sequential changes in trace metal, metallothionein and ...€¦ · 2.11.98). other metals found in...

J. Anat. (1999) 195, pp. 375–386, with 12 figures Printed in the United Kingdom 375

Sequential changes in trace metal, metallothionein and

calmodulin concentrations in healing skin wounds

A. B. G. LANSDOWN,1 B. SAMPSON1 AND A. ROWE2

"Departments of Clinical Chemistry, Imperial College School of Medicine and Charing Cross Hospital, and #Clinical

Pharmacology, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, UK

(Accepted 4 May 1999)

Metalloenzymes have an important role in repair and regenerative processes in skin wounds. Demands for

different enzymes vary according to the phase in the healing cascade and constituent events. Sequential

changes in the concentrations of calcium, copper, magnesium and zinc were studied in the incisional wound

model in the rat over a 10 d period. Copper levels remained low (! 10 µg}g dry weight) throughout, but

calcium, magnesium and zinc increased from wounding and peaked at about 5 d at a time of high

inflammation, granulation tissue formation and epidermal cell proliferation. Metal concentrations declined

to normal by 7 d when inflammation had regressed, re-epithelialisation of the wound site was complete and

the ‘normalisation’ phase had commenced. Although the wound was overtly healed by 10 d, the epidermis

was still moderately hyperplastic. In view of competitive binding of trace metals at membrane receptors and

carrier proteins, the ratios or balance between these trace metals was examined and the significance is

discussed. Using immunocytochemistry, we demonstrated increases in metallothionein immunoreactivity as

an indication of zinc and copper activity in the papillary dermis and in basal epidermal cells near the wound

margin 1–5 d after wounding. This is consistent with metalloenzyme requirements in inflammation and

fibrogenesis. Calmodulin, a major cytosolic calcium binding protein was highest in maturing keratinocytes

and in sebaceous gland cells of normal skin; it was notably more abundant in the epidermis near the wound

margin and in re-epithelialising areas at a time when local calcium levels were highest.

Key words : Skin wounds; repair ; trace metalloenzymes; metal ion balance; zinc ; calcium; copper ; magnesium.

Wound healing in the skin and in other tissues of the

human body depends upon the availability of com-

petent cells to carry out repair processes, appropriate

activation by hormones, growth factors, cytokines

and chemotactic factors, and a microenvironment

favouring cell movement, proliferation and functional

maturation (Grotendorst, 1992; Hunt & Hussain,

1992). Metal ions and metalloenzymes feature promi-

nently in this wound healing environment and

experimental and clinical studies are available to show

that deficiencies in zinc, copper, calcium, iron or

magnesium are potential causes of abnormal homeo-

static mechanisms and impaired wound healing

(Moynahan, 1974; Lansdown, 1995). Although at-

Correspondence to Dr A. B. G. Lansdown, Department of Clinical Chemistry, Division of Investigative Sciences, Imperial College School of

Medicine, Charing Cross Campus, London, W6 8RP, UK.

tention recently has focused upon the role of metallo-

proteinases in the degradation of extracellular matrix

in the early phases of wound healing (Nwomeh et al.

1998), it is to be expected that the demands for these

and other metalloenzymes will vary greatly as the

profile of wound healing proceeds. Surprisingly, the

action and interaction of trace metals in the skin

under normal conditions is not well documented, and

sequential changes in the healing wound are not

published for any species (Lansdown, 1995).

Much of the trace metal requirement for the body is

derived from the diet, but many topical dressings,

healing creams and other commercial preparations

containing zinc and calcium are now available to

improve wound healing. There is a general lack of

published data to show how much ‘active ’ metal ion

is absorbed percutaneously from these preparations,

and we can only speculate on how much additional

ion is necessary to advance the wound healing process.

Much attention has centred upon the value of

supplementary zinc in wound healing; zinc is a

constituent of more than 70 enzyme systems, many of

which are involved in wound healing (Lansdown,

1993, 1996). However, whereas zincated creams can

be beneficial under some circumstances, excessive zinc

may retard healing (Lansdown & Sampson, unpub-

lished studies). This is attributed to the fact that zinc,

copper and calcium interact at binding sites on carrier

proteins. Excess of one ion is liable to inhibit essential

processes modulated by others (Klevay, 1975; Heng et

al. 1993). It follows that any xenobiotic ion absorbed

into the skin that impairs the availability of zinc or

other trace metals, is a potential cause of delayed or

nonhealing of wounds (Lansdown, 1996).

Recent studies have demonstrated that concen-

trations of trace metals in normal skin are specific for

the region of the body, its keratinisation patterns and

contact with the environment (Lansdown, 1985, 1995;

Lansdown & Sampson, 1997). Zinc, calcium and

magnesium concentrations are highest in areas of

pressure keratinisation and parakeratosis, which are

normally characterised by high epidermal cell turn-

over. In the healing wound, the demand for trace

metals, mainly in the form of metalloenzyme com-

plexes, can be expected to vary according to the

sequential events of haemostasis, inflammation}granulation tissue formation, cell proliferation and

normalisation of the wound site (Lansdown, 1995).

Whereas demands for calcium will be high during

haemostasis and periods of keratinocyte proliferation

and maturation (Menon et al. 1985), zinc metallo-

enzymes will feature in matrix metalloproteases, RNA

and DNA polymerases and enzymes involved in

protein synthesis, mitosis, collagenesis and tissue

remodelling (Halstead et al.1974). Copper exhibits a

ubiquitous role in cellular metabolism in the skin, but

is notable as a component in lysyl oxidase necessary in

elastic and collagen fibre cross-linking (Chou et al.

