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Fiziologia - Physiology • 2015.25.1(85)FIZIOLOGIA

physiology
CONTENTS
1. Markers of Angiogenesis in Rectal Cancers .................................................................................................................................. 4
Goldis D, Tarta C, Lazar F, Neamtu C, Totolici B
2. Bacteriological Profile of Nosocomial Infections Identified in the Orthopedic Ward of County Clinic Emergency
Hospital of Braşov. Correlations with the Microorganisms from Hospital Environment ............................................................... 9
Rautia C, Nemet C, Idomir M, Godri D
3. Temporal Aspects of Full-Field ERG in Patients with Diabetes without Diabetic Retinopathy ................................................... 12
Alexandru DO, Corîci AC, Corîci OM, Sas TN, Ştefănescu-Dima A, Iancău M
4. Antibiotic Resistance of Enterococcus Spp. Strains Isolated from Outpatients with Urinary Tract Infections ......................... 17
Pepelea L, Simon L, Serban O, Junie M
5. Autofluorescence Imaging Video Endoscopy System: Principles, Technique, Colour Patterns of Early Laryngeal Cancer ..... 21
Dobre M, Doros CI, Balica NC, Poenaru M
6. Placental Stem Cells Modulate Tumor Formation in Mouse Model of Uterine Sarcoma ............................................................. 26
Anastasiu MD, Cean A, Bojin F, Gavriliuc O, Gluhovschi A, Anastasiu D, Craina M, Tatu C, Tanasie G, Panaitescu C, Paunescu V
7. Epithelial Markers Expressed by Human Mesenchymal Stem Cells upon In Vitro Induction ..................................................... 31
Ivanescu RA, Lazarovicz RA, Bojin F, Panaitescu C, Paunescu V, Tanasie G
8. Evaluation of Large and Giant Incizional Hernia Repair: a 5-Years Retrospective Observational Study ................................... 35
Noditi G, Lazar C, Noditi Ghe, Lazar F
9. Polyurethane Nanostructures Incorporating Ursolic and Oleanolic Acids: In Vitro Antiproliferative Evaluation ...................... 39
Oprean C, Borcan F, Cristea M, Bojin F, Ivan A, Mioc M, Trandafirescu C, Soica C, Paunescu V

CUPRINS
1. Markeri ai angiogenezei în cancerele rectale ................................................................................................................................. 4
Goldis D, Tarta C, Lazar F, Neamtu C, Totolici B
2. Profilul bacteriologic al infecțiilor nosocomiale depistate în Secția de Ortopedie a Spitalului ClinicJudețean de Urgență Brașov. Corelații cu flora circulantă din mediul de spital ......................................................................... 9
Rautia C, Nemet C, Idomir M, Godri D
3. Aspecte temporale ale ERG full-field la pacienții cu diabet fără retinopatie diabetică ............................................................... 12
Alexandru DO, Corîci AC, Corîci OM, Sas TN, Ştefănescu-Dima A, Iancău M
4. Rezistența la antibiotice a tulpinilor de Enterococcus Spp. izolate din infecții urinare la pacienții din ambulator ................... 17
Pepelea L, Simon L, Serban O, Junie M
5. Sistemul video de endoscopie cu autofluorescență: principii, tehnică, modele de culoare
pentru cancerul laringian incipient ............................................................................................................................................ 21
Dobre M, Doros CI, Balica NC, Poenaru M
6. Celulele stem derivate din placentă modulează formarea tumorilor în modelul animal de sarcom uterin ................................. 26
Anastasiu MD, Cean A, Bojin F, Gavriliuc O, Gluhovschi A, Anastasiu D, Craina M, Tatu C, Tanasie G, Panaitescu C, Paunescu V
7. Markeri epiteliali exprimați de celulele stem mezenchimale umane în condiții de diferențiere in vitro ..................................... 31
Ivanescu RA, Lazarovicz RA, Bojin F, Panaitescu C, Paunescu V, Tanasie G
8. Evaluarea reparării herniilor incizionale mari și gigante: studiu retrospectiv observațional pe 5 ani ....................................... 35
Noditi G, Lazar C, Noditi Ghe, Lazar F
9. Nanostructuri poliuretanice incorporând acizii ursolic și oleanolic: evaluarea efectelor antiproliferative in vitro ................... 39
Oprean C, Borcan F, Cristea M, Bojin F, Ivan A, Mioc M, Trandafirescu C, Soica C, Paunescu V
Fiziologia - Physiology • 2015.25.1(85)3MARKERS OF ANGIOGENESIS IN RECTAL CANCERSDAN GOLDIS1, CRISTI TARTA2, FULGER LAZAR2, CARMEN NEAMTU3,
BOGDAN TOTOLICI3
1Department of Anatomy, Faculty of Medicine, Pharmacy and Dental Medicine“VasileGoldiș” Western University Arad
2Department of Surgery, “Victor Babeș” University of Medicine and Farmacy Timisoara
3Department of Surgery, Faculty of Medicine, Pharmacy and Dental Medicine “VasileGoldiș” Western University Arad

