Protein J (2010) 29:8192

DOI 10.1007/s10930-009-9225-9

Amino Acids from Mytilus galloprovincialis (L.) and Rapana

venosa Molluscs Accelerate Skin Wounds Healing
via Enhancement of Dermal and Epidermal Neoformation
Diana L. Badiu Rafael Luque Elena Dumitrescu

Anca Craciun Danut Dinca

Published online: 21 January 2010

Springer Science+Business Media, LLC 2010

Abstract Wound healing consists of re-epithelialization, for collagen IV, CD 34 and CD 117 antibodies. According
contraction and formation of granulation and scar tissue. to the obtained results, as expressed by histological studies,
Amino acids from proteins are involved in these events, but the most abundant blood vessels, collagen fibres, basal and
their exact roles are not well understood. The present study stem cells were found only for treated animals with amino
was undertaken to investigate the anti-inflammatory effects acids from Rapana venosa extracts. The rich composition
of some amino acids from two molluscs, Mytilus gallo- of amino acids from the two molluscs merits consideration
provincialis (L.) (Mediterranean mussel) and Rapana as therapeutic agents in the treatment of skin burns.
venosa (hard shell-clam) employed in induced skin burn
injuries in Wistar rats. The treatment was evaluated in Keywords Amino acids
Histological
Skin

terms of essential amino acids composition which rendered Mytilus galloprovincialis (L.)
Rapana venosa
the extracts very efficient in healing skin burns. The
healing process was examined by periodic acid Schiffs,
Abbreviations
Verhoeffs Van Gieson and immunohistochemistry stains
Ser Serine
Ala Alanine
D. L. Badiu (&) Gly Glycine
Department of Biology-Biochemistry, Faculty of Natural and Pro Proline
Agricultural Sciences, Ovidius University of Constanta, Val Valine
124 Mamaia Blvd., 900527 Constanta, Romania Leu Leucine
e-mail: dianabadiu@yahoo.com
Ile Isoleucine
R. Luque Thr Threonine
Departament of Organic Chemistry, Cordoba University, Met Methionine
Campus de Rabanales, Edificio Marie Curie (C-3), Lys Lysine
Ctra Nnal IV, Km 396, 14004 Cordoba, Spain
e-mail: q62alsor@uco.es Phe Phenylalanine
Tyr Tyrosine
E. Dumitrescu Trp Tryptofan
National Institute of Recovery, Physical Medicine and Clinical Asp Aspartic acid
Balneary, 11 A, Ion Mihalache Blvd., Bucharest, Romania
Glu Glumatic acid
A. Craciun Hypro Hydroxyproline
Phatological Anatomy Laboratory, Constanta Clinical GCMS Gas Chromatography-Mass Spectrometry
Emergency Hospital, 145 Tomis Blvd, 900591 Constanta, CD Cluster designation
Romania
NBF Neutral buffered formalin
D. Dinca DAB Diaminobenzidine
Otorhinolaryngology Department, Constanta Clinical HRP Horseradish peroxidise
Emergency Hospital, 145 Tomis Blvd., 900591 Constanta, LPS Lipopolysaccharide
Romania