1968).

Present studies were conducted with a view to

Fig. 1. Metallothionein distribution in the basal layer of the epidermis of the back skin of a young adult Sprague–Dawley rat. Immuno-

cytochemistry using antimetallothionein antibody (DAKO, Copenhagen). ¬150.

Fig. 2. Calmodulin distribution in the outer layers of the epidermis of the back skin of a young adult Sprague–Dawley rat. Immuno-

cytochemistry using anticalmodulin polyclonal antibody (Santa Cruz Biotechnology Inc, CA.) ¬150.

Fig. 3. Incisional wound site in the rat after 24 h showing the inflammatory cell infiltrate at the wound margin and debris in the wound core.

Note lack of inflammation in adjacent tissue. H & E. ¬75.

Fig. 4. Narrow tongue of epidermal cells migrating between the acute inflammatory cells and wound debris at deeper aspects of the 48 h

incisional wound. H & E. ¬150.

identifying sequential changes in the mineral and trace

metal content of incisional wounds in the rat model,

and correlating these with cellular events charac-

terising the major events of the wound healing cascade

(Mast, 1992). The rat has been used for many years to

study wound healing profiles in the skin and has

proved a valuable model in which to evaluate the

influence of intrinsic and environmental influences on

repair processes (Lansdown & Pate, 1993). In this

work, we sought to define our experimental model for

use in the evaluation of dietary constituents, topical

preparations or medicated dressings which are po-

tentially able to alter trace metal ion uptake and

metabolism in the wound environment. From earlier

studies, we know that silver can interact with zinc to

advance experimental wound healing in the rat

(Lansdown et al. 1997). We are unclear at this stage,

whether the effect is achieved through applying silver

nitrate or silver sulphadiazine throughout the wound

healing period, or only during phases of high zinc

metalloenzyme activity. In another situation, the

xenobiotic metal cadmium was shown to induce a

marked increase in ‘bound’ zinc in intact skin

(Lansdown & Sampson, 1996). In wounded skin, this

would be expected to seriously impair wound healing

through inhibiting zinc metalloenzymes involved in

matrix degradation, cell proliferation and connective

tissue formation.

Animals

Adult male Sprague-Dawley rats of the CFY strain

(180–200 g body weight) were used in all sections of

this study. They were bred under barrier-maintained

specified pathogen free conditions at the Charing

Cross Campus of Imperial College School of Medi-

cine. They were housed under conditions of 22³2 °C,

45–55% relative humidity, and 12 h d}night cycles as

specified in the Animals [Scientific Procedures] Act,

1986. The animals were housed in groups of 5 in

plastic solid bottomed cages and provided with sterile

dust free sawdust as bedding. Pelletted small rodent

diet (CRM, Special Diet Services, Witham, Essex)

376 A. B. G. Lansdown, B. Sampson and A. Rowe

1 2

3 4Fig. 1. For legend see opposite.

Trace metals and skin wound healing 377

and tap water was provided ad libitum. The mineral

content of this diet includes copper (19 mg}kg),

zinc (65 mg}kg), magnesium (0.22%), and calcium

(0.80%) (Special Diet Services, Information Service,

2.11.98). Other metals found in this diet either as

trace metals or as contaminants are not present in

sufficiently high levels to significantly influence the

metabolism of zinc, calcium, copper or magnesium in

wound repair (Lansdown, 1995).

Experimental details

Incisional full thickness skin wounds (15 mm long)

were made surgically with a scalpel in the closely

shaved middorsal skin of fully anaesthetised rats. A

single middorsal wound was made in each animal.

Skin sites were cleansed with 70% ethyl alcohol and

animals anaesthetised with a single intraperitoneal

injection of Hypnorm (fentanyl}fluanisone) (Janssen

Animal Health) and midazolam combination an-

aesthetic (0.27 ml}100 g body weight). This provided

anaesthesia for " 30 min with good muscle relaxation

(Flecknall, 1987). Wounds were sutured at 3 equi-

distant sites using polyamide monofibre (Ethicon,

Edinburgh, Scotland). To standardise wounding, all

procedures were conducted during the period 09.00–

10.30 h.

Groups of 10 rats received wounds as above and

were euthanised by carbon dioxide asphyxiation after

1, 2, 3, 5, 7 and 10 d. Earlier studies have demonstrated

that rat wounds heal well within 10 d (Lansdown &

Pate, 1993). At autopsy, wound sites were excised,

with normal tissue to within 1.5 mm of the incision

line and samples taken for trace metal analysis,

histology and immunocytochemistry. Wounds from

5 animals for each time period, were cryopreserved at

–20 °C for metal analysis, 2 were preserved in ice-

cold paraformaldehyde (4% w}v in phosphate

buffered saline, PBS) for immunocytochemistry, and 3

fixed in cold (®4 °C) 10% PBS for paraffin wax

embedding and routine histology. Samples (5 mm¬5 mm) of intact skin from an uninjured area of back

skin were cryopreserved for metal analysis as no

useful data exist on trace metal content in normal rat

skin. Histological sections were stained with haema-

toxylin and eosin, or by Masson’s trichrome for

connective tissue.

To examine early changes in the wound, 2 wound

bearing rats were euthanised 12 h after surgery and

the wound site preserved in 10% phosphate buffered

formalin for histology and haematoxylin and eosin

stained sections.

