Biological Research for Nursing

000(00) 1-9
The Role of Doxycycline as a Matrix ª The Author(s) 2009
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Metalloproteinase Inhibitor for the Treatment DOI: 10.1177/1099800409346333
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of Chronic Wounds

Joyce Stechmiller, PhD, ACNP, BC, FAAN,1 Linda Cowan, ARNP,2 and
Gregory Schultz, PhD3

Abstract
Many chronic wounds fail to heal with conventional therapy, resulting in disability and impaired quality of life. New technologies
using recombinant growth factors, autologous growth factors, or bioengineered skin–tissue substitutes have been shown to be
effective, but these treatments are costly. An effective, low-cost treatment to improve healing of chronic wounds is needed. The
molecular environment of chronic wounds, like many other chronic inflammatory diseases, contains abnormally high levels of
proinflammatory cytokines (tumor necrosis factor [TNF]-a and interleukin [IL]-1b]) and matrix metalloproteinases (MMPs),
which impair normal wound healing. In animal models and clinical studies of ulcerative diseases, doxycycline, an inexpensive and
Food and Drug Administration (FDA)-approved antibiotic, appears to inhibit members of the MMP superfamily like MMPs and
TNF-a-converting enzyme (TACE). This article provides an overview of the roles of MMPs and intrinsic tissue inhibitors of metal-
loproteinases (TIMPs) in wound healing and the damaging effects of chronically elevated levels of MMPSs in chronic wounds. It also
explores the use of topical doxycycline, a synthetic MMP inhibitor (MMPI), to enhance healing of chronic wounds.

Keywords
metalloproteinase inhibitors, doxycycline, inflammation, chronic wounds

Wound healing is accomplished by an orderly sequence of
three interrelated phases: inflammation, proliferation and
remodeling, and the resulting healed tissue (Armstrong & Jude,
2002; Nwomeh, Yager, & Cohen, 1998; Suh & Hunt, 1998).
A healthy wound environment depends on the interaction of
the extracellular matrix (ECM), epidermal cells, dermal cells,
and local proteolytic enzymes and their natural inhibitors
(Armstrong & Jude, 2002; Medina, Scott, Ghahary, & Tredget,
2005). Numerous studies have described proteolytic enzymes,
such as matrix metalloproteinases (MMPs), as key regulators
for both normal and abnormal functioning in wound healing
(Sang et al., 2006). MMPs degrade many substances in the
ECM, including fibrin, tendons, and cartilage, to facilitate the
migration of cells and the development of new tissue and ECM.
However, as depicted in Figure 1, with repeated trauma, infec-
tion, hypoxia, and/or malnutrition, an acute wound is at risk for
chronic inflammation and abnormal functioning of MMPs. In a
chronic wound, the activation of macrophages and increased
neutrophils result in the increased expression of inflammatory
cytokines and reactive oxygen species. The molecular environ-
ment of chronic wounds, like many other chronic inflammatory
diseases (arthritis, tumor growth, atherogenesis, emphysema),
contains abnormally high levels of proteases (MMPs and neutro-
phil elastase) that prevent normal wound healing (Tarnuzzer &
Schultz, 1996; Trengove et al., 1999). When MMPs are
aberrantly expressed in chronic wounds, the prospect for syn-
thetic MMP inhibition for therapeutic benefit may be promising
(Chin, Thigpin, Perrin, Moldawer, & Schultz, 2003; Sang et al.,
2006). Research on MMP inhibitors (MMPIs) has been con-
ducted, but few clinical trials have evaluated outcomes in non-
healing chronic wounds.
Doxycycline is an antibiotic of the tetracycline family of
drugs and has been tested in numerous conditions associated
with elevated MMP activity including arthritis (Lauhio et al.,
1994), periodontitis (Golub et al., 2001), and chronic wounds
(Chin et al., 2003; Siemonsma et al., 2003; Smith, Mickler,
Hasty, & Brandt, 1999). In several animal studies, oral treatment
with doxycycline or analogs of tetracycline has enhanced heal-
ing outcomes (Ramamurthy, Jucine, McClain, McNamara, &
Golub, 1998; Seedor et al., 1987; Siemonsma et al., 2003).
1
College of Nursing, University of Florida, Gainesville, Florida.
2
North Florida/South Georgia Veterans Health Systems, University of Florida,
Gainesville, Florida.
3
Department of Obstetrics and Gynecology, University of Florida, Gainesville,
Florida.
Corresponding Author:
Joyce Stechmiller, College of Nursing, University of Florida, PO Box 100187,
Gainesville, FL 32610. E-mail: stechjk@ufl.edu