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82
i.p. Intraperitoneal
H2O2 Hydrogen peroxide
K2SO4 Potassium sulphate
CuSO4 Copper sulphate
H2SO4 Sulphuric acid
KOH Potassium hydroxide
NaCl Sodium chloride
Na2SO4 Sodium sulphate
HIO4 Periodic acid
FeCl3 Ferric chloride
O2 Oxygen
N2 Nitrogen
I Iodine
1 Introduction
The response to injury, known as the hypermetabolic
response, occurs after a severe thermal injury. Increases in
cardiac output, O2 consumption, N2 loss, body weight loss,
and metabolic rate are directly proportional to the size of
the burn. The energy requirements are met by mobilization
of carbohydrates, fat, and protein reserves. However,
energy reserves particularly glycogen, are quickly depleted
and the energy needs are met by increased glycogenesis
from amino acids mobilization originating from the skel-
etal muscle, which leads to the loss of muscle tissue and
malnutrition [6].
Bioactive substances with anti-inflammatory activity
(e.g. proteins) have been recently isolated from molluscs,
as well as crabs, shrimps, frogs and many fish including
halibut, sharks and seahorses. Fish oils and broad proteins
were successfully used to treat wounds and burns, with
accelerated regeneration and wound healing [26].
Previous studies from our laboratory showed that burn
injuries from five groups of Wistar rats with second-degree
burns could be healing using lipid extracts from the similar
molluscs [4].
The size of the pool for each amino acid is the result of a
balance between input and removal. The metabolic outlets
for amino acids are protein synthesis and amino acid
degradation, whereas the inputs are no novo synthesis for
non essential amino acids and protein breakdown. For
example, amino acid homeostasis and protein metabolism
can be altered in response to malnutrition and/or various
forms of trauma such as thermal burns [3]. In this regard,
especially essential amino acids (i.e. Leu, Lys and Thr) but
also non essential amino acids (i.e. Pro) are required to
adjust physiological functions that are involved in the
defence of and adaptation to amino acid limitation.
Williams and Barbul [23] found that Leu maintaining
nitrogen balance in conditions such as sepsis, trauma and
burns and together with other branched-chain amino acids
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D. L. Badiu et al.
(i.e. Val and Ile) support protein synthesis after injury and
decrease muscle proteolysis.
Zaets and Borisova [25] showed that incorporation of
Lys into total proteins of most organs of the burned animals
was significantly increased above normal. Furthermore,
Cynober et al. [9] measured plasma concentration of some
amino acids (i.e. Lys, Thr and Pro) in 12 severely burned
adults (20% of body surface or more) and he showed that
this remain unchanged until the third week, then growth
markedly.
Therefore, a therapy based on administration of Ser,
Ala, Gly, Pro, Val, Leu, Ile, Thr, Met, Lys, Phe, Tyr, Trp,
Asp and Glu amino acids, which are expected to be found
in analysed proteic extracts, could be able to promote
protein anabolism or slow down protein degradation.
In addition to the reported metabolic changes of dif-
ferent organs yielding trauma, the studies presented here
were undertaken to examine the anti-inflammatory effects
of essential and/or non-essential amino acids from proteic
extracts of two molluscs, Mytilus galloprovincialis (L.)
(Mediterranean mussel) and Rapana venosa (hard shell-
clam) on induced skin injuries in rats.
2 Materials and Methods
2.1 Sample Characterization
Samples were collected from the medium seacoast from
Constantas area (between Mamaia and Constanta Port). We
are aware of the viabilities due to sampling season as
reported by other authors [7, 12, 15]. In this regard, molluscs
were collected in May (2008 and 2009) as spring is ideal due
to the accumulation of bioactive (lipids) and structural
compounds (proteins) as the marine organisms prepare for
reproductive season as well as to the maximum concentra-
tions of organic substances found in plankton [17, 18, 22].
The two Mytilus galloprovincialis (L.) and Rapana
venosa proteic extracts were obtained after total N2 method
[2]. Total organ proteic content was determined. 510 mg
of tissue was added into a Kjeldahl glass. 0.11.0 g cata-
lytic agents [K2SO4 and CuSO4 in ratio 3:1 (v/v)] and
15 mL concentrated H2SO4 were then added and the
mixture was boiled until colourless solution.
Amino acids from proteic extracts were analysed by
GCMS using an Agilent GC 6890N model (1 lL sample
injected, split 1:50 column flow 1.6 mL/min, program
temp. 100290 C (rate 10 C/min) coupled with a quad-
rupolar MS 5973. Samples were injected according to
methylation method.
The methylation comprises several steps: 1 g extract
(Mytilus galloprovincialis (L.) and Rapana venosa proteic