Immunocytochemistry

Dissected skin wound samples were processed through

several changes of sucrose (15% w}v in PBS) over

36 h and then frozen in ‘Cryo-M-Bed’ (Bright Ltd, St

Ives, Cambridge, UK) at ®60 °C. Tissues were

sectioned at 6 µm and immunocytochemistry per-

formed (Rowe et al. 1997) using Vectastain ABC

Elite reagents (Vector Laboratories, Burlingham,

CA, USA) according to the supplier’s protocol. Rat-

adsorbed biotinylated antimouse IgG (Vector Labora-

tories) was used for the detection of IgG primary

antibodies. The chromagen employed was 3,3«-di-

aminobenzene (Vector Laboratories) with Harris’s

haematoxylin counterstain. Monoclonal mouse anti-

metallothionein antibody was kindly donated by

DAKO Laboratories (Copenhagen, Denmark). It was

supplied in liquid form in 0.05 Tris HCl, 15 m

NaN3, pH 7.5, 1% bovine serum albumin (BSA).

DAKO-metallothionein antibody is inhibited specifi-

cally and equally well by glutaraldehyde polymerised

human, horse, sheep or rat metallothioneins (MT-I

and MT-II) suggesting that its is directed against a

single and hightly conserved epitope ((DAKO Infor-

mation Sheet). It was diluted 1:150. Anticalmodulin

goat polyclonal antibody (Santa Cruz Biotechnology,

Santa Cruz, CA, USA) was diluted 1:100. Negative

control sections were incubated with no primary

antibody.

Metal analysis

Tissue samples were dried to a constant weight at

120 °C and then dissolved in concentrated nitric acid

(Aristar Grade, BDH, Poole, Dorset, UK) by heating

overnight at 90 °C. The resulting solutions were

diluted to an appropriate volume using ultrapure

water. Appropriate reference standards were analysed

at the same time (see Lansdown & Sampson, 1997).

Calcium, magnesium and zinc concentrations were

measured by flame atomic spectrophotometry (Uni-

cam 939, ATI Unicam) after dilution with lanthanum

chloride (for calcium and magnesium) or 6% butan-

1-ol (for zinc). Copper was measured by electro-

thermal atomisation flame atomic spectrophotometry

(Unicam 939 with GF90 electrothermal atomiser).

The skin on the back of an adult rat is densely haired

and is devoid of sweat glands, although sebaceous

glands are prominent in association with the nu-

merous hair follicles. The epidermis is 4–6 cells thick

and comprises characteristic layers of basal, spinous,


granular and keratinised cells. Under normal con-

ditions, basal cells express cytoplasmic metallo-

thionein and low levels of calmodulin (Figs 1, 2).

Minimal amounts of metallothionein are present in

more superficial layers of the epidermis, but cal-

modulin is more noticeable in maturing keratinocytes.

The dermis comprises collagen-rich connective

tissue interspersed with a modest vascular supply and

nervous tissue. Under normal conditions this tissue

displays low levels calmodulin and metallothionein.

Macrophages, fibroblasts and the paniculus carnosus

muscle (PCM) in the deeper aspects of the zona

reticularis exhibit immunoreactivity for both proteins.

Healing profiles in the skin wound

Haemostatic and early post wound period (0–12 h)

All animals recovered well from anaesthesia and

exhibited no signs of ill health at any time. No

fatalities occurred. Following surgery, there was a

minimal haemostatic phase (! 30 min). Wounds

dried quickly and showed no appreciable adverse

response to the suture material used. Histological

examination of 12 h wounds showed local oedema in

the region of the incision line but minimal evidence of

inflammatory cell infiltration in marginal areas. The

epidermis at the wound margin was necrotic. Aggre-

gations of red blood cells were evident at all levels

from the surface of the skin to the hypodermis.

Inflammatory and proliferative phase (1–7 d)

Crusting and scab formation occurred along the

wound suture line within 24 h and this persisted for

approximately 5 d. After this time, we noted a

progressive loss of sutures and prominent hair growth.

Where sutures had been lost, insertion marks re-

mained.

By 24 h the wounds exhibited a prominent central

core of cell debris with haemolysed erythrocytes

extending through the epidermis and dermis into the

hypodermis. In the dermis, this mass was separated

from mildly oedematous but otherwise histologically

normal tissue by a zone of acute inflammatory cells

(mainly neutrophils). Initially, this infiltrate was more

pronounced in the reticular zone than in the papillary

area (Fig. 3). The epidermis at the wound margin

consisted of a cap of fragmented and necrotic tissue

adjacent to histologically normal keratinocytes. Mi-

totic activity was not noted at this stage in histo-

logically normal-looking keratinocytes adjacent to the

wound margin. In the deeper reticular dermis, the

severed PCM was associated with infiltrates of

neutrophils, monocytes and a few eosinophils. At this

stage, epidermal concentrations of calmodulin near

the wound margin had increased appreciably and

were present in basal cells, which in intact skin are

usually low in this protein.

Metallothionein levels in the epidermis and dermal

fibroblasts near the wound margin after 24 h exhibited

a marginal increase.

After 48 h, there was a pronounced increase in

the dermal inflammatory cell infiltrate in the wound

margin. Importantly, by this stage mitotic activity was

present in the epidermis at the wound margin and

tongues of epithelial cells had begun to migrate

downward with the effect of separating the central

core of tissue debris and inflammatory cells from the

‘peripheral ’ zone of histologically normal tissue (Fig.