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2 Biological Research for Nursing 000(00)

endothelial cells, neutrophils, fibroblasts, macrophages, mast

Tissue injury cells, and eosinophils (Lobmann et al., 2002). They are able to
degrade most extracellular and basement membrane proteins,
including cartilage, tendons, and fibrin (Armstrong & Jude,
Repeated trauma, infection,
2002; Nagase & Woessner, 1999; Soo et al., 2000; Wall
hypoxia, malnutrition et al., 2002). MMPs play essential roles in all phases of nor-
mal wound healing (Figure 2). For example, MMPs control
the removal of damaged ECM components, which allows new
collagen molecules to integrate correctly into the ordered
Chronic
alignment of collagen fibrils in the wound bed. MMPs also
inflammation
degrade basement membranes of capillaries through which
endothelial cells can emerge and generate capillary buds,
leading to angiogenesis. Contraction of initial scar tissue by
Activation of Increased myofibroblasts requires controlled cleavage of ECM by
macrophages neutrophils MMPs, as does the migration of epithelial cells over the pro-
visional wound matrix, which leads to closure of the wound.
Finally, MMPs mediate the remodeling of scar tissue that can
Increased Increased occur several months after wound closure by removing the
inflammatory reactive oxygen loosely deposited collagen fibers that are first laid down in the
cytokines species scar, which are slowly replaced by ECM that more closely
resembles a normal dermal basket-weave structure of col-
lagen fibrils in healthy ECM (Schultz, Chin, Moldawer, &
Increased matrix Diegelmann, 2007).
degrading proteases, The normal activity of MMPs is controlled at three levels.
decreased protease The first is the gene level by transcriptional control. For exam-
inhibitors
ple, growth factors, including epidermal growth factor,
platelet-derived growth factor, interleukin 1 (IL-1), and TNF-
a, induce transcription of MMP genes, leading to increased
Excessive matrix levels of their messenger RNAs (mRNAs). The second is the
degradation, degradation of molecular level. MMPs are initially synthesized as inactive
growth factors, impaired
epithelialization
precursors, or proenzymes, and are activitated by proteolytic
cleavage of the propeptide portions. The third level is the nat-
ural inhibitors of MMPs, or the tissue inhibitors of metallopro-
teinases (TIMPs), which binds to active MMPs and form stable
inactive MMP/TIMP complexes (Murphy, 1995; Murphy &
Chronic
wound Willenbrock, 1995).

Biochemical Subclasses of MMPs

Figure 1. Pathway to a chronic wound.
Findings suggest that impairment of wound healing due to an
exacerbated inflammatory condition and proteolytic environ-
ment may be responsive to topical doxycycline as an MMP and
tumor necrosis factor a (TNF-a)–converting enzyme (TACE)
inhibitor. In this article, following a discussion of the roles of
MMPs and intrinsic tissue inhibitors of metalloproteinases
(TIMPs) in normal wound healing, we review the pathophysiol-
ogy of chronic wounds and the exacerbated proteolytic processes
of MMPs. We then explore the use of topical doxycycline, a syn-
thetic MMPI, to enhance healing of chronic wounds.
Role of MMPs in Normal Wound Healing
MMPs are zinc-dependent endopepidases that are part of the
superfamily of MMPs and are produced by keratinocytes,
MMPs are subdivided into four subclasses based largely on
their preferred substates or cellular localization: collagenases,
gelatinases, stromelysins, and membrane-type MMPs (see
Table 1).
Collagenases
MMP-1, MMP-8, and MMP-13 are members of the collagenase
subclass of MMPs. Collagenases are distinct from the other
MMPs because of their unique capability to make a single cut
in the native, intact triple helix of fibrillar collagen Types I and
III. This cut results in a small area of denaturation (unwinding)
of the native, tight, triple helix structure of fibrillar collagens
and permits the further, extensive denaturation (cutting) of the
triple helix into small peptides by the gelatinases. Collagenases
are also secondarily active against other materials in the ECM
(Schultz et al., 2007). For example, MMP-1 has direct action

2
Stechmiller et al. 3

Figure 2. Functions of matrix metalloproteinases (MMPs) in wound healing. ECM ¼ extracellular matrix.