extracts) dissolved in 10 mL MeOH (0.2 mL KOH were
Amino Acids Accelerate Skin Wounds Healing
also added to the mixture) was heated under reflux for
15 min, cooled down and subsequently extracted with
5 mL heptane and 10 mL saturated NaCl solution. The
organic phase was dried on Na2SO4 and subsequently
analysed by GC and GCMS.
2.2 Animal Model
Twenty male Wistar rats, weighing 200250 g, were ran-
domly divided into four groups (five rats per group), three
groups for burn experiments; the remaining group as nor-
mal control. Animals were housed in standard polycar-
bonate cages with free access to water and were maintained
on a 12:12 h light:dark cycle in a climatised room.
All animals used in this study were maintained in a
facility accredited by Helsinki Declaration and guidelines
of the Ethics Committee of the International Association
for the Study of Pain. They were approved by the Animal
Care Committee of the Faculty of Natural and Agricultural
Sciences, Ovidius University, Romania. Also, animals
received human care in accordance with the National
Institutes of Healths Guide for Care and Use of Laboratory
Animals.
2.3 Induction of Local Cutaneous Thermal Injury
Male Wistar rats were prepared *24 h before experi-
mentation. For this, they were anesthetized with sodium
pentobarbitone (50 mg/Kg i.p.; May and Baker Ltd. Essex,
UK) and the dorsum (around 2 cm diameter) skin area was
shaved. Local cutaneous thermal injuries were induced on
the dorsum skin using a 1 cm diameter temperature con-
trolled skin heater (Moor Instruments Ltd. Devon, UK) as
previously described [21]. The probe temperature was
maintained at 50 C for 10 min (contact duration). Anes-
thesia was maintained throughout the experiment and the
animals were killed by cervical dislocation 14 h in the last
day of experiment. Control skin was taken from an adja-
cent, contra-lateral area of the dorsum in the same animal
at the same time point under investigation. The location of
skin used was alternated to remove any bias relating to
regional skin heterogeneity. Also, the dorsum skin was
removed and thermally injured or control skin was excised
with a 16 mm diameter punch. The tissues were fixed in
NBF for periodic acid Schiffs, Verhoeffs Van Gieson
stains and immunohistochemical examination.
2.4 Treatments and Protocols
The groups of Wistar rats were divided according to the
different treatments, as follows:
Group 1 (control; n:5): Rats with an induced skin burn
without any treatment.
83
Group 2 (n:5): Rats with an induced skin burn treated
with amino acids from Rapana venosa proteic extracts
(0.3 mg/kg, twice a day).
Group 3 (n:5): Rats with an induced skin burn treated
with amino acids from Mytilus galloprovincialis (L.) pro-
teic extracts (0.3 mg/kg, twice a day).
Group 4 (n:5): Rats with an induced skin burn treated
with a mixture of amino acids from Mytilus galloprovin-
cialis (L.): Rapana venosa proteic extracts (1:1 ratio, w/w),
(0.3 mg/kg, twice a day).
Experiments were terminated after 24 days, after
achieving comparable healed wounds between the untreated
animals (control) and the animals treated with amino acids
from both Mytilus galloprovincialis (L.) and Rapana venosa
molluscs, as observed from the histological and immuno-
histological results and established through different scores.
2.5 Histological Evaluation
The specimens were immediately fixed in 10% NBF
solution overnight at room temperature. Subsequently,
longitudinal sections taken perpendicular to the anterior-
posterior axis of the wound were dehydrated in graded
ethanol series, cleared in xylene, and embedded in paraffin
by conventional procedures. Paraffin sections were cut to a
thickness of 5 lm, mounted on glass slide, deparaffinized
with xylene, rehydrated through a grade series of alcohols
to distilled water, and stained on periodic acid Schiffs and
Verhoeffs Van Gieson procedures. All tissue sections
collected for light microscopy were examined by a
pathologist without knowledge of the previous treatment.
The following histological criteria were used as score for
each animal: epithelial proliferation, granulation tissue
formation and organization, number of newly formed
capillaries (identified by the presence of erythrocytes in
their lumen).
The microscope employed in the sample analysis was
Nikon Eclipse E600 adapted with a Dikon Digital Camera
and the visualization was 40 9 10. Number of epithelial
layers, crusting, spongiosis, interstitial edema, intravascu-
lar fibrin deposition, congestion, haemorrhage and degree
of inflammation were also evaluated and considered for
scoring. The histological score system ranged between one
and three.
2.6 Periodic Acid Schiffs Procedure
Sections were deparaffinized and hydrated in distilled
water. Slides were placed into 0.5% HIO4 for 5 min and
rinsed in distilled water. Schiffs reagent was added and