4). There was minimal evidence of an epidermal

basement membrane in the early stages of re-

epithelialisation. A diffuse inflammatory cell infiltrate

and some oedema persisted in the dermis near the

wound margin, this being more obvious in the deeper

reticular region, where vascular dilation was noted.

By 48 h postwounding, dermal inflammatory cell

infiltrates included monocytes, some fibroblasts and

macrophages. Minimal evidence of collagenesis was

seen until after 72 h when the fibroblast population of

the wound site had markedly increased both in the

reticular and papillary regions. We noted a follicular

cell hyperplasia in the region of the wound, such that

follicles cut during surgery were contributing to the

re-epithelialisation process.

The re-epithelialisation process continued until

approximately 5 d after surgery, when the wound

site resembled a crater lined by epithelial cells and

containing a central core of wound debris and

inflammatory cells. In deeper aspects of the crater the

epidermis was not well defined and lacked a clear

basement membrane (Fig. 5). A few chronic inflam-

matory cells were present. Except in these deeper

layers, keratohyalin granules were prominent in the

reformed epidermis and in places extended over up to

60% of the epidermal thickness. In the 5 d wound, the

reformed epidermis was up to 3 times the normal

thickness and mitoses were numerous in the basal

layer, both in the new epidermis and in adjacent areas.

The stratum corneum was identifiable as a thin strand

in the early re-epithelialisation process but by 5 d

extended across the wound site. Some parakeratosis

was present in the deeper regions of the wound crater.

At this time, the re-epithelialised epidermis showed

minimal evidence of the columnar epidermal cell

organisation although more superficial cells were

flattened parallel with the skin surface.


5 6

7 8Fig. 5. Reepithelialised incisional skin wound in the rat after 5 d. Base of the crater to demonstrate the residual inflammatory cell infiltrate

(mainly lymphocytes), fibroblasts and early fibrogenesis. Note the lack of basement membrane at the epidermal-dermal interface. Masson’s

trichrome method. x150.

Fig. 6. Metallothionein distribution in the epidermis and papillary dermis of the incisional skin wound after 24 h. ¬150.


9 10Fig. 9. Rat incisional wound site after 10 d demonstrating the fibrous scar tissue, minimal inflammatory cell infiltration and modestly

hyperplastic epidermis. H & E. ¬75.

Fig. 10. Increased metallothionein concentrations in macrophages in the hypodermis under an incisional wound site in the rat 10 d following

surgery. Immunocytochemistry using DAKO antibody. ¬75.

The dermal inflammatory cell population after 5 d,

was predominantly of fibroblasts and lymphocytes

with macrophages, eosinophils and a few mast cells

present at deeper layers and in the region of the PCM.

Collagenesis was active beneath the wound and in the

papillary epidermis adjacent to the wound site.

Granulation tissue with prominent vascular dilatation

was notable, particularly in the deep dermis and

hypodermis.

The inflammatory and proliferative phases of

wound healing were marked by an increase in

metallothionein in epidermal cells at the wound

margin and in regenerating tissue (Fig. 6). Interest-

ingly, we noted a pronounced increase in metallo-

thionein in fibroblasts and macrophages of the

papillary epidermis in wounds examined up to 5 d.

Fig. 7. Increases in calmodulin activity in the wound margin of the 24 h skin wound. Note the presence of calmodulin reaction product in

basal cells and in all postmitotic cells of the epidermis. Calmodulin antibody is seen in sebaceous gland cells. ¬150.

Fig. 8. Rat incisional wound site after 7 d showing the hyperplastic epidermis, with a prominent granular layer, early evidence of realignment

of epidermal cells and hyperkeratinisation. Scar tissue formation is shown. Masson’s trichrome. ¬75.

Calmodulin in contrast was essentially epidermal in

distribution. It increased markedly over the first 4 d of

re-epithelialisation in the tongues of migrating keratin-

ocytes (Fig. 7). Increased epidermal calmodulin

persisted in the re-epithelialised epidermis well into

the normalisation phase.

Normalisation phase (7–10 d)

Healing in the rat wound from 5 d was associated

with a pronounced decline in the inflammatory cell

infiltrate in and near the wound margin, and a

progressive reduction in the crater formation with loss

of the wound debris and a realignment of epidermal

cells. Thus after 7 d, the wound site was characterised


Fig. 11. Sequential changes in zinc, calcium, copper and magnesium

in the healing wound site.

by a pit-like configuration and lined by a hyperplastic

epidermis with a prominent keratohyalin granulation

and hyperkeratinisation (Fig. 8). We noted a pro-

nounced formation of rete pegs at the epidermal–

dermal interface of the new epidermis, which was up

to 3 times the normal thickness. There was no evidence

of repair in the PCM but collagen deposition}scar

tissue formation was evident at this site. After 10 d,

the normalisation process in the rat wound was well

advanced (Fig. 9), but the epidermis was still

marginally thicker than normal and cellular realign-

ment incomplete. Epidermal mitoses were appreciably

less obvious than during the 3–5 d period but some

follicular cell hyperplasia persisted. Dermal cellularity

was greatly reduced, and blood vessels appreciably

less obvious than at earlier stages. Scar tissue was

prominent.

From 5 d after wounding, metallothionein levels

declined in the epidermis, papillary dermis and

follicular epithelium to normal levels. A slightly

increased level of immunoreactivity was seen in the

region of the PCM and in macrophages in the

hypodermis (Fig. 10). Calmodulin concentrations

were appreciably higher than normal in the hyper-

Fig. 12. Sequential changes in the ratio of zinc :calcium, calcium:

magnesium, and zinc:copper in the healing wound site.

plastic epidermis in maturing keratinocytes but not in

most basal cells.

