Delayed wound healing in aged skin rat models after thermalinjury is associated with an increased MMP-9, K6 and CD44expression

Simonetti Oriana a,1,*, Lucarini Guendalina b,1, Cirioni Oscar c, Zizzi Antonio d,Orlando Fiorenza e, Provinciali Mauro e, Di Primio Roberto b,Giacometti Andrea c, Offidani Annamaria a

aDepartment of Dermatology, Universita Politecnica delle Marche, Ancona, ItalybDepartment of Clinic and Molecular Sciences – Histology, Universita Politecnica delle Marche, Ancona, Italyc Institute of Infectious Diseases and Public Health, Universita Politecnica delle Marche, Ancona, Italyd Institute of Pathology, Universita Politecnica delle Marche, Ancona, ItalyeExperimental Animal Models for Aging Unit, Scientific Technological Area, I.N.R.C.A., I.R.C.C.S., Ancona, Italy

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7

a r t i c l e i n f o

Article history:

Accepted 16 September 2012

Keywords:

Burn

Aged skin

MMP-9

Collagen IV

K6

CD44

a b s t r a c t

Age-related differences in wound healing have been documented but little is known about

the wound healing mechanism after burns. Our aim was to compare histological features

and immunohistochemical expression of matrix metalloproteinase-9 (MMP-9), collagen IV,

K6 and CD44 in the burn wound healing process in aged and young rats.

Following burns the appearance of the wound bed in aged rats had progressed but slowly,

resulting in a delayed healing process compared to the young rats.

At 21 days after injury, epithelial K6, MMP-9 and CD44 expression was significantly

increased in aged rats with respect to young rats; moreover, in the aged rat group we

observed a not fully reconstituted basement membrane. K6, MMP-9 and CD44 expression

was significantly increased in wounded skin compared to unwounded skin both in young

and aged rats.

We hypothesise that delayed burn skin wound healing process in the aged rats may

represent an age dependent response to injury where K6, MMP-9 and CD44 play a key role. It

is therefore possible to suggest that these factors contribute to the delayed wound healing in

aged skin and that modulation could lead to a better and faster recovery of skin damage in

elderly.

# 2012 Elsevier Ltd and ISBI. All rights reserved.

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/burns

1. Introduction

Thermal injury is one of the most severe forms of trauma that

affects an organism [1]. Even with the development of

* Corresponding author at: Oriana Simonetti Clinica Dermatologica, Osfax: +39 0715963446.

E-mail address: o.simonetti@univpm.it (S. Oriana).1 These authors contributed equally to this work.

0305-4179/$36.00 # 2012 Elsevier Ltd and ISBI. All rights reserved.http://dx.doi.org/10.1016/j.burns.2012.09.013

improved burn patient care, many problems which arise

for burn patients are associated with the cutaneous wound

healing process. Burn wound healing is highly dynamic

with complex interactions between numerous cell types,

cytokines and proteins. Pathogenic abnormalities, ranging

pedali Riuniti, 60020 Torrette, Ancona, Italy. Tel.: +39 0715963494;

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7 777

from disease-specific intrinsic flaws in blood supply, angio-

genesis and matrix turnover to extrinsic factors due to

infection and trauma, contribute to interfere in the mecha-

nisms underlined the healing of the wounds [2]. In addition, it

is well known that ageing is associated with a delay in the rate

of the healing process [3,4]. The cutaneous wound healing

process goes through several sequential phases including cell

migration and proliferation, and extracellular matrix (ECM)

deposition and remodeling. Initiation of wound repair is

associated to the release of signalling molecules, such as

cytokines, chemokines and growth factors [5]. Furthermore,

the ability of keratinocytes to migrate into the wound area is

extremely crucial during this process [6] and several hours

following an injury to the skin, re-epithelisation is initiated,

with keratinocytes migration from the wound edge into the

wound clot [7]. In healthy epidermis, keratinocytes are not

activated and they slowly proliferate in the basal layer and

differentiate in the suprabasal layers. When an injury occurs,

keratinocytes are prepared to respond very quickly by produc-

ing sentinel molecules ready to signal promptly that the tissue

needs to become activated. Keratinocytes are capable of

following an alternative differentiation route, known as

regenerative differentiation, as a physiological adaptation of

epidermal homeostasis. Regenerative differentiation is char-

acterised by an overexpression of several proteins and, in

particular, K6 is tightly associated with the keratinocyte

phenotype of regenerative differentiation in vivo [8].

