further supported by recent findings

showing that genetically marked bone marrow

stromal cells serve as a source of progenitor cells forvarious mesenchymal tissues. Most importantly,


10/26

Hakkinen et al.

these cells apparently translocate to the target tissue

where they acquire the phenotype of the resident

cells (183, 193). These peripheral blood fibrocytes

accumulate in the wound already in the early acute

phase but are also present in the scar tissue (15).

Fibrocytes function as antigen presenting cells and

can prime naive T cells (25) and secrete a number of

cytokines involved in immune response, hematopo-esis and extracellular matrix synthesis (25, 26), sug-

gesting that fibrocytes may play a role in the regula-

tion of the acute inflammatory reaction as well as in

the matrix deposition in wounds.

Taken together, the matrix-producing cells that

populate wound granulation tissue originate from

diverse tissue sources and, depending on their ori-

gin, they may serve different functions.

Integrin expression and function in fibroblasts

during wound repairIn the connective tissue, fibroblasts are surrounded

by a matrix that contains collagen and cellular

fibronectin as the major components. Consequently,

quiescent fibroblasts express collagen receptors

a1b1 and a2b1 and the major fibronectin receptor

a5b1 integrin which they use for adhesion to the ma-

trix (74, 271, 281). Fibroblasts express a3b1 (281) and

integrin heterodimers of the av subfamily (Hakkinen

et al., unpublished) that can also be used to bind

fibronectin (4). Although cultured fibroblasts are

able to express avb1, avb3 and avb5 integrins (70,

73, 92), it is not completely clear with which b sub-

units av actually combines to form integrin hetero-

dimers in quiescent fibroblasts in vivo. In cell cul-

ture, fibroblasts also express a4b1 integrin that binds

to fibronectin (72), but it is unclear whether this

integrin is actually expressed by fibroblasts in vivo.

The b1 integrins in quiescent fibroblasts are par-

tially in an inactive state and become activated after

wounding in fibroblasts that are located near the de-

fect (Hakkinen et al., unpublished). Several lines of

evidence show that integrin activation and integrin-

mediated cell adhesion to extracellular matrix is im-portant for induction of DNA synthesis (179). Speci-

fic integrin-ligand binding is required to commence

DNA synthesis of cells. Cell adhesion to extracellular

matrix via integrins strongly influences the ability of

normal cells to respond to soluble mitogens (43, 76,

109, 221). Activated integrins can physically complex

with growth factor receptors (see above) which pro-

vides a mechanism of how different signaling mol-

ecules may be targeted to cell-matrix interaction

sites inside the cell (161, 218). It is, therefore, prob-

136

able that the decision of the fibroblast to enter the

cell cycle depends on the repertoire of integrins that

actively interact with specific extracellular matrix

proteins and on the combination of growth factors

that the cells are exposed to at the particular phase

of wound repair.

Cell migration is needed for fibroblasts to enter

the wound provisional matrix. Fibroblast migrationinto the blood clot occurs only after a lag period of

3 to 5 days, the time required for cells to proliferate

(32, 157). The lag phase may also be needed for the

cells to be activated from quiescent state to be fully

active migratory cells and to be recruited from their

original location in the tissue or bone marrow to the

wound site. It is believed that, in order to detach

from a collagen-rich matrix, fibroblasts have to

downregulate their expression of collagen receptors

and upregulate integrins that bind fibronectin, fi-

brin, fibrinogen and vitronectin in the provisional

wound matrix. Accordingly, about 3 to 5 days afterinjury, fibroblasts that migrate into the blood clot ex-

press the primary fibronectin receptor a5b1 integrin

and a3b1 integrin while they show downregulation

of collagen-bindinga1 and a2 integrin expression at

the wound margin (74, 281). Platelet-derived growth

factor, which is abundantly expressed during early

wound repair, stimulates cell migration towards

fibronectin by increasing the synthesis ofa5 and a3

integrins (74, 117). On the other hand, platelet-de-

rived growth factor downregulates a1 (74) while it

stimulates a2 integrin expression by fibroblasts in

culture. Therefore, the regulation of a2 integrin ex-

pression in vivoand in vitrois different, possibly be-

cause of the multiple signals in addition to platelet-

derived growth factor that modulate integrin ex-

pression in the tissue. One of the factors that modu-

lates integrin expression during wound repair is the

composition of the extracellular matrix. If fibroblasts

are cultured in fibrin and fibronectin-rich matrices

that resemble the early wound matrix, they show

selective upregulation of a3 and a5 integrin mRNA

as opposed to cells that are in collagen-rich matrix,

which show upregulation of a2 integrin (281). Thissuggests that signals initiated by growth factors and

extracellular matrix molecules collaborate to induce

appropriate integrin expression to support cell mi-

gration on fibrin-fibronectin matrix in the early

wound repair and to allow cell adhesion on collagen

later during scar formation.