Trace metal analyses

The mean concentrations of zinc, copper, calcium and

magnesium in uninjured shaved back skin of the

young adult Sprague Dawley rat are illustrated in

Figure 11. In the skin wound site, we noted a

progressive increase in zinc, calcium and magnesium

up 5 d postwounding and then a decline to normal

values by 7 d. Zinc concentrations were marginally

higher than normal 10 d after wounding. Copper

concentrations were very low at all stages in the

wound healing profile, but did show a marginal

increase in the first 2 d.

Because certain metals compete for binding sites on

carrier proteins, it was of interest to examine changes

in the ratio of metal concentrations in the period of

study (Fig. 12). Thus, there was a progressive decline

in the zinc:calcium ratio over first 5 d after wounding.

The zinc:calcium ratio had normalised by 7 d but had

increased further by 10 d. In contrast, the relative

concentration of calcium to magnesium increased to

5 d after wounding but had declined to near normal

levels by 7 d. Although copper levels were low for the

wound healing period studied, the ratio of zinc to


Table. Trace metal concentrations in the middorsal skin of a

young adult Sprague–Dawley rat

Zinc 63.94³11.73 µg}g dry tissue weight

Copper 5.13³1.06 µg}g

Calcium 0.59³0.01 mg}g

Magnesium 0.46³0.03 mg}g.

copper appeared to decline in the early postwound

period (C 1–2 d). They were increased at later stages.

Wound healing in acute skin wounds is a well defined

sequence of events involving cell–cell contact and

cell–macromolecular interactions leading to haemo-

stasis, inflammation (granulation tissue formation),

fibroplasia, neovascularisation, contraction, and re-

epithelialisation of the wound site (Cai et al. 1991).

Repair processes and their associated biology have

traditionally involved the use of in vitro systems and

laboratory animal models which allow the constituent

processes to be studied under controlled conditions

with the avoidance of such variables as genetic

configuration, nutrition and environmental factors

(Mulder, 1991). With new technology and the avail-

ability of specific antisera, it now possible to in-

vestigate more closely the role of cytokines, growth

factors and other modulators in cellular morpho-

genesis, extracellular matrix protein synthesis, and

membrane receptor activity (Mast, 1992; Nickoloff et

al. 1991; Kilcullen et al. 1998). Although the par-

ticipation of metalloenzymes is appreciated, the

present work is the only evaluation to our knowledge,

where sequential changes in trace metal concen-

trations have been correlated with cytological events

in the healing cascade. We assume here that largest

part of the metals present will be as constituents of

metalloenzyme systems (Lansdown, 1995; and Table).

Through sequential histological observations over

the wound healing period, we can appreciate the

duration of phases of haemostasis, inflammation and

granulation tissue formation, proliferation and nor-

malisation (remodelling) in the rat model. However,

whereas we can identify periods of high}maximal

inflammatory activity, fibrogenesis or epidermal cell

proliferation, our model can only provide an approxi-

mate guide to the time of onset of and ‘cut-off’

points for the 4 main phases.

The complex sequence of events leading to repair in

the skin wound commences at the time the tissue is

damaged and the ‘negative feedback’ mechanism

induced (Gelfant & Candelas, 1972; Bullough, 1975;

Argyris, 1981). Vascular damage following trans-

epidermal wounding leads to haemorrhage with the

release of platelets (Wahl & Wahl, 1992). Aggregation

of the platelets promoted through contact with type

IV and V collagen molecules of the subendothelium

and the release of platelet derived growth factor

(PGDF), thromboxane A#, ADP, fibronectin, sero-

tonin, factor VIII (von Willebrand’s factor) (Lynch,

1991) achieves haemostasis. Calcium performs a key

role in this haemostatic process, probably as a primary

catalyst in platelet aggregation and in the production

of clotting factors VIII, IX and X (Born & Cross,

1964; Blair et al. 1990). Calcium is also necessary in

neutrophil activation and epidermal cell proliferation

at slightly later stages. In the rat wound model,

haemostasis is accomplished within 30 min but after

12 h minimal evidence is seen histologically of im-

pending repair mechanisms. Nevertheless, the im-

portance of calcium at these early stages in wound

healing is recognised by the 100% increase in

local concentrations of the metal in the 24 h after

wounding. The importance of calmodulin as a major

calcium binding protein in keratinocyte maturation is

demonstrated in this study by heavy deposits in the

regenerating epidermis at and near the wound margin.

Inflammation and granulation tissue formation are

promoted largely by secretions released from activated

platelets (Wahl & Wahl, 1992). In the rat, this phase

has commenced by 24 h after wounding and persists

until at least 5 d. There is considerable overlap

between this phase and the period of epithelial cell

proliferation and fibrogenesis, which also seems to

peak at about 5 d postwounding. By this time, local

concentrations of calcium, zinc and magnesium have

increased greatly over prewounding levels. Although

zinc metalloenzymes play a major role in immune and

inflammatory responses and are essential in tissues

subject to proliferation and metabolic activity (Lans-

down, 1996), our analyses show that demands for

calcium are notably higher at all stages in the wound

healing profile. Whereas calcium is required for the

activation of many intercellular reactions, zinc tends

to act as an inhibitor (Chvapil et al. 1975; Brewer

et al. 1979). Zinc is now known to regulate calcium

uptake and its contribution to the calmodulin-c-AMP

pathway. Zinc injected intradermally has been shown

to reduce calmodulin and calcium concentrations in

the skin, and raise cAMP levels (Heng et al. 1993).