Metalloproteinases (MPs) are indispensable for the com-

pletion of the wound healing process, and increased levels of

MMP-9 have been identified in many chronic wound types [9].

It has also been observed that MMP-9 (gelatinase B), a zinc-

dependent endopeptidase, is up-regulated in burn wounds

[9,10]. Interestingly, one of the major substrates of MMP-9 is

collagen IV, an essential component of basement membranes

(BMs). Studies in mice suggest that type IV collagen is

indispensable for structural integrity and functions of the

BM [11] and for the interaction of BMs with cells [12].

CD44 molecules are a family of transmembrane cell

adhesion glycoproteins that are involved in cell–cell and

cell–matrix interactions, leucocyte homing and activation,

cellular migration, ECM assembly and cytokine binding and

activation [13,14]. It has been observed that CD44 showed

the highest expression levels on the plasma membranes of

the hyperplastic cells [15]. In addition, it has been suggested

that CD44 may have functions in the epidermis other than

those related to hyaluronan (HA), such as binding growth

factors [16] or MPs [17]. HA has been shown to influence

keratinocyte proliferation and migration, and epidermal

wound healing through its cell surface receptors such as

CD44 [18].

Animal models make it possible to study the wound

healing process in great detail and allow to us overcome

several ethical considerations that limit the use of human

volunteers. Considering that full-thickness wounds, like

third degree burn wounds, heal leading to excessive

scarring.

Since age-related differences in wound healing have been

clearly documented [19] but little is known about wound

healing mechanisms after burns, our aim was to investigate

the burn wound healing process in the aged and young rats.

We compared the histological features and the immunohisto-

chemical expression of markers for proliferation, differentia-

tion, re-epithelialisation and dermal repair (MMP-9, collagen

IV, K6 and CD44).

2. Materials and methods

2.1. Animals

Twenty young (7–10 months, weighting 220–320 g) and 20

old (19–28 months, weighting 340–420 g) male Wistar rats

were selected for this study. All animals were housed in

individual cages under constant temperature (22 8C) and

humidity with 12-h light/dark cycle, and had access to

chow and water as much as desired throughout the

study. The study was approved by the animal research

Ethics Committee of the I.N.R.C.A. – I.R.R.C.S., Ancona,

Italy.

2.2. Experimental protocol

Twelve young and 12 old rats were used as models of wound

healing burned skin and eight young and eight old animals

were used as controls of unwounded skin of each age.

Rats were anaesthetised by intra-muscular injection of

ketamine/xylazine (40 and 13 mg kg�1, respectively) and the

back was shaved and washed with povidon iodide propa-

nolol solution. A copper bar (12 mm � 12 mm) heated in

boiling water (100 8C for 10 min) was placed on the

paraspinal site of each animal for 40 s without pressure.

Only the weight of the block was used to create the burns.

No pressure was added. After reheating the probe in boiling

water, a second burn was made symmetrically on the other

side of the back. Bar application resulted in two full-

thickness burns (zone of coagulation). In order to avoid

variations, one person (FO) created all the burns. The rats

were then resuscitated with 1 ml of Ringer’s lactate solution

administered by an intra-peritoneal injection and returned

to their cages. In order to avoid infections and/or damages,

the wounds were protected by a band of cloth.

To mimic the clinical situation in burned patients, 48 h

after the injury surgical debridement was performed in all

the animals with isoflurane general anaesthesia to reduce

the high risk of wound colonisation. During the surgery,

the animals were maintained under body controlled

temperature (36/37 8C) using a homeothermic blanket

(Harvard apparatus). The wounds were left to heal by

secondary intention (i.e., the wound edges were not closed

by sutures). To protect the wounds from outside contami-

nation and infection, a sterile hydrated gauze was used. The

animals were returned to individual cages and examined

daily.

Each dressing change was made during general anaesthe-

sia with isoflurane. After each measurement, the dressing is

removed and the wound is flushed with sterile bandages

physiology. At 7, 14 and 21 days after burns, tissue specimens

were collected from each treated area. Moreover, specimens

were collected from the unwounded rat group. All the

samples were processed for morphological analysis. All the

Table 1 – Score of morphological features.