ABSTRACT
Surgery reach its limits in treating rectal cancers. Old fashion chemotherapy has so many adverse effects and thesurvival rates became steady for the last decades. Efforts were made in two other directions prevention of rectal
cancers by large population screenings, and the other was research of intimate mechanism of rectal cancer
progression and identifying new markers for better targeted new anti-cancer drugs. We intend to emphasize the role ofangiogenesis markers in actual stage of research in order to settle new goals for our research. We identified threepossible paths in evaluation of the rectal cancer angiogenesis suitable for our research: microvessel density quantified
by CD34 and CD105, morphometric analysis of the intratumor and peritumor vessel marked with CD34 and CD105 inorder to achieve new patterns of progressing for rectal cancer, and a combined analyses of VEGF and podoplanin.The
biological behaviour of rectal cancers is tailored by many factors, most of them activating different pathways of vascular
growth. Reasearch should focus on the relation between these factors and finding new ways of targeting multiple
factors during chemotherapy in order to restrict tumor blood supply by slowing the angiogenesis.
Keywords: rectal cancer, angiogenesis markers, CD34, CD105, microvessels

INTRODUCTION
Surgery reaches its limits in treating rectal cancers. Old
fashion chemotherapy has so many adverse effects andthe survival rates became steady for the last decades.
Efforts were made in two other directions prevention ofrectal cancers by large population screenings, and theother was research of intimate mechanism of rectal
cancer progression and identifying new markers forbetter targeted new anti-cancer drugs. We intend toemphasize the role of angiogenesis markers in actual
stage of research in order to settle new goals for our
research. We identified three possible paths in evaluationof the rectal cancer angiogenesis suitable for our
research: microvessel density quantified by CD34 andCD105, Morphometric analysis of the intratumor andperitumor vessel marked with CD34 and CD105 in orderto achieve new patterns of progressing for rectal cancer,
and a combined analyses of VEGF and podoplanin.
Immunohistochemistry assay and evaluation of the
impact of microvessel density in rectal cancers
CD34 is an endothelial marker used in
immunohistochemistry testing of the blood vessel both innormal and tumor tissues. Microvessel density (MVD)
represents one of the markers for the blood vessel of thethrectal malignant tumors. Its expression was studied
intense and quantified for colon cancers and colon cancer
metastasis, but not so often for rectal cancers. The studyof CD34 in rectal tumors was associated with local
inflammation and tumor intravascular emboli. Though,being a marker for all endothelial cells, malignant andbenign, without specifity for malignant tissue blood vessel,
its expression did not gain general acceptance as a major
prognostic factor for the evolution and treatment of rectal
cancers [1].This is the reason why CD34 usage as amarker for rectal cancer needs association of anothermarker, which should be more specific in assessing
activated endothelial cells from the tumor vessels. Today,
the most frequently used marker for quantifying activatedendothelial cells of the malignant tumors, and obtaining
MVD at this level, is endoglin (CD105). CD105 has an
expression at the level of activated endothelial cells of thetumor, but no expression in the endothelial cells of thenormal tissue [2]. Endothelial cells have many markers
with variable specificity and sensibility, depending on thedevelopment stage of these cells, pathological or
physiological state, and on the local organ environment.
When analyzed separately these markers did not show
any prognosis importance, but we believe that a combined
analyze of both markers might lead to an improvement intheir role as prognosis factors.