Table 1. Matrix Metalloproteinases (MMPs)

Protein Names Actions

MMP-1 Interstitial collagenase; fibroblast collagenase Types I, II, III, VII, and X collagens
MMP-2 72 kd gelatinase; gelatinase A; Type IV Types IV, V, VII, and X collagens
collagenase
MMP-3 Stromelysin-1 Types III, IV, IX, and X collagens; Types I, III, IV, and V gelatins;
Fibronectin, laminin and procollagenase
MMP-7 Matrilysin; uterine metalloproteinase Types I, III, IV and V gelatins; Casein, fibronectin and procollagenase
MMP-8 Neutrophil collagenase Types I, II and III collagens
MMP-9 92 kd gelatinase; gelatinase B; Type IV Types IV and V collagens; Types I and V gelatins
collagenase
MMP-10 Stromelysin-2 Types III, IV, V, IX, and X collagens; Types I, III, and IV gelatins;
Fibronectin, laminin, and procollagenase
MMP-11 Stromelysin-3 Not determined
MMP-12 Macrophage metalloelastase Soluble and insoluble elastin
MMP-13 Collagenases Collagen types I, II and III
MMP14, 15, 16, 17 Membrane-type matrix metalloproteinases Bind to other MMPs

against Type III collagen while MMP-8 has preferential action
for Type I collagen. MMP-13 has the ability to cleave Types I,
II, and III collagen (Armstrong & Jude, 2002). In addition,
MMP-1 and MMP-8 can degrade other ECM proteins, includ-
ing Types VII, VIII, and X collagen and partially degraded col-
lagen (gelatin).
In practical terms, it is hypothesized that within hours after
wounding, neutrophils releasing MMP-8 infiltrate the wound
during inflammation, resulting in higher concentrations of
MMP-8 in relation to MMP-1 (Armstrong & Jude, 2002).
Higher levels of MMP-8 are required for wound debridement
and lysis of damaged Type 1 collagen present in the wound
during the inflammatory phase. During the proliferative phase
of healing, MMP-1 is active against Type III collagen but at
lower levels. Because the normal wound is experiencing a
maturing ECM and epithelialization, MMP-1 must be tran-
scribed by the appropriate gene to be activated. As a result,
there may be a 12-hr delay before MMP-1 secretion occurs.
By contrast, MMP-8 is readily available and activated in sec-
onds because it is stored in granules within nearby neutrophils
in the wound environment.
Gelatinases
MMP-2 and MMP-9 are gelatinases that cleave partially dena-
tured collagen fibrils of both Types I and III collagen that have
been previously cleaved by collagenases. Gelatinases degrade
(cut) other types of nonfibrillar collagens and ECM