Score Re-epithelialisation Granulation tissue Collagen deposition

0 Trace and focal migrating Trace None

1 Migrating Hypocellular and none vessel Trace

2 Partial Many cells and few vessels Slight

3 Hypertrophic and partial stratum corneum Many fibroblasts, some fibers and some vessels Moderate

4 Complete More fibers, few cells Marked

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7778

rats were sacrificed at the end of the experiment with excess

anaesthetic.

2.3. Excision of skin biopsies

All skin samples were frozen in liquid nitrogen and stored at

�70 8C. Five micrometer tissue sections, including the epider-

mis, the dermis and the subcutaneous panniculus carnosus

muscle, were cut by a microtome, air-dried overnight and then

fixed in acetone for 10 min.

2.4. Histological examination

In order to assess at the histologic level the healing process

of burn wounds, the sections were stained with haematox-

ylin and eosin and evaluated at 7, 14 and 21 days after

injury. We also examined histological features between

aged and young unwounded skin. All subsequent analyses

were performed simultaneously by two investigators (GL

and AZ), blinded to treatment, using a double-headed light

microscope. Images were captured with a Nikon DS-Vi1

digital camera (Nikon Instruments, Europe BV, Kingston,

Surrey, England).

The specimens were assessed for progression of new

epithelium, inflammation, vascular responses and the forma-

tion of collagen in the wound defect. The following morpho-

logical features, such as degree of re-epithelialisation,

granulation tissue formation and collagen organisation, were

scored as described in Table 1. Scores were given according to

a system reported previously to evaluate maturity of wound

repair [20].

2.5. Immunohistochemistry

The unwounded skin and the healing process levels at 21 days

after burns were evaluated for all samples also by immuno-

histochemistry.

Sections were incubated overnight at 4 8C with the

following monoclonal antibodies: anti-collagen type IV (dilut-

ed 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-

MMP-9 (diluted 1:300, Abcam, Cambridge, UK), anti-K6 (NCL-

CK6, diluted 1:20, Vision Biosystems Novocastra, Newcastle,

UK) and anti-CD44 (diluted 1:50, Vector Laboratories, Burlin-

game, CA, USA).

The reaction was revealed using the streptavidin–biotin–

peroxidase technique according to the manufacturer’s

instructions (Dako-Envision Plus/HRP peroxidase kit, Dako

SpA, Milan, Italy).

Sections were incubated with 3,30-diaminobenzidine (Sig-

ma–Aldrich, Milan, Italy) (0.05 diaminobenzidine in 0.05 M Tris

buffer, pH 7.6 and 0.01% hydrogen peroxide) and counter-

stained with Mayer’s haematoxylin (Bio-Optica SpA, Milan,

Italy). Negative controls were performed by substituting the

primary antibody with non-immune serum.

We evaluated: MMP-9 immunostaining in epithelial cells

and connective cells, K6 and CD44 immunostaining in

epithelial cells and collagen type IV at the BM level. Type IV

collagen expression was evaluated on the basis of linearity

and continuity of the BM surrounding the epithelium. The

number of MMP-9, K6 and CD44 positive cells was counted

using NIS Elements BR 3.22 imaging software (Nikon Instru-

ments, Europe BV, Kingston, Surrey, England) (field: 0.07 mm2,

magnification: 400�) in at least 10 fields per sample and

quantified as a percentage of the total counted cells. The fields

were randomly selected evaluating the most positive, moder-

ately and less positive areas. All counts were performed

simultaneously by two investigators blinded to the treatment

(GL and AZ), using a double-headed light microscope. Both had

to agree on the count of cell positivity. Images were captured

with a Nikon DS-Vi1 digital camera (Nikon Instruments,

Europe BV, Kingston, Surrey, England).

2.6. Statistical analysis

Statistical analyses were performed using Statistical Package for

Social Sciences (SPSS) 16 (SPSS Inc., Chicago, IL, USA). Signifi-

cance was set at P < 0.05. We evaluated the score of morpho-

logical and features related to wound repair and the

immunohistochemical staining. Continuous variables that were

normally distributed were compared by the analysis of variance

(ANOVA) test and presented as means and standard deviations.

Other continuous variables (wound healing scores) that depart-

ed from a normal distribution were presented as means and

standard deviations (median, minimum–maximum) and com-

pared by the non-parametric Mann–Whitney U test.