thReceived January 15 2015. Accepted February 10 2015. Address for correspondence: Goldis Dan, MD, PhD, First Surgical
Clinic, Arad County Hospital, 2-4 AndrenyKaroly Street, RO-310037; phone: +40724251243; e-mail: goldisdan@yahoo.com

4

Fiziologia - Physiology • 2015.25.1(85)MVD CD34 for the tumoral tissue is reported to haveelevated numbers in literature [3, 4]. MVD CD34 use as asingle marker for rectal cancer and its metastasis is not
accepted anymore [5]. We supposed that analyzing the
MVD CD34 ofintratumor (IT) area and peritumor(PT) area
and associating these values with TNM staging mightbring some better results in assessing MVD CD34 role inrectal cancer. Differences between MVD IT and PT in
rectal cancers and association of these with TNM staging
was based upon ultrasound methods of determining thetumor blood supply, showing that MVD CD34 did notcorrelate with any TNM parameters [6]. Others
demonstrated that there is a correlation between MVD
value and N parameter [7]. Zhou et al showed that
vascular spreading with CD34 and CD105 positive
vessels was a factor of bad prognosis for rectal cancers
in the first and second TNM stages [8]. Li et al
determined the plasmatic of CD105 and correlated theselevels with MVD CD 105 finding a statistical significancebetween tissue expression, plasmatic levels and survival
rates after Duke classification showing once more that
CD105 is an independent prognosis factor for survival inrectal cancer [9].
Morphometric analysis of the intratumor andperitumor vessel marked with CD34 and CD105
MVD was previously a widely used parameter for the
study of microscopic tumor angiogenesis in various typesof tumors. The method used for the first time by Weidner
et al [10] has proved to be insufficient over time in theevaluation of different types of tumor angiogenesis in thetumor. For this reason to the assessment of tumor
vessels was attempted the addition of other parameters,such as the study of endothelial cell proliferation [11],microscopic analysis of the bifurcation of the tumor
vessels, and the vascular area [12]. The combination of
all these factors appears to have a significant correlation
with clinical and biological factors and prognosis invarious tumor types. Quantification of endothelial area isa useful parameter in the evaluation of tumor vessels, but
at the same time is an extremely laborious method
despite the existence of computerized systems for suchprocedures. For this reason, studies which include theassessment of tumor vessels are sparse in thecolo-rectal
lesions, which are nominated in less than 120 articles.
Furthermore, correlations with TNM system parameters,
as well as tumor grade or the presence of anemia andleukocytosis are extremely rare and only sporadically
being mentioned as studies conducted only in a few
countries.Wedid not find a comparative analysis ofvascular area between CD34 and highlighted vessels
identified by endoglin (CD105).For the above reasons we
considered useful reassessment of the tumor vessel area
highlighted with CD34 in rectal tumors and more, acomparison between vascular area identified by CD34
Fiziologia - Physiology • 2015.25.1(85)and CD105, given that endoglinintratumoral andperitumoral highlights activated vessels.
Usually the study of tumor angiogenesis is achieved byusing a single marker for quantifying endothelial MVD,
the CD34. Currently there are only 9 studies reporting ofa single marker to quantify MVD strictly for rectal tumors(without including those of the colon). We recommend
the use of two comparative quantificationswith CD34 andCD105 in order to identify patients who may apply for
individualized therapy. For rectal tumors there are
currently in the literature studies that compare the MVD
measured by CD34, and CD 105 in close interrelation
with the expression of VEGF, suggesting that VEGF
inhibition is effective in reducing the vascular density [13].
We believe that the method of quantifying the positive
CD105 vessel area can be a useful parameter for
determining the optimal dose of humanized anti CD105
that could be applied to rectal tumors in the future giventhat currently we did not identified in the literature data on