microwaved for 4560 s until deep margenta. Slides were
then washed in running tap water for 5 min and counter-
stained in haematoxylin for 3 min. Water was added and
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blue haematoxylin, rinsed in distilled water, dehydrated in
alcohol, cleared and coverslipped was added in the end. All
three polysaccharides (glycogen, starch, and cellulose)
gave a positive Periodic Acid Schiffs reaction [5].
2.7 Verhoeffs Van Gieson Procedure
Slides were deparaffinized and hydrated in distilled water.
Verhoeffs haematoxylin was added for 30 min and the
solution was kept until stain was completed. Sections were
then washed in tap water, differentiated in 2% FeCl3
solution and checked microscopically for black fibres on a
grey background. The slides were again rinsed in water,
hyped for 1 min to remove I and counterstained in Van
Giesons for 5 min. This was followed by dehydratation,
cleaning in xylen and eventually coverslipping. All elastic
fibers gave a positive Verhoeffs Van Gieson reaction [5].
2.8 Immunohistochemical Staining
For the detection of collagen IV and CD 34, mouse
monoclonal antibodies (Dako Cytomation) and for CD 117
a polyclonal rabbit anti-human (Dako Cytomation), were,
respectively used.
The antibodies, clone, dilutions, staining, pre-treatment
conditions and sources of immunohistochemical studies are
listed in Table 1.
Tissue was fixed in NBF solution, processed to paraffin,
and subsequently immunostained at room temperature in
10% formalin-fixed deparaffinized sections, using strepa-
tavidin biotin system with HRP and DAB chromogen
(Dako, MA). Optimum primary antibody concentrations
were predetermined by use of known positive control tissue
LPS. Paraffin section were cut at 4 lm on rotary micro-
tome, mounted on positively charged glass slides (capillary
glass slides) and air-dried overnight. Sections were incu-
bated in unlabeled blocking serum for 15 min to block non-
specific binding of the secondary antibody. All slides were
then incubated at the same time for 25 min with buffer
alone as a negative reagent control. An isotope control and
negative reagent control were run at each time point and
for each organ to ensure specificity. After washes in buffer,
Table 1 Antibodies used for immunohistochemistry
Antibodies Clone Source Dilution Staining Pre-treatment
Type IV C IV Dako 1:20 C H
collagen
CD 34 QBEnd 10 Dako 1:20 M H
CD 117 C-kit Dako 1:20 M H
C cytoplasmic staining; M membrane staining; H heating citrate
buffer (pH = 6)
123
D. L. Badiu et al.
sections were incubated for 25 min with a biotinylated
polyvalent secondary antibody solution (containing goat
anti-rabbit immunoglobulins). Next, sections were washed
with buffer, incubated in HRP-conjugated streptavidin
biotin complex for 15 min, washed again in buffer, and
then incubated with two changes, 5 min each, of a freshly
prepared mixture of DAB and H2O2 in buffer, followed by
washing in buffer and then water. Sections were then
counterstained with hematoxylin, dehydrated in a graded
series of ethanol and xylene, and coverslipped.
Slides were reviewed by light microscopy, and positive
reactions with DAB were identified as a dark brown
reaction product [20, 24]. Scoring was done according to
Colburn et al. [8]: 0, normal, inactivated tissue; 1?, mild
activation; 2?, moderate activation, and 3?, intense
activation.
2.9 Immunohistochemical Analysis
The slides from each tissue samples of Wistar rats skin
(n:20) were confocally imaged, taken 25 images, one every
2 lm, using the same microscope and visualization
employed for the histological slides. Non-overlapping
fields from each section were selected and then quantifi-
cation of the chosen sections was made.
2.10 Statistical Analysis
Data were analyzed by one-way ANOVA. In the case of
the presence of significant F values, the Student Newmal-
Keuls test was used to compare the groups. A p \ 0.05
level of significance was accepted.
3 Results and Discussion
3.1 Properties of the Amino Acids
Quantitative GCMS analyses of the proteic extracts from
the two molluscs were performed in order to identify the
different compounds in the extracts following a similar
methodology to that reported elsewhere [2]. Several amino
acids were identified and quantified (mg%).
The results of the content of proteic extracts from
Mytilus galloprovincialis (L.) and Rapana venosa molluscs
are summarised in Table 2. Several interesting differences
could be found in the extracts. The two extracts had similar
values of Ala, Val, Phe and Trp (Table 2, entries 1, 2, 12
and 15). Nevertheless, the differences between samples in
terms of content and distribution of amino acids were
significant.
Thus, the Mytilus galloprovincialis (L.) proteic extract
had up to almost 50% weight (of the amino acids content),
Amino Acids Accelerate Skin Wounds Healing 85