We can appreciate that the critical ratio of zinc:

calcium will change as the inflammation}granulation

tissue formation progresses, and proliferative activity

gathers momentum in the epidermis at the wound

margin. Thus the zinc:calcium ratio declined from the

time of wounding until approximately 5 d. Thereafter,


metal concentrations and ratios of zinc:calcium

tended to return to prewounding levels by 7 d.

The final phase in the wound healing cascade is

characterised by a marked decline in the cellularity of

the wound site with reduction in the inflammatory cell

infiltrate and granulation tissue stage, and consoli-

dation of the fibrous scar tissue. The re-epithelial-

isation process is complete and the collagen IV-rich

basement membrane reformed. Once this is achieved,

the epidermal ‘normalisation’ process involves a

realignment of postmitotic cells from a lateral or

semilateral migration pathway (as seen in the re-

epithelialisation process) to the vertical movement

(Pinkus, 1970). In the rat, this occurs over the 7–10 d

postwounding period. But, even after 10 d the wound

exhibited an epidermis 2 to 3 times thicker than

normal. By 10 d, we saw a fractionally higher zinc

concentration and increased zinc:calcium ratio. It

is conceivable that in this situation, zinc ions are

involved in suppressing calcium motivated epidermal

cell proliferation and maturation (Hennings et al.

1980; Yuspa et al. 1989).

The modulation of trace metal ions in normal

mammalian systems is not well documented but is

likely to involve carrier proteins, growth factors and

cytokines (Hembry et al. 1985; Winge & Nielson,

1985). In the present study, we have examined only

calmodulin and metallothionein as markers. Metallo-

thionein occurs as 2 major isoforms, MT-I and

MT-II, which may be expressed in a tissue-dependant

manner (Pauwels et al. 1994; Yang et al. 1994). There

are also 2 other isoforms, MT-III which occurs

primarily in neural tissue (Palmiter et al. 1992), and

MT-IV which has been identified in squamous

epithelial tissue (Quaife et al. 1994). The antibody

used in the present study is known to react with MT-

I and MT-II (DAKO, Copenhagen), but it is unclear

if it reacts with MT-IV also. Although we noted that

calmodulin was present in most tissues, it was highest

in differentiating keratinocytes reflecting the import-

ance of calcium in this process. Metallothioneins on

the other hand, related well to zinc and possibly

copper involvement in dermal fibrogenesis and scar

tissue formation. Thus we saw increases in metallo-

thionein reaction product in the papillary dermis at

about the time at a time of early inflammatory cell

infiltration and fibrogenesis (collagenesis) with a

decline at later stages in wound healing. These changes

are consistent with the demand for zinc dependant

RNA and DNA-polymerases in essential repair

processes (Lindeman & Mills, 1980). Metallothioneins

are induced by and bind copper, such that changes in

their distribution are likely to reflect areas of increased

copper metabolism and the requirement for copper in

lysyl oxidase in collagenesis and elastic tissue for-

mation (Chou et al. 1968; Madden & Peacock, 1968).

Therapeutically, advantage is taken of the need for

additional calcium in wound healing in the devel-

opment of calcium alginate products (Jarvis et al.

1987; Blair et al. 1990; Lansdown, & Payne, 1994).

Thus, when calcium alginate fibres contact the blood,

calcium ions exchange for sodium in the wound

exudate and are released into the wound environment

to be available for haemostatic and other calcium

dependant processes. Zinc-containing products have

been developed variously for treating diaper rash and

other minor skin lesions (Lansdown, 1993, 1996).

Experimental studies have demonstrated that high

concentrations of zinc applied to the skin in an

amphiphilic cream to aid absorption, can be det-

rimental in wound healing, presumably because the

excess zinc absorbed is inhibiting calcium dependant

pathways (Lansdown & Sampson, unpublished).

Although copper may have a role in stabilising

epidermal cells and as Solcoderm is claimed to be

successful in treating epidermal cysts (Ronnan et al.

1993), excess copper is contraindicated in wound

healing (Barranco, 1972; Sucvi et al. 1981; Fisher,

1986). Our studies show that demands for copper are

particularly low in normal wound healing in the rat,

suggesting that excess concentrations are likely to

prove toxic through upsetting critical balances with

other divalent trace metal ions.

The skin in the rat is overtly different from human

skin but the constituent tissues are of a similar origin.

They will undergo comparable morphogenetic

patterns and be subject to the influence of similar

chemotactic systems, growth regulators, cytokines,

and nutritional factors. In the presence of injury, we

can expect cellular responses and homeostatic mech-

anisms to respond in a similar fashion in rats and

humans, differences being more in the rate and

magnitude of reaction, than of type. In mechanistic

studies the rat wound model is presented as an initial

screen in which to evaluate the potential value of a

dietary or therapeutic means of advancing tissue

repair.

Toxicologically, we can predict that any situation

liable to impair the uptake or metabolism of trace

metals, or alter the critical ratios of one trace metal to

another will be detrimental in wound healing in the

skin. This will include the application of medicated

dressings and topical creams designed to deliver

increased levels of zinc or calcium. More importantly,

percutaneous absorption of environmental pollutants

containing lead, cadmium or other xenobiotic min-


erals which are known to interact with zinc or other

essential trace element are likely to be detrimental in

wound healing (Lansdown, 1995). On the other hand,

silver which is known to induce metallothionein and

through preferential binding releases zinc, has been

shown to advance wound healing in the skin.