3. Results

3.1. Histological examination of excised tissues

3.1.1. Unwounded young and aged rat skinThe unwounded skin of aged rats showed major differences in

the epidermis, which was thinner compared to that of young

rats (Fig. 1(a) and (b)). Moreover, the papillary ridges became

flattened with age, resulting in decreased surface contact

(Fig. 1(c) and (d)). In the dermis, we did not find significant

differences between the two groups.

3.1.2. Wounded young rat skinAt 7 days after burn, biopsies showed severe damage

extending through the dermis to the panniculus carnosus

Fig. 1 – Histology of unwounded skin in young and aged rats. The skin of aged rat showed major differences in the epidermis

(haematoxylin and eosin; scale bars: 100 mm).

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7 779

and the surface of the burn was filled with inflammatory cells.

The dermis also showed full-thickness damage (Fig. 2(a)). We

observed poor re-epithelialisation, persistence of abundant

fibrinous exudation and a reduced accumulation of granula-

tion tissue with few cells and vessels (Fig. 2(b)). The

connective tissue of the upper portion of the dermis

demonstrated denaturation of collagen fibers. Wound heal-

ing score 1.

At 14 days after burns, biopsies showed incomplete re-

epithelialisation, with hypertrophic epidermis, a reduction of

fibrinous exudation, an increase in the granulation tissue with

many cells and some fibers and moderate deposition of

collagen (Fig. 2(c) and (d)). Wound healing score 2/3.

At 21 days after burns, biopsies showed robust epidermal

coverage with an increased degree of stratification (Fig. 2(e)).

The epithelium was poorly hypertrophic, almost everywhere

with stratum corneum. We observed thick granulation tissue

and regular collagen deposition (Fig. 2(f)). Wound healing score

3/4.

3.1.3. Wounded aged rat skinAt 7 days after burns, the injury was evident by the

detachment of the epidermis from the dermis. In addition,

the ECM in the wound area was disrupted (Fig. 3(a)). We

observed abundant fibrinous exudation and poorly organised

granulation tissue with a robust inflammatory response

(Fig. 3(b)). Wound healing score 0/1.

At 14 days after burns, biopsies displayed an overall

impairment of the wound healing process (Fig. 3(c)), highlight-

ed by poor re-epithelialisation, fibrinous exudation, a greater

accumulation of granulation tissue with many cells and few

fibers, a robust inflammatory response and trace of collagen

organisation (Fig. 3(d)). Wound healing score 1/2.

At 21 days after burns, biopsies showed a continuous

epithelial lining (Fig. 3(e)) but poorly organised in well

differentiated layers (Fig. 3(f)). We observed a sensible

reduction of fibrinous exudation, an increase in the granula-

tion tissue and a higher degree of collagen deposition.

3.1.4. Wound healing score 2/3In summary, haematoxylin and eosin staining of the wound

tissue of young and old rats showed that wound closure was

markedly progressed from the day 7, but in old rats it

progressed more slowly (Table 2). Regeneration and migration

of epithelial cells in young rats progressed noticeably before

day 21; however, in old rats, it was not observed at the same

time.

At 21 days in old rats, the epithelium appeared less organised

in comparison with young rats (P = 0.019), while granulation

tissue formation and collagen deposition were similar (Table 3).

3.2. Immunohistochemistry

The results are summarised in Table 4.

3.2.1. Presence of BMTo assess the presence of BM, the sections were stained

with antibodies directed against collagen type IV. In

unwounded (Fig. 4(a) and (b)) and wounded skin of young

rats (Fig. 5(a)) collagen type IV was similarly localised at the

dermo-epidermal junction and in the basal lamina of

capillaries: we observed an interrupted BM. Conversely,

wounded aged rats showed focal collagen IV expression at

the dermo-epithelial junction: so the BM was not fully

reconstituted.

3.2.2. MMP-9In unwounded young and aged skin, we detected faint MMP-9

staining at the basal layer (23.7 � 2.6 and 22.0 � 3.3 respec-

tively, P = 0.067) and in dermis (25.2 � 2.3 and 24.3 � 1.98

Fig. 2 – Histology of the young rat burn wound at 7, 14 and 21 days after burns (haematoxylin and eosin; scale bars: 100 mm).