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4 Biological Research for Nursing 000(00)
components including Type IV collagen, Type V small-
diameter fibrillary collagen, Type VII epithelial anchoring col-
lagen, Type X collagen, and elastin (Birkedal-Hansen, 1995).
Gelatinases also further degrade Types I, II, and III collagen
after they have been removed from the triple helix (Agren,
1994). Fibroblasts are responsible for the secretion of MMP-
2. MMP-9, however, is produced by leukocytes and keratino-
cytes (Pilcher et al., 1999).
Gelatinases may also play a role in matrix remodelling and
scar formation. Specifically, MMP-9 may not only function
during inflammation, but also, because it is secreted by kerati-
nocytes, play an important role in the later stages of wound
healing, including cell migration, detaching of keratinocytes
from the basement membrane, and remodeling of the ECM.
Stromelysins
MMP-3, MMP-7, MMP-10, MMP-11, and MMP-12 are classi-
fied as stromelysins and play many roles in the degradation
of the ECM incuding action on collagen Types IV, V, IX, and
X; elastin; fibronectin; and certain proteoglycans (Murphy,
Murphy, & Reynolds, 1991; Quantin, Murphy, & Breathnach,
1989). There is little research describing the structural and
functional aspects of stromelysins and wound healing. Because
stromelysins are found mostly in the clot matrix, they may also
play a key role in clot dissolution early in wound healing.
Membrane-Type MMPs
MMP-14, MMP-15, MMP-16, and MMP-17 are considered
membrane-type MMPs and differ structurally from the other
subsets. MMPs in this subclass are located on cell membranes
and function by binding to other MMPs, activating them or
assisting in their activity. For example, MMP-14 binds to and
activates MMP-2 and MMP-9 (Ueno et al., 1997). MMP-15 and
MMP-16 also bind to the gelatinases (Takino, Sato, Shinagawa,
& Seiki, 1995).
MMP-18 and MMP-20
MMP-18 and MMP-20 are uniquely different from the MMPs
in the other subclasses in both structure and function (Llano
et al., 1997; Pendas et al., 1997). MMP-18 is active against a
synthetic stromelysin substrate (Llano et al.,1997). However,
MMP-20 performs proteolytic actions on the destruction of
enamel proteins during tooth development (Bartlett, Simmer,
Xue, Margolis, & Morena, 1996).
Roles of MMP Subclasses in Healing
In the inflammatory phase of healing, proteolysis of the ECM
proteins such as collagen debris must occur so that newly
synthesized collagen molecules in the wound matrix can allow
migration of epidermal cells and fibroblasts into the wound
bed. Removal of damaged collagen molecules begins with the
collagenases. MMP-1 secretion is induced largely by growth
factors (transforming growth factor [TGF]-a, TGF-B1,
4
epidermal growth factor [EGF], and hepatocyte growth factor)
and is responsible for cell migration and wound re-
epithelialization, along with MMP-9 and MMP-10 (Parks,
1999). MMP-1 (collagenase-1) is produced by keratinocytes
in normal and in nonhealing chronic wounds (Assoian, 1997;
Pilcher, Gaither-Ganim, Parks, & Welgus, 1997; Sudbeck, Pil-
cher, Welgus, & Parks, 1997). Next, gelatinases further
degrade collagen molecules into smaller fragments that are
then removed from the injured area by macrophages and neu-
trophils (Stechmiller & Schultz, 2008). MMP-2 (gelatinase)
plays a key role in angiogenesis and matrix remodeling, first
by degrading the basement membrane that surrounds the vascu-
lar endothelial cells (VEC). This degradation permits the
sprouting of capillary buds that make channels through the
ECM through which the VECs migrate. This process eventu-
ally creates new capillary arcs. Another gelitanase, MMP-9
plays an important role in tissue repair by dissolving ECM pro-
teins such as gelatin, collagens, and elastin. MMP-2 also
cleaves Types I and III collagens and has effects on fibronectin,
elastin, laminin, and Type IV collagen. Finally, MMP-3 (stro-
melysin) is required for myofibroblasts to contract the ECM
during the maturation and remodeling phases.
Tissue Inhibitors of MMPs in Normal Wound
Healing
The release of MMPs into the ECM is controlled by a number
of mechanisms that limit proteolysis of the ECM. A family of
TIMP proteins consists of four members, TIMP-1, -2, -3, and
-4, that inhibit MMPs by binding to their active sites (Table 2).
TIMP-1 is synthesized by keratinocytes, fibroblasts, smooth
muscle cells, and endothelial cells and most often inhibits
MMP-1; however, it is also capable of inhibiting the activity
of all known MMPs. In chronic wounds, TIMP-1 is absent or