In the early wound, fibroblast migration seems to

be primarily mediated by fibronectin because

fibroblast migration from a three-dimensional colla-

gen matrix into a fibrin clot depends on the presence


11/26


of fibronectin in the matrix and can be blocked with

antibodies against a5b1 and avb3 integrins in vitro

(84). Fibroblasts can also use avb3 and avb5 inte-

grins for cell adhesion on vitronectin present in the

clot (70) and avb3 for cell adhesion and migration

on fibrin and fibrinogen, respectively (71, 84). Cell

migration is regulated by the structural organization

of the fibronectin matrix. In the normal connectivetissue, cells deposit and arrange fibronectin into a

fibrillar network between the cells and other matrix

molecules using primarily a5b1 and possibly avb3

integrins (279, 280, 287). In contrast, in the blood

clot, plasma fibronectin is cross-linked with fibrin to

form a three-dimensional scaffold (224). The vari-

ation in the architecture of the fibronectin network

can regulate multiple cell functions, suggesting that

fibronectin in the connective tissue and in the blood

clot may elicit different signals (224, 233). During

wound repair, cells are induced to deposit alterna-

tively spliced isoforms of cellular fibronectin con-taining either the EIIIA or EIIIB modules (see above).

The EIIIA or EIIIB modules have different properties

to support cell adhesion, migration, and prolifer-

ation (95, 150, 151), suggesting that they may have

different functions in wound repair.

Wounding induces deposition of extracellular ma-

trix proteins that modulate cell adhesion. Among

these molecules, tenascin-C, thrombospondin and

secreted protein, rich in cysteine/osteonectin

(SPARC) are multifunctional molecules that can

serve as ligands for multiple cell surface receptors

(83). Typically, the expression of tenascin-C and

thrombospondin is potently stimulated early during

granulation tissue formation, while SPARC is ex-

pressed from middle to late stages of repair. Express-

ion of tenascin-C and SPARC remains elevated dur-

ing tissue reorganization while the expression of

thrombospondin is only transient (50, 144). Interest-

ingly, wounding specifically induces the expression

of the large tenascin-C splice isoform (144, Hakkinen

et al., unpublished) that is normally expressed pre-

dominantly during embryonic development (156,

192). Tenascin-C contains both adhesive andcounteradhesive domains, although mostly it is be-

lieved to reduce the adhesion of cells to fibronectin

and therefore enhance cell migration (253). The ef-

fect of tenascin-C on cell adhesion to fibronectin can

also further modulate gene expression by the cells

(253). Interestingly, the large tenascin-C splice iso-

form functions differently in regulation of cell ad-

hesion and in binding fibronectin as compared with

the more ubiquitously expressed small isoform (29,

63, 64, 113, 169, 184). Tenascin-C can serve as ligand

137

for several cell surface receptors, including avb3

integrin in fibroblasts that binds to the RGD se-

quence found in the third fibronectin type repeat of

the tenascin molecule. Ligand binding by integrins

to this repeat is modulated by other counteradhesive

domains in the molecule (40).

Thrombospondin can also, like tenascin-C, modu-

late cell adhesion, migration and proliferation (83).It contains an RGD sequence that is recognized by

avb3 integrin expressed by fibroblasts (240, 255), but

it also contains, like tenascin-C, additional binding

sites for other cell surface receptors (240). SPARC is

also able to interfere with integrin-mediated cell ad-

hesion on extracellular matrix, and this is believed

to occur via interaction with other cell surface mol-

ecules that are able to bind SPARC and not with

steric disruption of integrinextracellular matrix in-

teraction (168). Because of their different temporal

expression pattern during wound repair, it is poss-

ible that tenascin-C, thrompospondin and SPARCregulate different cellular functions. Indeed, gene

knockout animal studies have indicated that throm-

bospondin plays a role in regulation of the activity

of transforming growth factor-b, collagen fiber or-

ganization and vascularization in the connective

tissue (39, 126), which are all important processes in

the physiology of wound repair.

Wound contraction

One of the tasks of fibroblasts in granulation tissue

is to bring the wound margins closer together to

allow rapid wound closure. It is not completely clear

how wound contraction actually happens in vivo.