We observed: at 7 days (a, b) severe damage extending through the dermis to the panniculus carnosus and a poor re-

epithelialisation; at 14 days (c, d) incomplete re-epithelialisation with hypertrophic epidermis and an increase in the

granulation tissue; at 21 days (e, f) a robust epidermal coverage with an increased degree of stratification, thick granulation

tissue and regular collagen deposition.

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7780

respectively, P = 0.315); we observed no age-associated change

for MMP-9 staining in epithelium and in dermis between the

two groups (Fig. 4(c) and (d)).

In wounded skin of young rats, we observed faint MMP-9

expression at the basal layer level (30 � 2.15) (Fig. 5(c)). In old

rats, epithelial MMP-9 expression was significantly increased

with respect to the young rats (60.6 � 1.6; P = 0.000) and was

localised at basal and spinous layers (Fig. 5(d)). At the dermal

level, we observed no differences between the two groups

(30.2 � 4.6 vs. 30.0 � 3.2).

Fig. 3 – Histology of the old rat burn wound at 7, 14 and 21 days after burns (haematoxylin and eosin; scale bars: 100 mm). We

observed: at 7 days (a, b) evident detachment of the epidermis from the dermis and the extracellular matrix disruption in

the wound area; at 14 days (c, d) overall impairment of the wound-healing process highlighted by a poor re-

epithelialisation; at 21 days (e, f) a continuous epithelial lining but poorly organized, an increase in the granulation tissue

and a higher degree of collagen deposition.

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7 781

We observed significant increased epithelial and dermal

MMP-9 expression in wounded skin compared to unwounded

skin both in young and aged rats (P = 0.000).

3.2.3. K6In unwounded young and aged skin, there was no K6

expression except in the hair follicle where it was faint and

focal (Fig. 4(e) and (f)).

At 21 days after burns, young and oldrats showed cytoplasmic

K6 expression in suprabasal keratinocytes (50.2 � 5.20) (Fig. 5(e)).

In old rats, K6 expression was significantly increased with respect

to young rats (70.3 � 3.2, P = 0.000) (Fig. 5(f)).

3.2.4. CD44In unwounded aged skin, CD44 expression (28.6 � 3.5) was

significantly lower (P = 0.000) than in the young rats (38 � 2.0)

Table 2 – Wound-healing score (according to the criteriashown in Table 1 in young and old rats after burns.

Rats 7 daysafter burn

14 daysafter burn

21 daysafter burn

Young 1 2/3 3/4

Old 0/1 1/2 2/3

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7782

(Fig. 4(g) and (h)), and the majority of basal cells exhibited a

membranous staining pattern. On the contrary, the lower

spinous layer showed a more cytoplasmic staining pattern

(Fig. 4(g)).

At 21 days after burns, in the old rats CD44 expression was

significantly increased (60.5 � 1.85; P = 0.000) compared to

young rats (40.1 � 2.50) where the expression was found

mainly in the basal layer of epidermis at the membrane level

(Fig. 5(g)), while in the old rats it was detected both in the basal

and spinous layers (Fig. 5(h)).

When we compared wounded skin to unwounded skin, we

observed a significantly increased CD44 expression both in

wounded young (P = 0.042) and aged rats (P = 0.000).

4. Discussion

Burn wound healing is a complex process consisting of a stage

of inflammatory phase, delayed cell death, formation of

Table 4 – Immunohistochemical expression of Collagen type Iyoung and old rat.

Rat model Collagen type IV MMP-9 (% positi

Basementmembrane

Epithelial cells(mean � S.D.)

D(m

Unwounded young skin Regular 23.7 � 2.6 25

Unwounded old skin Regular 22.0 � 3.3 24

Wounded young skin Regular 30.0 � 2.15 30

Wounded old skin Not fully regular 60.6 � 2.5 30

ANOVA test Wound young vs.

wound old

P = 0.000

U

vs

P

Unwound skin vs.

wound skin

P = 0.000

–: no staining; wound: wounded skin; unwound: unwounded skin.