its levels are significantly lower than in normally healing
wounds (Trengove et al., 1999). TIMP-2 is produced by the
basal keratinocytes in normally healing wounds and is also
capable of inhibiting the activity of all known MMPs. It prefer-
entially acts as an effective inhibitor of the pro and active
MMP-2. TIMP-3, which inhibits the activity of MMP-1, -2,
-3, -9, and -13, is expressed during human hair and bone devel-
opment and in skin, mammary, and colon cancers as well as in
intestinal ulcers (Soo et al., 2000; van der Stappen, Hendriks, &
de Boer, 1989). It is expressed by macrophage-like cells of the
granulation tissue and by endothelial cells. TIMP-4 inhibits the
invasion of breast cancer cells in vitro and tumor growth and
metastasis in mice in vivo (Savage, Lacombe, Boulos, &
Hembry, 1997). It also inhibits MMP-2 and -7 and has a lesser
effect on MMP-1, -3, and -9.
Pathophysiology of Chronic vs. Acute
Wounds
The nature of acute and chronic wounds differ greatly
(Stechmiller & Schultz, 2008). In acute wounds, such as a cut,
a sudden and quick insult to the skin occurs. Immediately
Stechmiller et al.
Table 2. Tissue Inhibitors of Metalloproteinases (TIMPs)
Protein Name
TIMP-1 Tissue inhibitor of metalloproteinases-1
TIMP-2 Tissue inhibitor of metalloproteinases-2
TIMP-3 Tissue inhibitor of metalloproteinases-3
TIMP-4 Tissue inhibitor of metalloproteinases-2 and -7
thereafter, blood clotting and platelet degranulation induce the
inflammatory response, which lasts for a few days. However, in
chronic wounds, a persistent inflammatory stimulus such as
repeated tissue trauma, bacterial contamination, foreign bodies,
or ischemia causes skin breakdown and prolongs the inflamma-
tion phase. The affected area becomes a good medium for bac-
terial growth. An inflammatory response is initiated by the
influx of neutrophils and macrophages into the wound. Acti-
vated, overexpressed MMPs begin degrading growth factors
and matrix as soon as they are secreted at the cell surface. A
vicious cycle occurs as inflammatory cells secrete the cyto-
kines TNF-a and IL-1b, which in turn attract more inflamma-
tory mediators (Mast & Schultz, 1996). Furthermore, tissue
damage ensues as the inflammatory phase is prolonged. It has
been proposed that the prolonged inflammatory phase is due to
the presence of inflammatory leukocytes, typically neutrophils,
and their production of proinflammatory cytokines, which per-
petuate inflammation. The activation and overexpression of
tissue-damaging MMPs, which degrade newly formed tissues
thereby preventing the normal wound-healing processes to pro-
ceed, further delays healing.
A comparative study of acute and chronic wound fluid
demonstrated high concentrations of TNF-a, IL-1 and IL-6 in
nonhealing chronic leg ulcers (Trengove, Stacey, McGechie,
& Mata, 1996). As the ulcers began to heal, the level of proin-
flammatory cytokines decreased dramatically, indicating a
reduced inflammatory state. In addition, mitogenic activity
in the nonhealing samples was significantly less than in the
healing wounds. Furthermore, the rate of cell proliferation
and differentiation in the nonhealing wounds decreased as
compared to the levels in the acute wounds, retarding growth
(Trengove, Bielefeldt-Ohmann, & Stacey, 2000). In another
study (Tarnuzzer & Schultz, 1996), late mastectomy wound
fluids contained up to 100-fold higher TNF-a and IL-1b levels
and significantly higher IL-6 levels than early mastectomy fluids.
These findings suggest that proinflammatory cytokines are less
strictly regulated in chronic wounds than in acute wounds.
Role of MMPs in the Pathophysiology of
Chronic Wounds
Another difference between acute and chronic wounds is the
elevated levels of MMPs and decreased levels of TIMPs in
chronic wounds. TNF-a and IL-1b secreted by activated
inflammatory cells stimulate the production of MMPs and the
suppression of TIMP-1, -2, and -3. For instance, venous ulcers
are characterized by chronic inflammation and lysis of
5
collagen. The exudate in chronic venous ulcers contains
elevated expression of gelatinases (MMP-2 and MMP-9) and
a reduction in the expression of TIMPs (Medina et al., 2005;
Trengove et al., 1999; Weckroth, Vaheri, Lauharanta, Sorsa,
& Konttinen, 1996; Wysocki, Staiano-Coico, & Grinnell,
1993). Nonhealing venous ulcers also present with elevated
expression of TNF-a, IL-1B, and IL-6, which are responsible
for upregulating MMP-2 and other MMPs (Medina et al.,
2005). Moreover, in diabetic ulcers, there is an increased con-
centration of a number of MMPs (MMP-1, MMP-2, MMP-8,