The most widely held concepts suggest that wound

closure follows when central granulation tissue is

contracted either by myofibroblasts pulling the new

collagen matrix (69) and/or by fibroblasts changing

the organization of the wound matrix by migrating

through it (54, 67). On the other hand, experimental

removal of central granulation tissue does not seem

to prevent wound contraction, suggesting an alter-

native mechanism in which polarized coordinatedmigration of a rim of densely packed fibroblasts pull

forward the wound edges (89).

Myofibroblasts are likely to play a role in wound

contraction and matrix deposition. This is supported

by findings that myofibroblasts are typically present

in tissues that are under mechanical stress but they

are only occasionally found in normal tissue (46).

Myofibroblasts become especially prominent in

pathological conditions including wound repair,

tissue fibrosis and tumor stroma (42, 46, 201). These


12/26

Hakkinen et al.

cells are characterized by presence of abundant

cytoplasmic microfilament bundles and a-smooth

muscle actin. They differ from normal fibroblasts

morphologically, in growth potential, expression of

proteoglycans and collagen, responsiveness to trans-

forming growth factor b, organization of focal ad-

hesions, and in their ability to organize cellular

fibronectin in culture (51, 93, 94, 212). Fibroblastscultured from wound granulation tissue are rich in

myofibroblasts and express a similar repertoire of

integrins as normal connective tissue fibroblasts.

They are, however, phenotypically unique in that

they show reduced cell spreading on fibronectin as

a result of altered interaction of a5b1 integrin and

fibronectin (92).

The differentiation of myofibroblasts occurs be-

tween 6 and 15 days after wounding (42, 217). After

15 days, about 70% of fibroblasts in granulation

tissue express a-smooth muscle actin. Differen-

tiation of myofibroblasts coincides with wound con-traction, which has led to the idea that these cells

are actually involved in this process. The differen-

tiation of myofibroblasts is induced by transforming

growth factor b in vivo and in cell culture but not

with other profibrotic growth factors and cytokines

(47, 204). Importantly, myofibroblasts differentiation

is controlled by integrin mediated mechanisms and

depends on the composition of the extracellular ma-

trix and the tensional forces mediated from the ma-

trix. Recent studies have shown that the induction of

myofibroblast differentiation by transforming

growth factor-b requires deposition of cellular EIIIA

fibronectin in the pericellular matrix (226). In tissue,

differentiation of wound fibroblasts into myofibro-

blasts follows induction of EIIIA fibronectin express-

ion (226). The induction of the expression of a-

smooth muscle actin in fibroblasts by transforming

growth factor b is also regulated by the changes in

the collagen matrix around the cells and on the de-

velopment of intracellular tension (5, 171). This is in

turn regulated by interaction between a2b1 integrins

and collagen (171). The induction of a-smooth

muscle actin expression during wound repair isthought to provide a mechanism to stop fibroblast

migration when cells reach their destination. Ter-

mination of migration and formation of prominent

focal adhesions may be needed to promote stable

(200) cell adhesion to matrix, which is required for

myofibroblasts to acquire a matrix-synthesizing and

contractile phenotype.

In ideal wound repair, scar tissue is remodeled and

reorganized to form structurally and functionally nor-

mal connective tissue. When contraction stops, the

138

myofibroblasts disappear because of apoptosis and

the scar becomes less cellular and new fibroblasts

with properties typical to normal connective tissue

fibroblasts emerge (42, 48, 217). Apoptosis of myo-

fibroblasts begins at day 12, peaks at day 20 and re-

solves by day 60 after wounding (48). Factors that in-

hibit a-smooth muscle actin expression and differen-

tiation of myofibroblasts are not well characterized. Ithas been noted that interferon-g has an antifibrotic

effect, which probably results from its ability to inhibit

fibroblast differentiation into myofibroblasts (217).

Additionally, myofibroblasts are more sensitive to

basic fibroblast growth factorinduced apoptosis

than normal connective tissue fibroblasts (68), which

may provide a mechanism to reduce the amount of

myofibroblasts in the tissue when they are no longer

needed. Interestingly, myofibroblasts persist in cer-

tain pathological conditions, including hyperthroph-

ic and fibrotic lesions of many organs, suggesting that

they are involved in the accumulation of extracellularmatrix that is characteristic for these lesions (45, 217).

Therefore, for normal wound repair it is important

that the myofibroblasts undergo apoptosis and are re-

placed by normal fibroblasts when wound contrac-

tion is completed.