Table 2 The concentrations of

No. Amino-acid Concentration (mg%) Concentration (mg%) from
the amino acids found in the two
from Mytilus galloprovincialis (L.) Rapana venosa proteic extract
proteic extracts
proteic extract

1. Alanine (Ala) 21.66 22.76

2. Valine (Val) 6.50 4.78
3. Leucine (Leu) 1.20 6.60
4. Isoleucine (Ile) 7.45 4.90
5. Proline (Pro) 1.25 4.66
6. Glycine (Gly) 7.56 3.07
7. Serine (Ser) 8.10 4.10
8. Threonine (Thr) 6.77
9. Aspartic acid (Asp) 8.03 3.80
10. Methionine (Met) 4.57 2.67
11. Glutamic acid (Glu) 17.26 11.80
12. Phenylalanine (Phe) 6.72 7.78
13. Lysine (Lys) 8.98
14. Tyrosine (Tyr) 4.63 2.65
15. Tryptophan (Trp) 3.53 2.67
Total [98 [98

(Table 2, entries 4, 6, 7, 9 and 14) compared to amino acids
from Rapana venosa extract.
Similarly, non-essential amino acids (Table 2, entries 1,
5, 6, 7, 9, 11 and 14) were found to be distributed in higher
content (over 30% of weight) in Mytilus galloprovincialis
(L.) proteic extract compared to those from Rapana ven-
osa, excepting Ala and Pro (Table 2, entries 1 and 5).
It is worth mentioning at this stage that the content of
Leu, Pro, Thr and Lys from Rapana venosa proteic extract
(Table 2, entries 3, 5, 8 and 13) is significant (p = 0,004)
and almost non existing in Mytilus galloprovincialis (L.)
extract, which could offer specific properties for the
Rapana venosa proteic extract.
3.2 Histological Studies of Regenerative Properties
of the Amino Acids
Histological analysis by periodic acid Schiffs technique
showed that glycogen quantification did not differ statisti-
cally among the three groups of animals involved in the
treatments. Table 3 summarises the main scoring employed
Table 3 The histological score of the four groups of Wistar rats
distinguished by periodic acid Schiffs stain
Group Histological
score
Group 1, Control 2?
Group 2 2?
Group 3 1?
Group 4 2?
for the periodic acid Schiffs stain of the wound tissue
samples. It may be supposed that animals, after massive skin
injury, used the majority of exogenous glutamine in the tis-
sues, intending to improve its absorptive capacity, with less
glycogen absorption and subsequent synthesis into hepato-
cyte. A summary of the main results is presented in Fig. 1.
The Verhoeffs Van Gieson stain shows that elastic
fibres regenerate, in both the dermis and the epidermis, in
spontaneously healed partial-thickness burns but that they
do not reach the number present in normal skin. Table 4
summarises the main scoring employed for the Verhoeffs
Van Gieson stain of the wound tissue samples. The sig-
nificantly higher number of elastic fibers in normotrophic
scars compared with hypertrophic scars suggests a regu-
latory role for the skin elastic system in the outcome of
burn wound healing. A summary of the main results is
presented in Fig. 2.
Skin sections from control animals consisted of edem-
atous and swollen granulation tissue with no epithelial
covering by periodic acid Schiffs stain (Fig. 1, Group 1,
Control, B, 2? score).
On the other hand, microscopic examination of treated
rats disclosed good histological scorings in wound healing
(Table 3, Groups 2, 3 and 4, 2?, 1? and 2? scores).
Moderate to complete re-epithelialization, minimal inter-
cellular or subepithelial edema, but no crusting were
observed in Group 2 (Fig. 1, C, 2? score). Granulation
tissues were characterised by dense collagen matrix depo-
sition, slight edema, and few scattered inflammatory infil-
trates mainly confined in deep dermis and to the wound
margins. The number of capillaries was significantly higher

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86 D. L. Badiu et al.