(Lansdown et al. 1997).

We are grateful to Mr Obah Ojarikre for his assistance

with statistics and preparation of the tables used in

this paper.

ARGYRIS TS (1981) The regulation of epidermal hyperplastic

growth. CRC Critical Reviews in Toxicology 9, 151–200.

BARRANCO VP (1972) Eczematous dermatitis caused by internal

exposure to copper. New England Journal of Medicine 239,

386–388.

BLAIR SD, JARVIS P, SALMON M, MCOLLUM C (1990)

Clinical trialof calciumalginatehaemostatic swabs.BritishJournal

of Surgery 77, 568–570.

BORN GVR, CROSS MJ (1964) Effects of inorganic ions and of

plasma proteins on the aggregation of blood platelets by

adenosine phosphate. Journal of Physiology 17D, 397–414.

BREWER GJ, ASTER J, KNUTSEN CA, KRUCKEBERG WC

(1979) Zinc inhibition of calmodulin: a proposed molecular

mechanism of zinc action of cellular functions. American Journal

of Haematology 7 53–60.

BULLOUGH WS (1975) Mitotic control in adult mammalian

tissues. Biological Reviews 50, 99–127.

CAI J-P, HARRIS B, FALANGA V, EAGLESTEIN P, MERTZ

M, CHIN YH (1991) Recruitment of mononuclear cells into

wounded skin: mechanism and modulation. In Clinical and

Experimental Approaches to Dermal and Epidermal Repair:

Normal and Chronic Wounds (ed. Barbul A, Caldwell MD, Hunt,

TK, Marshall, D, Pines E, Skover G ), pp. 243–256. New York:

Wiley-Liss.

CHOU WS, SAVAGE JE, O’DELL BL (1968) Relation of

monoamine oxidase activity and cross-linking in copper deficient

and control tissues. Proceedings of the Society for Experimental

Biology and Medicine 128, 948–952.

CHVAPIL M, WELDY PL, STANKOVA L (1975) Inhibitory

effect of zinc ions on platelet aggregation and serotonin release

reaction. Life Sciences 16, 561–571.

FISHER AA (1986) Contact Dermatitis. Philadelphia: Lea and

Febiger.

FLECKNALL PA (1987) Laboratory Animal Anaesthesia, pp.

159–223. London: Academic Press.

GELFANT S, CANDELAS GC (1972) Regulation of epidermal

mitosis. Journal of Investigative Dermatology 59, 7–12.

GROTENDORST G (1992) Chemoattractants and growth factors.

In Wound Healing; Biochemical and Clinical Aspects (ed. Cohen

IK, Diegelmann RF, Lindblad WJ), pp. 237–246. Philadelphia:

W. B. Saunders.

HALSTEAD JA, SMITH JC, IRWIN MI (1974) A conspectus of

research on zinc requirements in man. Journal of Nutrition 104,

345–378.

HEMBRY RM, MURPHY G, REYNOLDS JJ (1985) Immuno-

localisation of tissue inhibitor of metalloproteinases (TIMP) in

human cells. Journal of Cell Science 73, 105–119.

HENG MK, SONG MK, HENG MCY (1993) Reciprocity

between tissue calmodulin and cAMP levels : modulation by

excess zinc. British Journal of Dermatology 129, 280–285.

HENNINGS H, MICHAEL D, CHENG C (1980) Calcium

regulation of growth and differentiation in mouse epidermal cells

in culture. Cell 19, 245–254.

HUNT TH, HUSSAIN Z (1992) Wound environment. In Wound

Healing: Biochemical and Clinical Aspects (ed. Cohen IK,

Diegelmann RF, Lindblad WJ), pp. 274–281. Philadelphia:

W. B. Saunders.

JARVIS PM, GALVIN DAJ, BLAIR SD, MCOLLUM C (1987)

How does calcium alginate achieve haemostasis in surgery?

Thrombosis and Haemostasis 58, 80.

KILCULLEN JK, QUAN P, CHANG TH, LEVENSON SM,

STEINBERG JJ (1998) Non-viable Staphylococcus aureus can

stimulate macrophage recruitment, angiogenesis, fibroplasia and

collagen accumulation in wounded rats. Wound Repair and

Regeneration 6, 149–156.

KLEVAY LJ (1975) The ratio of zinc to copper of diets in the

United States. Nutrition Reports International 11, 237.

LANSDOWN ABG (1985) Morphological variations in keratin-

ising epithelia in beagle dogs. Veterinary Record 116, 127–130.

LANSDOWN ABG (1993) The influence of zinc oxide on the

closure of open wounds: an experimental study. International

Journal of Cosmetic Science 15, 83–85

LANSDOWN ABG, PAYNE MJ (1994) An evaluation of the local

reaction and biodegradation of calcium sodium alginate (Kalto-

stat2) following subcutaneous implantation in the rat. Journal

of the Royal College of Surgeons, Edinburgh 39, 284–288.

LANSDOWN ABG (1995) Physiological and toxicological changes

in the skin resulting from the action and interaction of metal ions.

CRC Critical Reviews in Toxicology 25, 397–462.

LANSDOWN ABG (1996) Zinc in the healing wound. Lancet 347,

706–707.

LANSDOWN ABG, PATE P (1993) Animal models in wound

healing in the skin. In Proceedings of the 5th Symposium of

the Federation of European Laboratory Science Associations,

Brighton. pp. 395–397.