An interesting question is where the fibroblasts that

replace myofibroblasts and that eventually maintain

the connective tissue structure during tissue homeo-

stasis originate from. Because these cells have pheno-

typic properties characteristic to normal connective

tissue fibroblasts, it is possible that they are derived

from the connective tissuenext to thewound or, alter-

natively, they differentiate from more primitive

fibroblasts that initially populate the wound. Regard-

less of their origin, it seems that replacement of the

wound fibroblasts with cells of normal connective

tissue fibroblast phenotype is necessary for the com-

plete regeneration of the tissue.

Mechanical tension

The interactions between cells and extracellular ma-

trix during granulation tissue formation can bestudied in cell culture using cell-populated three-di-

mensional fibrin or collagen gels (86, 256). In prin-

ciple, the fibrin gels correspond to the situation in

early wound repair when fibroblasts have migrated

into the fibrin containing provisional matrix while

the collagen gel model resembles later phase of

wound repair when granulation tissue fibroblasts re-

side in a newly deposited collagen-rich matrix that

undergoes remodeling. In these models, fibroblasts

attach to the proximal fibrin or collagen fibers and


13/26


in an attempt to migrate pull the fibers together to

form a dense tissue-like structure. When fibroblasts

are allowed to grow in fibrin gels they eventually re-

place the fibrin matrix with newly deposited colla-

gen (256). If the gel is not relaxed, fibroblasts trying

to pull the fibers experience high tension from the

matrix that resembles wound granulation tissue.

Contraction of three-dimensional collagen gel is me-diated by integrins and can be stimulated by serum

and growth factors present in wound, including lyso-

phosphatidic acid, transforming growth factor b and

platelet-derived growth factor (34, 87, 90, 164, 197,

250). Currently, there is some controversy over which

specific integrin-ligand interactions actually mediate

collagen contraction. Binding between fibroblasts

and collagen fibrils is evidently the first and most

important step in the collagen gel contraction. Most

data show that in human connective tissue fibro-

blasts, collagen gel contraction is mediated by the

collagen receptor a2b1 integrin (214, 238). The rateof contraction depends on the amount ofa2b1 inte-

grins expressed by the cells (197). This is in accord-

ance with simultaneous induction ofa2 integrin ex-

pression by wound fibroblasts with formation of col-

lagenous scar and beginning of wound contraction

in vivo (282).

Role of integrins in regulation of cell proliferationand survival in granulation tissue

During the formation of granulation tissue, fibro-

blasts that emerge into the provisional wound matrix

stop migrating and undergo cell proliferation. Ac-

cordingly, about 7 to 10 days after wounding, the

granulation tissue becomes hypercellular containing

abundant fibroblasts and endothelial cells (157, 271).

After that, during tissue maturation, the cell number

in the granulation tissue gradually decreases (48,

157, 271). It appears that the organization of collagen

and mechanical tension mediated from the extracel-

lular matrix are important factors that modulate

DNA synthesis and cell survival. This is evidenced by

the finding that fibroblasts cultured under mechan-ical tension proliferate actively, but after stress relax-

ation the DNA synthesis is rapidly downregulated

and cells start to undergo apoptosis (66, 87, 139,

162). Stress relaxation also downregulates autophos-

phorylation of growth factor receptors, which con-

tributes to the growth reduction (139, 159, 241). In a

mechanically stressed collagen gel, cells develop an

organized cell surface fibronectin network (162, 251)

that disappears after stress relaxation. Since a5b1

integrin can probably serve as a mechanoreceptor

139

and mediate signals that promote cell growth (269,

294), differences in fibronectin receptor engagement

may be a mechanism of how cell proliferation is

regulated by mechanical signals. Therefore, reduc-

tion of mechanical strain during tissue maturation

may provide a mechanism to downregulate cell

growth and induce apoptosis to normalize the cellu-

larity in the tissue. Additionally, in the later phasesof wound repair the activity of growth factors is

gradually downregulated (231, 288), which will

further reduce the number of growth stimulatory sig-

nals for fibroblasts. In the regulation of cell growth

and apoptosis in the granulation tissue, integrins

probably play an important role because they me-

diate the signals initiated by mechanical tension and

collaborate with growth factor signaling and mediate

signals that regulate cell survival (109, 205, 206, 228).

Interestingly, fibroblasts undergo apoptosis in con-

tractile collagen gels but not in fibrin gels (66), sug-

gesting that specific types of integrinextracellularmatrix interactions are needed for initiation of

apoptosis. This may provide a mechanism to prevent

fibroblasts from undergoing apoptosis in the early

fibrin-rich provisional wound matrix and to allow

their proliferation, which is needed to populate the

granulation tissue with fibroblasts.