Fig. 1 Microscopy images (40 9 10, periodic acid Schiffs stain) of
the tissue sections of a healthy rats before the skin burn was induced;
b untreated rats with induced skin burns after 22 days (Group 1,
Control); c rats with induced skin burns treated with amino acids from
Rapana venosa proteic extract postburn day 10 (Group 2); d rats with
induced skin burns treated with amino acids from Mytilus gallopro-
vincialis (L.) proteic extract postburn day 14 (Group 3); e rats with
induced skin burns treated with a mixture of amino acids from Mytilus
galloprovincialis (L.): Rapana venosa proteic extracts (1:1 ratio, w/w)
postburn day 16 (Group 4)

Table 4 The histological score of the four groups of Wistar rats The treatments of rats with amino acids improved colla-
distinguished by Verhoeffs Van Gieson stain gen deposition, reduced the hyalinization, and increased the
Group Histological vascularity and glycogen density in Groups 2 and 3 (Fig. 2, C
score and D, 3? and 1? scores) showed by Verhoeffs Van Gieson
stain. Amino acids showed to increase collagen deposition in
Group 1, Control 2?
normal animals and improve the overall histological picture
Group 2 3?
in all groups of Wistar rats under the treatments.
Group 3 1?
In our study, microscopic examination of the histopa-
Group 4 1?
thological slides prepared after the completion of treat-
ment, revealed that the wounds treated with amino acids
than that found in untreated rats, with no evidence of fibrin from Rapana venosa proteic extract (Figs. 2, 3, Group 2, C,
deposition, hemorrhage or vascular congestion in Group 4 2? and 3? scores) contained fewer inflammatory cells
(Fig. 1, E, 2? score). than the untreated control (Figs. 2, 3, Group 1, Control, B,

123
Amino Acids Accelerate Skin Wounds Healing 87

Fig. 2 Microscopy images

(40 9 10, Verhoeffs Van
Gieson stain) of the tissue
sections of a healthy rats before
the skin burn was induced;
b untreated rats with induced
skin burns after 22 days (Group
1, Control); c rats with induced
skin burns treated with amino
acids from Rapana venosa
proteic extract postburn day 10
(Group 2); d rats with induced
skin burns treated with amino
acids from Mytilus
galloprovincialis (L.) proteic
extract postburn day 14 (Group
3); e rats with induced skin
burns treated with a mixture of
amino acids from Mytilus
galloprovincialis (L.): Rapana
venosa proteic extracts (1:1
ratio, w/w) postburn day 16
(Group 4)

2? score for both). The majority of cells infiltrating the
cutaneous wounds were shown to originate from blood,
except for the tissue mast cells, which are abundant in the
dermal and hypodermal compartments of rodent skin and
may migrate to the wound site from the local sources.
Results pointed out the untreated wounds required a
time to heal over 3 weeks (2224 days) to achieve a 2?
score for both periodic acid Sciffs and Verhoeffs Van
Gieson stains (Figs. 1, 2, Tables 3, 4). The use of amino
acids was found to remarkably reduce such time of healing
to 12 days (with a daily treatment with amino acids from
Rapana venosa proteic extract) and 1516 days (daily
treatment with amino acids from Mytilus galloprovincialis
(L.) extract) to achieve 2? and 1? scores for periodic acid
Schiffs and 3? and 1? scores for Verhoeffs Van Gieson
stains, respectively. Interestingly, the mixture of the two
amino acids from extracts did not significantly improve the
time of healing (1718 days, Figs. 1, 2, Group 4).
The results of this study have shown that amino acids
from Rapana venosa proteic extracts accelerated wound
healing by at least 10 days as compared to the control.
Thus, it is highly significant that this amino acid content is
able to increase the rate of wound healing as well as to
decrease the collagen content. We believe that amino acids
from the Rapana venosa proteic extract stimulate the pro-
liferation of differentiating keratinocytes and it is reason-
able to expect that this feature could assist the process of
wound re-epithelization.
3.3 Immunohistochemistry
Immunostaining results for collagen IV, CD 34 and CD
117 antibodies are depicted in Figs 3, 4, 5. Table 5 and
Figs. 6, 7, 8 summarise the main scoring employed for the
immunohistochemistry data.