LANSDOWN ABG, SAMPSON B (1996) Dermal toxicity and

percutaneous absorption of cadmium in rats and mice. Lab-

oratory Animal Science 46, 549–554.

LANSDOWN ABG, SAMPSON B (1997) Trace metals in

keratinising epithelia in beagle dogs. Veterinary Record 141,

571–572.

LANSDOWN ABG, SAMPSON B, LAUPATTARAKASEM P,

VUTTIVIROJANA A (1997) Silver aids healing in the sterile

skin wound: experimental studies in the laboratory rat. British

Journal of Dermatology 137, 728–735.

LINDEMAN RD, MILLS BJ (1980) Zinc homeostasis in health

and disease. Mineral and Electrolyte Metabolism 3, 223–236.

LYNCH SE (1991) Interactions of growth factors in tissue repair.

In Clinical and Experimental Approaches to Dermal and Epidermal

Repair: Normal and Chronic Wounds (ed. Barbul A, Caldwell

MD, Eaglestein WH, Hunt TK, Marshall D, Pines E et al.),

pp. 3412–337. New York: Wiley-Liss.

MADDEN JW, PEACOCK EE (1968) Studies on the metabolism

of collagen during wound healing. I. Rate of collagen synthesis

and deposition in cutaneous wounds in the rat. Surgery 64,

228–294.

MAST B (1992) The skin. In Wound Healing: Biochemical and

Clinical Aspects (ed. Cohen IK Diegelmann RF, Lindblad WJ),

pp. 344–355. Philadelphia: W. B. Saunders.

MENON GK, GREYSON S, ELIAS PM (1985) Ionic calcium

reservoirs in mammalian epidermis : ultrastructural localisation

by ion-capture cytochemistry. Journal of Investigative Derma-

tology 84, 508–512.


MOYNAHAN EJ (1974) Acrodermatitis enteropathica: a lethal

inherited human zinc deficiency disorder. Lancet 1, 399–400

MULDER GD (1991) If wounds could talk. In Clinical and

Experimental Approaches to Dermal and Epidermal Repair:

Normal and Chronic Wounds (ed. Barbul A, Caldwell WH,

Hunt TH, Marshall D, Pines E et al.), pp. 55–66. New York:

Wiley-Liss.

NICKOLOFF BJ, VARANI J, MITRA RS (1991) Modulation

of keratinocyte biology by gamma-interferon: relevance to cu-

taneous wound healing. In Clinical and Experimental Approaches

to Dermal and Epidermal Repair: Normal and Chronic Wounds

(ed. Barbul A, Caldwell MD, Eaglestein WH, Hunt TK, Marshall

D, Pines E, Skover G), pp. 141–154. New York: Wiley-Liss.

NWOMEH BC, LIANG H-X, DIGELMANN RF, COHEN IK,

YAGER DR (1998) Dynamics of the matrix metalloproteinases

MMP-I and MMP-8 in acute open human dermal wounds.

Wound Repair and Regeneration 6, 127–134.

PALMITER RD, FINDLEY SD, WHITMORE TE, DURNHAM

DM (1992) MT-III, a brain specific member of the metallo-

thionein gene family. Proceedings of the National Academy of

Sciences of the USA 89, 6333–6337.

PAUWELS M, VAN-WEYENBERGH J, SOUMILLION A,

PROOST P, DE LEY M (1994) Induction by zinc of specific

metallothionein isoforms in human monocytes. European Journal

of Biochemistry 220, 105–110.

PINKUS H (1970) The direction of growth of human epidermis.

British Journal of Dermatology 83 556–564.

QUAIFE CJ, FINDLEY SD, ERICKSON JC, FROELICK GJ,

KELLY EJ, ZAMBROWICZ BP et al. (1994) Induction of a new

metallothionein isoform (MT-IV), occurs during differentiation

of stratified epithelia. Biochemistry 33, 7250–7259

RONNAN M, SHUSTER S, KLIN B (1993) Treatment of

epidermal cysts with Solcoderm (a copper ion and acid solution).

Clinical and Experimental Dermatology 18, 500–503.

ROWE A, FARRELL AM, BUNKER CB (1997) Constitutive

endothelial and inducible nitric oxide synthase in inflammatory

dermatoses. British Journal of Dermatology 136, 18–23.

SUCVI I, PRODAN L, LAZAR V (1981) Research on copper

poisoning. Medical Law 3, 190.

WAHL GF, WAHL SM (1992) Inflammation. In Wound Healing:

Biochemical and Clinical Aspects (ed. Cohen IK, Diegelmann RF,

Lindblad WJ), pp. 40–62. Philadelphia: W. B. Saunders.

WINGE DR, NIELSON KB (1985) Distinct metal binding

configurations in metallothionein. In Trace Elements in Man and

Animals (ed. Mills CF, Bremner I, Chesters JK), pp. 307–311.

Slough: CAB.

YANG YY, WOO ES, REESE CE, BAHNSON RR, SAIJO N,

LAZO JS (1994) Human metallothionein isoform gene ex-

pression in cisplatin-sensitive and resistant cells. Molecular

Pharmacology 89, 453–460.

YUSPA SM, KILKENNY AE, STEINERT PM (1989) Expression

of murine epidermal cell markers is tightly regulated by extra-

cellular calcium concentrations in vitro. Journal of Cell Biology

109, 1207–1217.


sequential changes in trace metal, metallothionein and ...€¦ · 2.11.98). other metals found in...

Documents