An additional mechanism that is likely to regulate

cell growth in the granulation tissue is the structural

organization of the extracellular matrix. During

granulation tissue formation, fibroblasts first syn-

thesize fibronectin and then type I collagen that is

gradually organized from monomeric molecules to

form a fibrillar collagen matrix (125). Monomeric

collagen through a2b1 integrin promotes cell pro-

liferation while fibrillar collagen downregulates it

(123). Additionally, studies using fibronectin de-

ficient cells have shown that assembly of fibronectin

matrix by the cells is required for fibronectin to pro-

mote cell growth on different extracellular matrix

molecules (233). Therefore, it is probable that the de-

position and organization of extracellular matrix by

fibroblasts provides a feedback mechanism to regu-

late cell growth during wound repair.

Regulation of protein synthesis and matrixdegradation in the granulation tissue

Remodeling of tissue during wound repair requires

controlled synthesis and degradation of extracellular

matrix. The most important factors that regulate

synthesis and secretion of these molecules include

growth factors and signals from the extracellular ma-

trix. There is evidence that integrin binding to its


14/26

Hakkinen et al.

ligand can directly induce gene expression (102, 254,

272). Most importantly, there seems to be a specific

integrin-ligand interaction that is needed for stimu-

lation of specific type of protein synthesis (254, 272).

In this process, the signals mediated by integrins are

further modulated by growth factors, the structural

architecture of the extracellular matrix as well as by

tensional forces (109, 202, 228).During granulation tissue formation, fibroblasts

are stimulated by transforming growth factor-b to

deposit new extracellular matrix proteins. Interest-

ingly, early matrix deposition seems to occur first at

about day 7 after injury in the granulation tissue im-

mediately under the newly formed epithelium (178).

This coincides with the peak in activation of latent

transforming growth factor-b from storages in the

blood clot provisonal matrix and in the matrix of the

granulation tissue (81, 288) and with induction of

epithelial avb6 integrin which may regulate the acti-

vation of latent transforming growth factor-b (seeabove, 91, Hakkinen et al., unpublished). Once acti-

vated, transforming growth factor-b can induce its

own production by fibroblasts (139), which may be a

mechanism to maintain high activity of transforming

growth factor-b in the granulation tissue.

As discussed earlier, a potent modulator of protein

synthesis is mechanical tension. Generally, when

cells are grown in collagen gels under tension they

show a relatively high protein synthesis rate, while

after stress relaxation the protein synthesis is down-

regulated (162). The signals initiated by tension in

a three-dimensional collagen matrix require specific

integrin-collagen interactions and may lead to speci-

fic gene activation. Additionally, platelet-derived

growth factorinduced upregulation of a2 integrin

expression is stimulated if fibroblasts are cultured in

collagen gel while expression ofa5 and a3 integrins

is attenuated (281). Transforming growth factor-b

can also potently stimulate expression ofa2 integrin

in fibroblasts that interact with collagen, and this de-

pends on the tensional forces that the cells experi-

ence (5). It seems that tension mediated from the

matrix can effectively regulate the balance betweenmatrix production and degradation. For example,

upon stress relaxation the expression of type I colla-

gen is downregulated while that of matrix metallo-

proteinase-1 and -13 is upregulated (30, 195, 197,

252). The regulation of collagen and matrix metallo-

proteinase genes by collagen matrix is partially regu-

lated by different collagen receptors. The downregu-

lation of collagen synthesis after stress relaxation is

mediated by a1b1 integrin and the upregulation of

matrix metalloproteinase-1 and matrix metallopro-

140

teinase-13 expression occurs by a2b1 and by both

a1b1 and a2b1 integrins, respectively (195, 197).

Integrin a1b1 seems to be able to function as a feed-

back regulator of collagen synthesis in fibroblasts

(75).

Gingival wound repair: similarities to scarless fetalwound repair

There has been a general observation that wounds

in the oral mucosa heal faster and with less scarring

than extraoral wounds (Table 3). Scar tissue is char-

acterized by excessive accumulation of disorderly ar-

ranged collagen, mostly type I and III (219), proteo-

glycans (274, 289) and persistent myofibroblasts (42,

46, 201), which leads to aberrant function of the

tissue. Surprisingly, there are only a few reports that

have quantitatively tested this hypothesis. Neverthe-

less, based mostly on animal studies it seems that

wound healing in oral mucosa, indeed, is faster andresults in less scarring than in skin (223, 286). It is

probable that oral wound healing is enhanced partly

because of factors present in the saliva and by the

specific microflora of the oral cavity (see below). Ad-

ditionally, the properties of the cells involved in

tissue regeneration in oral mucosa are unique and

share properties of fetal cells (219). Scar formation is

developmentally regulated because, in early ges-

tation, fetal wound repair occurs without scar forma-

tion and the transition to healing with scar forma-

tion occurs late in the gestation (56, 104). Interest-

ingly, the initiation of scar formation parallels the

induction of myofibroblast differentiation during

wound repair at different stages of embryonal devel-

opment (56), suggesting that phenotypic modulation

of wound fibroblasts into myofibroblasts may be in-

volved in scar formation.