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88 D. L. Badiu et al.

Fig. 3 Microscopy images

(40 9 10) of the tissue sections
in immunostaining of type IV
collagen, positive cells:
a healthy rats before the skin
burn was induced; b untreated
rats with induced skin burn after
day 22 (Group 1, Control);
c rats with induced skin burn
treated with amino acids from
Rapana venosa proteic extract
postburn day 10 (Group 2);
d rats with induced skin burn
treated with Mytilus
galloprovincialis (L.) proteic
extract postburn day 14 (Group
3); e rats with induced skin burn
treated with a mixture of amino
acids from Mytilus
galloprovincialis (L.): Rapana
venosa proteic extracts (1:1
ratio, w/w) postburn day 16
(Group 4)

Light microscopic immunology with collagen IV antibody
(Fig. 3) shows new epithelium is being laid down in all
groups. Neo-vessels, collagen fibres, dilated existing vessels
and basal membrane can be visualized in provisional fibrin
matrix. Skeletal muscle cells form a border zone between
normal and wounded tissue, which is more accentuated in
Group 3 (Fig. 3, D, 1? score). In Group 2 (Fig. 3, C, 2?
score), the epithelial layer was completed on day 12 and
granulation tissue matured. Scar tissue is still visible for the
Groups 1, 3 and 4, while granulation tissues moved down to
the base of the wound in the first recovery tissues of Group 2
treated with amino acids from Rapana venosa proteic extract.
CD 34 antibody was detected in all blood vessels and
endothelial cells (Fig. 4), but with different frequency. In
granulated tissue and skeletal muscle cells at the base of
the wound at the first recovery tissue on day 12 of Group 2
(Fig. 4, C, 2? score). In others Groups (1, 3 and 4, Fig. 4,
B, D and E, 2?, 1? and 1? scores), this expression is
limited to neo-formation vessels from skeletal muscle cells
at the base of the wounds.
Expression of basal and stem cells by CD 117 antibody
(Fig. 5), showed the macrophages line, the re-epitheliali-
zation border and form the predominant part of provisional
fibrin matrix in all treated tissues. In Groups 3 and 4
(Fig. 5, D and E, 1? and 3? scores), macrophages have
spread throughout the mature granulation tissues and can
be visualised in high density. Basal and stem cells stained
appear predominantly on day 12 for Group 2 (Fig. 5, C, 3?
score, animals treated with amino acids from Rapana
venosa proteic extract).
3.4 Discussion Along the Wounds Healing Properties
of the Extracts
To date, information dealing with amino acids involved
in in vivo tasks in skin repair is clearly restricted. Thus,

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Amino Acids Accelerate Skin Wounds Healing 89

Fig. 4 Microscopy images

(40 9 10) of the tissue sections
in immunostaining of CD 34
antibody, positive cells:
a healthy rats before the skin
burn was induced; b untreated
rats with induced skin burn after
day 22 (Group 1, Control);
c rats with induced skin burn
treated with amino acids from
Rapana venosa proteic extract
postburn day 10 (Group 2);
d rats with induced skin burn
treated with Mytilus
galloprovincialis (L.) proteic
extract postburn day 14 (Group
3); e rats with induced skin burn
treated with a mixture of amino
acids from Mytilus
galloprovincialis (L.): Rapana
venosa proteic extracts (1:1
ratio, w/w) postburn day 16
(Group 4)