Table 3 – Summary of wound healing parameters at day 21 p

Rat model (number) Epithelialisation score Gr

Young rats (10) 3.20 � 0.50

(3.20, 2.30–4.10)

Old rats (10) 2.70 � 0.30

(2.83, 2.30–3.0)

Mann–Whitney U test P = 0.019

Values are shown as mean � standard deviation (median, minimum–ma

granulation tissue, matrix formation and remodeling [1]. In

a previous study, it was suggested that in the wound healing

process, age-related differences could be present [19]. Our

histologic results supported these data indicating that in old

rats the wound remodeling, after 2 weeks, still exhibited an

immature healing response, characterised by an incomplete

epidermal lining, persistence of abundant fibrinous exudation,

an immature granulation tissue, full of inflammatory cells. On

the contrary, the young rat group showed hypertrophic

epidermis overlying a granulation tissue that was still highly

cellular but moderately organised. At 21 days after burn, the

wounds in young rats were extensively resurfaced and the

neodermis progressively presented a regular collagen deposi-

tion. The appearance of the wound bed in old rats, at the same

time, had progressed but with an immature aspect, repre-

sented by a presence of epithelial coverage with no stratifica-

tion and still nascent neodermis, resulting in a delayed healing

process with respect to the young rat group.

It is well known that after injury keratinocytes undergo

changes in keratin expression with down-regulation of

differentiation-specific keratins and expression of inducible

keratins, such as K6 [21]. K6 exhibits a complex regulation

that includes constitutive expression in specific compart-

ments within epithelial appendages, but are excluded from

epidermis [22]. Together with its partner, K16, it shows

enhanced expression in stratifying keratinocytes during

hyperproliferative and malignant transformation and in

V, MMP-9, K6 and CD44 in unwounded and wound skin of

ve cells) K6 (% positive cells) CD44 (% positive cells)

ermal cellsean � S.D.)

Epithelial cells(mean � S.D.)

Epithelial cells(mean � S.D.)

.2 � 2.3 – 38.0 � 2.0

.3 � 1.2 – 28.6 � 3.8

.2 � 4.6 50.2 � 5.20 40.1 � 2.5

.0 � 3.2 70.3 � 3.2 60.5 � 1.85

nwound skin

. wound skin

= 0.000

Wound young vs.

wound old

P = 0.000

Unwound young

vs. old

P = 0.000

Wound young

vs. wound old

P = 0.000

Unwound skin

vs. wound skin

P = 0.000

ost-wounding in rat models.

anulation tissue Score Collagen organization Score

2.85 � 0.47 2.20 � 0.57

(2.88, 2.30–3.40) (2.20, 1.50–3.0)

2.77 � 0.52 1.70 � 0.68

(2.75, 2.0–3.80) (1.65,0.70–2.50)

P = 0.628 P = 0.078

ximum).

Fig. 4 – Immunohistochemical expression of collagen type IV, MMP-9, K6 and CD44 in unwounded skin of young and old rats

(immunoperoxidase; scale bars: 100 mm). We observed: interrupted basement membrane in young (a) and aged skin (b); no

age associated change for MMP-9 staining in epithelium and in dermis (c, d); no K6 expression, except in hair follicles, in

both rat groups (e, f); lower CD44 expression in aged rat skin (g) compared to the young (h) and especially localized in the

basal layer.

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7 783

Fig. 5 – Immunohistochemical expression of collagen type IV (a, b), MMP-9 (c, d), K6 (e, f) and CD44 (g, h) in the burn wound of

the young and old rats at 21 days after burns (immunoperoxidase; scale bars: 100 mm). Young rats (a) showed an

interrupted basement membrane while aged rats (b) showed focal collagen IV expression at the dermo-epithelial junction

and not fully reconstituted basement membrane. In old rats epithelial MMP-9 (d), K6 (f) and CD44 (h) expression was

significantly increased with respect to the young rats (c, e, g).

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7784

primary keratinocytes cultures [23–25]. According to the

literature, our results revealed the presence of K6 in

suprabasal cells in the wounded skin of young and old rats;

moreover, in old rats the cells positive for K6 were

significantly increased. This major expression could be

associated to the delay in the regeneration of epidermis with

respect to young rats. Oender et al. [26] demonstrated an up-

regulation of genes for K6 in aged human skin donors.