As compared with adult wounds, fetal wounds are

characterized by the absence of clot formation and

inflammatory reaction. They also show unique spa-

tial and temporal organization of extracellular matrix

and reduced expression of transforming growth fac-

tor-b (274). Several investigations have compared theproperties of adult skin and gingival fibroblasts with

fetal fibroblasts. The findings indicate that, unlike

adult skin fibroblasts, adult gingival fibroblasts

located in the papillary connective tissue share

many properties with fetal fibroblasts, including

their growth and migration properties, cell mor-

phology, production of migration stimulating factor,

and production and response to cytokines (219). Ad-

ditionally, similar to fetal fibroblasts, oral mucosal or

gingival fibroblasts are able to contract three-dimen-


15/26


Table 3. Special features of oral wound healing

Factor Mechanism

Saliva Moisture, ionic strengthGrowth factors (EGF, TGFb, FGF, IGF etc.)Unknown factors

Bacteria Stimulation of macrophage influx Direct stimulative action on keratinocytes and fibroblasts

Phenotype of cells Fetal-like fibroblasts with unique responseSpecialized epithelium and connective tissue

sional collagen matrix faster as dermal fibroblasts

(105, 237). They also populate experimental wounds

faster than their dermal counterparts in culture (2).

Gingival fibroblasts also differ from dermal fibro-

blasts in their ability to secrete proteolytic enzymes.

Matrix metalloproteinase-13, a potent collagenase

(273), is expressed in the granulation tissue during

acute wound repair at 7- and 14-day-old human gin-gival wounds but not in dermal wounds (296). Ad-

ditionally, when gingival fibroblasts are inoculated

into three-dimensional fibrin matrix they are able to

reorganize and degrade the matrix rapidly. This is

because of high expression of tissue plasminogen ac-

tivator. Matrix reorganization and fibrinolysis is less

advanced in dermal fibroblast-inoculated matrices

(141, 142). The regulation of fibrinolysis by gingival

fibroblasts depends on the tensional forces that the

cells experience from the matrix (140). The various

findings described above suggest that several cell

functions important in tissue repair are shared by

fetal and gingival fibroblasts and differ from dermal

fibroblasts. It is possible that gingival fibroblasts are

phenotypically unique cells in adult tissue that may

contribute to the rapid healing of oral wounds with

minimal scarring in the gingiva.

Role of saliva and gingivalcrevicular fluid in oral wound

healing

While it is clear that the excellent healing potential

of oral mucosa results to a large extent from the in-

trinsic tissue factors such as the presence of struc-

tural cells with potential for tissue regeneration,

dense vasculature and high turnover rate of connec-

tive tissue and epithelium, it is also apparent that

saliva provides a unique environment in the mouth

conducive for rapid tissue repair (Table 3). This be-

comes obvious from the studies showing delayed

141

healing of oral wounds in people with xerostomia

or sialadenectomized animals (12, 55, 103). Animals

instinctly lick their wounds, which appears to result

in faster healing (11). There are several physico-

chemical factors in saliva favoring gingival healing.

These include an appropriate pH, ionic strength, and

presence of ions such as calcium and magnesium

required for healing (53). Saliva also has an efficientcapacity to reduce redox activity caused by tran-

sitional metal ions and inhibit the production of free

radicals that may be beneficial for the healing pro-

cess (170). Lubrication of oral mucosa provided by

saliva is beneficial for wound healing. The advan-

tageous effects of maintaining a moist wound en-

vironment include prevention of tissue dehydration

and cell death, accelerated angiogenesis and in-

creased breakdown of fibrin and tissue debris.

Hence, the use of hydrocolloid occlusive dressing is

a useful adjunct in facilitating cutaneous wound

healing (61). The therapeutic effect of saliva on heal-

ing of skin wounds has been demonstrated exper-

imentally in calves. Compared to saline-treated

wounds, saliva-treated wounds had shorter in-

flammatory reaction and faster epithelial coverage

and connective tissue regeneration (268). It appears

that moisture and ionic strength are not the primary

factors in saliva that promote tissue repair. This po-

tential is probably due to the presence of several fac-

tors in saliva including various growth factors and

bacteria (293).