in vitro and in vivo experiments implicated that amino
acids contributes to granulation tissue formation by trig-
gering endothelial migration, proliferation and thus, par-
ticipate in capillary growth into the wound site during
restructuration of the tissue [13]. Interestingly, the
observed wound-related amino acids activity was exclu-
sively connected to viable macrophages at the wound site
in early studies. More recent data confirmed that wound
macrophages are a prominent source of amino acids
expression in the early inflammatory phases of tissue
reparation [19].
Pro and Gly are the amino acids necessary for the pro-
duction of collagen and cartilage for healthy joints, liga-
ments, tendons and the largest organ of the body, skin.
Aging, combined with the effects of sun and free radical
damage, results in older looking, wrinkled skin. Pro, along
with other amino acids such as Leu, Lys and Thr, helps
create new cell formation and can contribute to maintaining
younger looking skin. Ala has been shown to accelerate the
altered wound healing process in diabetic mice when
administrated via the i.p. route [1], but its effect on burns
has not yet been completely investigated.
Met and Leu have been reported to be capable of
accelerating the healing rate of wounds in injured rats to
the same extend when present in equivalent amounts.
When healing is thought to be essentially complete, i.e.
when the N2 content of wounded and corresponding normal
tissue is similar, the Leu level in the former is 2.5 times
that of the normal tissue, and the Met content has also
increased, but to a lesser degree [16].
Experimental injury causes a mobilization of Met from
muscle for use by the skin of the wounded rats. Met may
then be converted to Cys in the skin to meet the Cys
requirement of the regenerating wound. The data indicate
that injury produces a decreased specific activity with
respect to Cys in skin tissue [10].

123
90 D. L. Badiu et al.

Fig. 5 Microscopy images

(40 9 10) of the tissue sections
in immunostaining of CD 117
antibody, positive cells:
a healthy rats before the skin
burn was induced; b untreated
rats with induced skin burn after
day 22 (Group 1, Control);
c rats with induced skin burn
treated with amino acids from
Rapana venosa proteic extract
postburn day 10 (Group 2);
d rats with induced skin burn
treated with amino acids from
Mytilus galloprovincialis (L.)
proteic extract postburn day 14
(Group 3); e rats with induced
skin burn treated with a mixture
of amino acids from Mytilus
galloprovincialis (L.): Rapana
venosa proteic extracts (1:1
ratio, w/w) postburn day 16
(Group 4)

Table 5 The score of Type IV

Groups Type IV collagen CD 34 CD 117
collagen, CD 34 and CD 117 of
antibody antibody antibody
each group of Wistar rats
Group 1Control 2? 2? 2?
Group 2 2? 2? 3?
Group 3 1? 1? 1?
Group 4 3? 1? 3?

These data justify a potential therapeutic approach
aimed at reducing the exaggerated lipid peroxidation that
impairs the healing capacity of the wounds. Ser, Ala, Gly,
Pro, Val, Leu, Ile, Thr, Met, Lys, Phe, Tyr, Trp, Asp and
Glu amino acids are a family of compounds in which the
combination in the same molecule of a chain with the
reducing ability of thiol groups (dual antioxidants) may
result in powerful and peculiar biological action, especially
in those oxidative stress-mediated situations in which a
significant depletion of endogenous thiols is observed [14].
The amino acids used in this experiment demonstrated a
significant increase in the Hypro content of the granulation
tissue indicating increased collagen turnover. Collagen, the
major component which strengthens and supports extracel-
lular tissue is composed of amino acids and Hypro, which has
been used as a biochemical marker for tissue collagen [11].

123
Amino Acids Accelerate Skin Wounds Healing 91

30 with respect to Mytilus galloprovincialis (L.) extract. The

25 activities of such extracts were then investigated on
20 induced skin burns in twenty Wistar rats.
15 The proteic extracts were found to accelerate the time
10 of healing from 2224 days (untreated animals) to
5 1218 days (treated animals), based on periodic acid
0 Schiffs and Verhoffs Van Gieson stains. Furthermore, the
Group 1- Group 2 Group 3 Group 4 immunohistochemical staining confirmed the hypothesis
Control
that tissues from Group 2 (treated with amino acids from
Fig. 6 Immunostaining score of type IV collagen antibody the Rapana venosa proteic extract) were firstly healed. We
believe that Leu, Lys, Thr and Pro essential amino acids are
directly involved in the healing process, which enhanced
wound reparation by reducing inflammatory infiltration and
20
burn edema and by stimulating dermal and epidermal
15 regeneration, proliferation of fibroblasts, and formation of
new well-structured capillary vessels.
10
The results showed that the employed amino acids act as
5 key signalling agents throughout the phases of healing in
the skin and may be used in the management of wounds
0
Group1- Group 2 Group 3 Group 4
and exploited as targets for therapeutic development.
Control

Fig. 7 Immunostaining score of CD 34 antibody

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