However, mixed results have been obtained in previous

studies involving K6-null mice models. Unlike us, Wojcik

et al. [27] reported that loss of K6a causes a delay in the

epithelialisation of partial-thickness skin wounds in vivo and

the response of skin tissue to full-thickness injury was not

altered in the K6a/K6b-null animals able to survive in the

genetic background used [28]. Wong and Coulombe [29]

investigating the role of keratin intermediate filament (IF)

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7 785

during the epithelialisation of skin wounds using a keratin 6a

and 6b (K6a/K6b)-null mouse model showed that null kerati-

nocytes exhibited an enhanced epithelialisation potential due

to increased migration. They observed fragility of the K6a/K6b-

null epidermis that was confirmed when applying trauma to

chemically treated skin. K6a/K6b-null keratinocytes located at

the edge of acute wounds exhibited lysis typical of keratin

deficiency, presumably in response to stress. The enhanced

migratory potential exhibited by K6a/K6b-null skin keratino-

cytes, thus, appears counterintuitive considering that evolution

has selected for the involvement of K6 proteins after injury.

They proposed that the enhanced migratory properties shown

by K6a/K6b-null keratinocytes in the idealised setting of skin

explant culture, thus, appeared negated by their fragility once in

the harsher environment of a wound in vivo; therefore, the

alterations in IF gene expression after tissue injury fostered a

compromise between the need to display the cellular pliability

necessary for timely migration and the requirement for

resilience sufficient to withstand the rigours of a wound site.

In view of these considerations, we think that the over-

expression of K6, observed in our aged rats, could be considered

an attempt to ameliorate the delay in aged wound healing.

MMP-9 plays a complex role in wound healing interfering in a

series of responses that include inflammation, ECM remodel-

ing, angiogenesis and epithelial regeneration [30]. Immature

keratinocytes produce matrix metalloproteinases (MMPs) and

plasmin, which enables their dissociation from the BM and also

facilitates their migration. Previous study have documented

that MMP-9 increased in burned skin in response to injury and

the need for tissue remodeling [31]. Kyriakides et al. suggested

that MMP-9 is required for normal progression and wound

closure [32]. Our immunohistochemical investigation showed

no age-associated change for MMP-9 staining in the epithelium

and in the dermis between the unwounded young and aged

skin, but it was significantly increased and its localisation was

different in the epithelium of the wounded old rats compared to

the young ones. This result is consistent with our histological

findings of different grade of wound maturity, being signifi-

cantly delayed in old animals. In fact, in young rats we observed

MMP-9 expression only in the basal layer of the epidermis,

whereas it was present in basal and spinous layers in old

animals. It has to be considered that in the naturally aged skin of

old subjects in comparison with young skin, the expression of

procollagen a1(I) messenger RNA (mRNA) is lower, and that the

level of MMP-1 and the activities of MMP-2 and MMP-9 tend to be

higher [33]. Thus, we could hypothesise that, in the aged rats,

MMP-9 expression was dramatically increased not only in

response to burns but also to address to a specific need of tissue

remodeling that in the old rats progresses slowly. Ashcroft et al.

[34] created 4-mm punch biopsy excisional wounds and

observed their healing: in all their subjects, MMP-9 was

expressed during normal wound healing but, according to

our findings, they also noted that there was dramatically more

MMP-9 expressed in older patients, who healed more slowly,

than in younger patients. As Ashcroft et al. suggested we

typically associate advanced age with delayed wound healing,

and this is related with higher levels of MMP-9. The exact

mechanism by which MMP-9 increases in wound healing of

aged skin with consequent delayed healing is not well

established. Relative to the significant amount of research on

ageing and wound repair, little work has been done on the

impact of increasing chronological age on wound repair. Most of

these studies noted the common observation that wounds in

the elderly heal but they usually take longer. A number of

hypotheses are consistent with the delayed healing response in

older animals. Cells that enter wounds of older animals may

have impaired responses to normal stimuli may be reduced, cell

may have impaired migratory or proliferative capacity [35], or

there may be few cells capable of responding. Ageing-associat-

ed alterations in the physiologic environment and/or in the cells

themselves could result in a prolonged healing response if the

repairing cells fail to respond correctly or as rapidly to the

wound stimulus. The elevated activity of MMP-9 detected in

aged skin wound healing could represent an age-related

phenotypic shift that results in appropriate response to normal

stimuli.

Thus, the age-associated healing delay would seem to be a

function of the early phase of wound repair: inflammation.