Growth factors found in saliva are synthesized bysalivary glands or derived from plasma through gin-

gival crevice. Epithelial cells and connective tissue

cells also produce their own growth factors that act

either in a paracrine or autocrine manner. Because

many of the growth factors are transported to saliva

along with gingival crevicular fluid, it is conceivable

that their concentrations in gingival tissue are higher

than elsewhere in the oral cavity. Therefore the peri-

odontium is in a favorable position with respect to

tissue healing.


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Hakkinen et al.

As discussed above, wound healing is a complex

phenomenon involving increased proliferation, ad-

hesion and migration of cells of connective tissue

and epithelium, inflammatory reactions and re-

modeling of extracellular matrix. All these phenom-

ena are directed by growth factors (9, 10, 82). Differ-

ent growth factors have specific functions and target

cells in wound healing, and their delicate balance isrequired for optimal tissue repair. The actual roles of

each growth factor in saliva are not known. The best

studied salivary growth factor, and possibly the most

important one, is epidermal growth factor. In his

pioneer studies, Cohen found that a protein compo-

nent of mouse submandibular gland induced pre-

cocious eyelid opening and incisor tooth eruption.

Later it was discovered that the factor termed epider-

mal growth factor has a multitude of effects on cell

proliferation, cell migration and extracellular matrix

metabolism (21, 37, 220). It has now become obvious

that epidermal growth factor is needed for the nor-mal maintenance and repair of oral mucosa (174).

Interestingly, salivary epidermal growth factor also

plays a role in the maintenance of gastric and ileal

mucosal integrity (194, 213). In humans and many

other animals, salivary glands are the major epider-

mal growth factorproducing organs. In humans epi-

dermal growth factor is synthesized by both parotid

and submandibular glands. During oral wound heal-

ing, the concentration of epidermal growth factor is

increased in saliva (99, 180, 181).

Another growth factor found in saliva is the vascu-

lar endothelial growth factor (243). This protein is

important in many aspects of angiogenesis and in-

flammation such as endothelial growth, permeability

and leukocyte adherence (52). A number of other

growth factors are also present in saliva. These in-

clude nerve growth factor and members of trans-

forming growth factor b, fibroblast growth factor and

insulin-like growth factor families (230). As these

substances have specific regulatory roles in cell

growth and extracellular matrix formation, they are

important in maintaining health of the oral cavity

and in healing of oral mucosal tissues (293).The presence of growth factors in crevicular fluid

has received limited attention. The major focus of

the studies has been pro-inflammatory cytokines,

such as interleukin-1 and tumor necrosis factor a

(177). It is important to recognize that while one ac-

tion of the crevicular fluid cytokines is promotion of

inflammation, another function is to facilitate tissue

repair. Maintenance of the normal tissue turnover

results from a delicate balance of the cytokines. An

alteration in this balance results either in persistence

142

of inflammation and tissue destruction or increased

cell growth and tissue repair.

Integrin ligands such as collagen, vitronectin,

fibronectins and laminins are involved in directing

the adhesion and migration of cells (261). It is there-

fore possible that these molecules, when present in

gingival crevicular fluid or saliva, may modulate

wound healing of gingiva. Indeed, fibronectin hasbeen found both in crevicular fluid and saliva (112,

129, 257). Even though not yet reported, it is reason-

able to assume that other adhesion molecules may

be present in saliva.

Extracellular matrix remodeling is an essential

part of wound repair. In this process matrix metallo-

proteinases and other neutral proteinases have an

important function. During periodontal diseases

gingival crevicular fluid and saliva are rich in col-

lagenases, gelatinases and elastases (133136, 149,

232, 263265). Most of these enzymes appear to de-

rive from neutrophils migrating from inflamed peri-odontium. However, salivary gland cells and acti-

vated epithelial cells and fibroblasts of periodontium

produce their own matrix metalloproteases includ-

ing collagenase-1 (matrix metalloprotease-1), col-

lagenase-3 (matrix metalloprotease-13) and gela-

tinase A (matrix metalloprotease-2) (209, 211, 258).

In concert the matrix metalloproteases and elastases

are capable of cleaving all extracellular matrix pro-

teins and proteoglycans. As discussed earlier, proteo-

lytic enzymes are necessary for proper wound heal-

ing. These matrix metalloproteases could also re-

lease cryptic bioactive domains from matrix

molecules that may regulate cell proliferation and

migration (285). For example, fibronectin and colla-

gen fragments have been demonstrated in gingival

crevicular fluid (245) and saliva (77). Practically no

information presently exists on the possible role of

these extracellular matrix fragments in promoting

healing of oral wounds.