MMP-9) and reduced levels of TIMP-2, supporting the con-
tention that impairment of wound healing in diabetic ulcers
is due to a prolonged inflammatory and proteolytic wound
environment. Pressure ulcers also exhibit elevated expression
of MMP-1, MMP-2, MMP-8, and MMP-9 and lower concen-
trations of TIMP-1 compared with normally healing wounds.
The MMP-9/TIMP-1 ratio is significantly higher in pressure
ulcers when compared with normally healing wounds
(Medina et al., 2005; Stechmiller, Kilpadi, Childress, &
Schultz, 2006; Weckroth et al., 1996; Yager, Zhang, Liang,
Diegelmann, & Cohen, 1996).
Mast and Schultz (1996) summarized acute and chronic
wound environments at the molecular level. Based on fluid
analysis from acute healing wounds and chronic ulcers, they
discovered that healing wounds show high levels of mitogenic
activity, low concentrations of cytokines, low levels of pro-
teases, and high levels of growth factors and competent fibro-
blasts. In contrast, chronic wounds are characterized by low
mitotic activity, high levels of cytokines, high levels of pro-
teases, low levels of growth factors and senescent cells, and
suppression of angiogenesis.
Use of Doxycycline in Chronic Wound Care
Novel approaches to MMP inhibition are being considered in
chronic wound care (Murphy & Nagase, 2008). The use of bio-
logical reagents to block inflammatory cytokines also reduces
MMP expression in many chronic inflammatory states. For
example, the tetracyclines, which are inhibitors of MMP cata-
lytic activity, also blunt MMP synthesis and have been success-
fully trialed in rheumatoid arthritis. Preliminary findings
suggest that impaired wound healing due to an exacerbated
inflammatory condition and proteolytic environment may be
responsive to topical doxycycline, which acts as an MMP and
TACE inhibitor (Chin et al., 2003)
Doxycycline and other tetracyclines have been studied
extensively in human and animal diseases with high levels
of MMP activity. In humans, these include osteoarthritis
(Shlopov, Smith, Cole, & Hasty, 1999; Yu, Smith, Hasty &
Brandt, 1991), rheumatoid arthritis (Lindy et al., 1997), adult
periodontitis (Golub et al., 1995), and noninfected corneal ulcers
(Perry & Golub, 1985). In animals, tetracyclines have been eval-
uated in models of ulcerative diseases such as alkali-burned rab-
bit corneas (Burns, Stack, Gray, & Paterson, 1989; Seedor et al.,
1987) and alveolar bone loss in rats (Chang et al., 1994) and in
models of angiogenesis where MMP activity is required
5
6 Biological Research for Nursing 000(00)
Pro TNFα
MMPs TACE ⊗ of doxycycline at 50, 500, and 5,000 mM significantly (p < .05)
Doxycycline
⊗ TNFα
Destruction of growth Neutrophil elastase
factors, receptors, ECM α1PI
MMPs ⊗ 34 weeks indicated that topical doxycycline treatment signifi-
Inactive
α1PI
Destruction of growth
factors, receptors, ECM
Figure 3. Doxycycline model for wound healing. Doxycycline
inhibits MMPs and TACE, which impair wound healing when they
occur at high levels. ECM ¼ extracellular matrix; MMP ¼ matrix
metalloproteinase; TACE ¼ TNF-a-converting enzyme; TNF-a ¼
tumor necrosis factor a.
(Tamargo, Bok, & Brem, 1991). In each of these studies, the ben-
eficial effect of doxycycline or other tetracycline analogues were
not due to the direct antimicrobial effects of the medications. This
conclusion was dramatically demonstrated using a nonantimicro-
bial, chemically modified tetracycline in a rat model of bacterially
induced alveolar bone loss in vivo (Chang et al., 1994). When
dosed orally with doxycycline or the nonantimicrobial tetracy-
cline, both groups of rats infected with Porphyromonas gingivalis
showed completely inhibited bone loss.
The fact that the beneficial effects of these drugs are inde-
pendent of their antimicrobial activity suggests that they are
due instead to the inhibitory effects of tetracyclines on MMP
activity and TACE, as shown in Figure 3. Doxycycline inhibits
TACE and prevents TNF-a release. TNF-a is an important
inflammatory mediator that regulates the activity of neutro-
phils, eosinophils, and T and B lymphocytes and can impair
wound healing. It is also a cell-regulator protein whose activity
is largely determined by the cell type to which it binds (Bazzoni
& Beutler, 1996). Although the local effects of TNF-a on
wound healing depend greatly on the model in general, local
application of TNF-a impairs wound healing, in particular.
Doxycycline is traditionally administered intravenously or
orally and has broad inhibitory activity on metalloproteinases,