Trengove et al. [36] found that MMP-9 was massively elevated

in the chronic wound environment and recently Reiss et al.

hypothesised that it is the prolonged and excessive production

of this protease that leads to disordered wound healing [9]

associated to the tissue inflammation and a worse clinical

course. MMP-9 synthesis, activation and activity are, in part,

regulated by tumour necrosis factor-a (TNF-a). Normally,

MMP-9 facilitates re-epithelialisation but in situations where

inflammation continues, TNF and subsequently MMP-9 persist

resulting in the loss of keratinocytes, a shorter epithelial

tongue and a delay or failure of wound closure [37]. The

complicated interplay between matrix attachment molecules,

cytokines, inhibitors and activators in the wound environ-

ment becomes disjointed and unbalanced in the wound that

fails to heal.

It is well known that proper re-epithelialisation requires

the re-establishment of the BM that provides for the

demarcation and cohesion between the epidermal and dermal

layers. Collagen IV is an essential component of BMs and is one

of the major substrates of MMP-9. An intact BM has been

shown to be important for the adherence of the epidermis to

the dermis and for differentiation of keratinocytes [38].

Immunohistochemical staining with type IV collagen antibody

revealed in young rats the presence of an intact and

continuous BM while in the aged ones it was not fully

reconstituted. This finding suggested that in aged rats MMP-9

overexpression may prevent the reestablishment of the

dermal–epidermal junction and thereby it limits epithelial

migration and wound closure.

Since the expression process and the regulation mechanism

of MMP-9 have already been known, some therapeutics to

improve burn wound healing could be available by regulating

the expression and activation of MMP-9. In this regard, in a

previous study [39], we showed that in staphylococcal infected

burns tigecycline, a tetracycline derivative, exhibited a high

antimicrobial activity in association with a significant decrease

of MMP-9 expression, accelerating wound healing.

HA also plays a crucial role in wound healing and

inflammation. It has been shown that it influences prolifera-

tion and migration of other cell types through its cell surface

receptor CD44 [40]. CD44 expression is regulated at the same

time as the expression of HA [15] and the appearance of HA in

b u r n s 3 9 ( 2 0 1 3 ) 7 7 6 – 7 8 7786

the dermis and epidermis parallels the histolocalisation of

CD44. According with our results in unwounded aged skin, the

most dramatic histochemical change observed in senescent

skin is the marked decrease in epidermal HA [41]. This

decrease contributes to the apparent dehydration, atrophy

and loss of elasticity that characterises aged skin [42]. It is well

known that HA levels increase after skin injury and in early

phase of wound healing [43]. Moreover, the wounding-induced

up-regulation of HA synthesis is not limited to mesenchymal

cells, but is very prominent also in the epithelium [44] and in

healing wounds CD44 expression was found in migrating

keratinocytes [45]. Strong epidermal CD44 staining was seen

mostly in hyperplastic skin areas with signs of tissue damage.

Moreover it has been shown that, limiting the role of CD44, it

may improve healing parameters in adult tendon injury, [40]

and the absence of CD44 in skin wounds was associated with a

delayed early wound closure [46]. It is clear that the function of

HA in tissue repair is complex and cannot be specifically

attributed to any single one of its many properties. In burned

epidermis of our aged rats, the expression of CD44 was higher

and diffuse compared to the young rats. Its tissue distribution

was in the basal and spinous cell layers in aged tissue and only

in the basal layer in young tissue. Presumably, basal

keratinocyte HA is involved in cell-cycling events, whereas

the secreted HA in the upper outer layers of the epidermis in

mechanisms of disassociation and eventual sloughing of cells

[42]. The mechanisms by which HA and CD44 are involved in

the quality of the aged skin wound healing is however not

clear but it is very like to be a result of a persistent HA-rich

environment that may affect cell–cell and cell–matrix inter-

actions. This in turn may lead to different activation and

control of various cell populations in comparison to the young

skin environment when the HA-rich environment during

tissue repair is transient.

In conclusion, the baseline data from the present investi-

gation on unwounded skin could be useful to better under-

stand the overexpression of the wound healing markers,

MMP-9, K6 and CD44, found in the burned aged rats compared

to young animals. Our results underline that delayed wound

healing observed in old animals might represent an age-

dependent response to injury where K6, MMP-9 and CD44 play

a key role. Moreover, our data may help to elucidate aspects of

the human skin ageing healing process, suggesting that in the

aged tissue several factors contribute to the delayed wound

healing capacity, particularly after burns. It is therefore

possible to hypothesise that the modulation of these factors

could lead to a better and faster recovery of skin damage in

elderly and at last save money on national health care.

Conflict of interest statement

The authors have no conflict of interest to declare.

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