Role of bacteria in oral wound healing

The oral cavity harbors considerable amounts ofbacteria. More than 500 bacterial species have been

so far identified in the oral cavity (165). Recognizing

the limitation of the present detection methods, in

reality several times more species may colonize in

the mouth. It is clear that bacteria affect wound

healing in the oral cavity, and it is well established

that wounds colonized by pathogenic bacteria have

delayed healing (198, 249). Clinicians are aware of

the painful complications in extraction wound repair

that result from bacterial infection (38, 57). Much


17/26


less attention has been given to the fact that small

concentrations of bacteria may increase the rate of

wound healing.

In 1921, Carrel reported that wounds of dogs

treated with certain concentrations of Staphylococ-

cus aureus healed faster than untreated wounds.

Since then several studies have confirmed that find-

ing, also using other bacterial species. Many factorsmay contribute to this effect. Inflammatory reaction

that is a prerequisite for tissue repair is accentuated

by bacterial contamination. Bacteria present in a

wound will attract macrophages into the area and

induce their cytokine secretion. As a consequence,

blood supply and granulation tissue formation are

accentuated in the wound. Proliferation of mes-

enchymal cells is increased and synthesis rate of

connective tissue components is stimulated, leading

to greater tensile strength of the contaminated

wounds in the course of healing (115, 127, 128, 136).

Bacteria contain a variety of substances, somestimulating host cell proliferation and others

exerting toxic effects. In addition, the same sub-

stance can be either stimulatory or inhibitory, de-

pending on its concentration in tissue. Bacteria may

act directly on epithelial cells and connective tissue

cells in wounds and, depending on the type and

concentration, either accelerate or delay wound re-

generation. We found that proliferation of gingival

fibroblats in culture was increased by Prevotella in-

termediusbut decreased by the same concentrations

of Porphyromonas gingivalis (133). Interestingly,

there was a great variation in this effect between

fibroblast populations obtained from different pa-

tients. These findings imply that the potential for

periodontal repair depends both on the bacterial

flora and the individual cell populations of the peri-

odontal wounds. Some bacterial factors have direct

fibroblast and epithelial cell stimulating properties.

Lipopolysaccharide of both Actinobacillus actino-

mycetemcomitans and P. gingivalis slightly increases

cell growth in vitro (98, 189). At higher concen-

trations lipopolysaccharide from different bacterial

species inhibits cell proliferation (8, 134). Lipopoly-saccharide as well as bacterial plaque extracts in-

crease hyaluronan production in cultured gingival

fibroblasts (7, 130). Hyaluronan is a high-molecular-

weight polyanionic glycosaminoglycan that plays an

important role in wound healing through specific in-

teraction with other matrix molecules and cells (23).

S. aureus lipoteichoic acid and protein A induce

fibroblast production of hepatocyte growth factor,

which stimulates epithelial proliferation (6). There

are numerous other factors present in bacteria that

143

could modulate oral wound healing. For example, A.

actinomycetemcomitans GroEL-like heat-shock pro-

tein and phospholipase C are able to modulate epi-

thelial cell growth and cell migration (62, 80, 294).

Specific mechanisms of these effects remain to be

explored.

Conclusion

Wound healing in the oral cavity is a very complex

process. We are just starting to uncover the complex

interplay between various cell types, growth factors

and salivary components. The focus of this chapter

was to summarize the two major events of gingival

wound healing, namely re-epithelialization and the

formation of granulation tissue. Because of the

unique environment of oral cavity, we also reviewed

the special features of oral healing. The interplay be-

tween oral cells and their extracellular matrix, bac-teria, saliva and gingival crevicular fluid involves

myriad factors dictating the nature of the tissue re-

pair process. It would be an enormous if not imposs-

ible task to sort out all these factors. Some of the

factors are already relatively well understood and

could be used for practical applications. Active

studies on certain growth factors are underway in

order to provide new tools for periodontal therapy

(101). Similar studies utilizing other factors benefi-

cial for wound healing can be expected when more

basic research has been done elucidating their speci-

fic functions. Only then we can design precision

tools to speed-up (or slow-down) re-epithelializ-

ation, granulation tissue formation and scarless

wound healing. Modulation of celltoextracellular

matrix adhesion will be the key target for computer

designed drugs engineered to guide optimal tissue

repair.

Acknowledgments

We thank Colin Wiebe for critical reading of themanuscript. Wound-healing studies performed in

the laboratories of the authors are supported by the

Medical Research Council of Canada.

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