including MMPs and TACE. Previous studies (Barone et al.,
1998; Mackelfresh, Soon, & Arbiser, 2005; Nwomeh, Liang,
Cohen, & Yager, 1999; Nwomeh, Liang, Diegelmann, Cohen,
& Yager, 1998; Wysocki & Grinnell, 1990; Wysocki et al.,
1993; Yager & Nwomeh, 1999; Yager et al., 1996), confirmed
by Schultz et al. (1992) and Stechmiller et al. (2006), have
established that the molecular environment of chronic pressure
ulcers is uniformly characterized by elevated concentrations of
proinflammatory cytokines and proteolytic activities, espe-
cially the MMPs and the serine protease neutrophil elastase.
These studies suggest that elevated levels of cytokines and pro-
teases interfere with the healing of diabetic ulcers by destroy-
ing growth factors, receptors, and ECM proteins essential for
6
normal wound healing. Chin and colleagues (2003) examined
fluids from four diabetic foot ulcers and found that the addition
reduced protease activity by 44%, 75%, and 89%, respectively.
Also, all four chronic wounds treated with 1% topical doxycy-
cline healed (times to healing were 4, 7, 24, and 30 weeks). In
contrast, only one of the three patients treated with the placebo
hydrogel healed during the initial 20-week treatment period.
Chi square analysis of the seven patients’ healing outcome at
cantly (p ¼ .05) increased healing of the ulcers compared to
treatment with a placebo hydrogel. This pilot study also deter-
mined the safety of topical doxycycline treatment. No adverse
effects were noted for either the topical doxycycline treatment
or the placebo hydrogel.
Based on this study, complications are not anticipated with
the use of topical doxycycline. Skin reactions secondary to oral
doxycycline are rare and include redness, swelling, and blister-
ing rash. Common allergic reactions with oral doxycycline
include hives, shortness of breath, and swelling of the face, lip,
tongue, or throat. Common systemic reactions with oral doxy-
cycline include headache, dizziness, fever, chills, rash, nausea,
vomiting, diarrhea, thrush, or vaginitis. Oral doxycycline can
also cause photosensitivity. A much larger randomized clinical
trial must be conducted to establish conclusively the efficacy of
topical 1% doxycycline on the healing of chronic lower-
extremity ulcers in diabetic patients.
Another important effect of doxycycline in treating inflam-
matory diseases is its ability to indirectly inhibit serine pro-
teases (Lee et al., 1997). In addition to the elevated MMP
activities detected in diabetic foot ulcer fluids, levels of serine
proteases, especially neutrophil elastase, are often elevated
(Chin et al., 2003; Grinnell, Ho & Wysocki, 1996; Grinnell
& Zhu, 1996; Wysocki & Grinnell, 1990). Chin et al. (2003) 1
showed that when doxycycline gel was topically applied to dia-
betic ulcers, levels of serine proteases were decreased.
Neutrophil elastase has been implicated in the proteolytic
destruction of matrix proteins such as fibronectin and growth
factors essential for wound healing (Grinnell et al., 1996;
Wysocki & Grinnell, 1990; Figure 3). The primary endogenous
regulator of neutrophil elastase is a1-proteinase inhibitor (a1-
PI), also called 1-antitrypsin. In regulating elastase activity, the
a1-PI rapidly forms a 1:1 inactive complex with the enzyme.
Deficiencies of a1-PI levels in serum, either genetically
induced or acquired, result in several disease processes (eg,
pulmonary emphysema and periodontal bone loss) in which
elastase substrates (eg, elastic fibers, fibronectin, proteogly-
cans) are excessively degraded. a1-PI can be readily inacti-
vated by neutrophil collagenase (MMP-8) and gelatinase
(MMP-9) and by reactive oxygen species generated by acti-
vated inflammatory cells.
Conclusion
Diabetic foot ulcers pose a significant risk for lower-extremity
amputations, with an estimated 85% of patients with ulcers
 Stechmiller et al.
going on to amputation. To improve nursing care and quality of
life for these patients, we must advance our understanding of
the biomolecular basis of diabetes and diabetic wound healing
and translate this to practical applications. Although doxycy-
cline has been shown to be advantageous in treating chronic
wounds because of its ability to inhibit MMPs, future research
directed at identification of specific MMP targets unique to a
specific MMP profile are necessary for an increased under-
standing of their structure, regulation, and function.
Declaration of Conflicting Interests
2
Funding
3 tive arthritis. Annals of the New York Academy of Science, 732,
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