the use of this therapy in rectal cancers and to a lesser
extent in the colon. This couldlead to a tumor vasculature
original analysis of rectal tumors with a direct impact ontargeted therapy using humanized monoclonal antibodies.
Reverse significant correlation between CD34 positive
vessels area and the CD 105 positive vessels in the areaadjacent to rectal tumors supports the use of anti CD105
rectal tumors because it can target tumor vessels withminimal side effects on normal tissues. One study by
Takahashi et al demonstrated that serum levels ofendoglin in patients with metastatic colorectal cancer was
significantly increased compared with the group ofpatients with the same tumor type, but without
metastases. This suggests that endoglin is a systemic
factor involved in hematological and especiallyinflammatory tumor lesions (14).
Factors that influence the mechanisms of theangiogenesis in rectal tumors
VEGF is a therapeutic target in many types ofmetastatic cancer, bevacizumab therapy was applied as
first line treatment in metastatic colorectal cancer [15].
Despite approval of bevacizumab as adjuvant therapy
in colorectal cancer it is still controversial and its
effectiveness depends on a number of factors based onage [16] and ending with survival and disease-freeinterval of patients who benefited from such therapies[17, 18]. Polymorphism of VEGF gene in colorectal
cancers partly explains the controversial results
regarding the impact of bevacizumab on prognosis andsurvival in these patients [19, 20]. Most studies quantify
the expression of VEGF often limited to tumor cells
without having to quantify the expression of VEGF andinflammatory infiltration associated with themalignancies. Currently in the literature there are only
five possible studies of VEGF expression in5inflammatory cells describing the effect ofbemacizumabtherapy on the factors secreted by thestromal cells, especially macrophages [21]. Accordingly
to these studies blocking VEGF enhances an
inflammatory pathway and suggest that the use of
combinedtherapies,
anti-inflammatory
andbemacizumab together would be more beneficial in
optimizing the effect of neoadjuvant treatment. A recent
study by Caramanolis et al suggest that rectal tissue after
irradiation had a decrease of VEGF expression with MVD
growth that was attributed to inflammation that appeared
frequently post irradiation and this is considered anadverse effectradiotherapyon rectaltissue. These data
were reported in radiation proctitis [22]. Correlation of
VEGF expression with clinical and biological factors inrectal tumors suggest their usefulness in quantifying
serum of patients correlated with the amount ofleukocytes, platelets and carcinoembryonic antigen [23].
Kwon et al demonstrated increased serum levels of
VEGF in patients with colorectal cancer preoperatively
and reported that this increased levels were a factor ofpoor prognosis for overall survival of these patients [24].
These results obtained in 2010 were validated a year ago
by Wang et al after a metaanalysis, they certified the useof VEGF as prognostic biomarker, and also of the MVD
proving that they have a major impact on survival andprognosis in other types of cancer [25]. Based on theabove we decided that in our study we should quantify theexpression of VEGF differentiated between distance andnear normal rectal mucosa adjacent to the tumor andinflammatory infiltrate tumor bores of rectal tumors
included in the study. We also wanted to identify the impactof your MVD VEGF cuantifiacte by two markers (CD34 andendoglin).
All these data we considered to be useful for a better
quantification of angiogenesis in rectal cancers. The
blood vessels are not the only way of metastasingfor
rectal tumors. Lymphatic vessels are another way ofdisseminating tumor cells. Factors influencing the
progression and metastasis on the lymphatic pathways
are not fully studied rectal cancers. Except limphatic
vessels MVD in rectal cancer, lymph podoplanin
quantification was not a research priority. It is well known
that podoplanin is expressed differently in different tumor
cell types [26] and is a factor that favors tumor
progression and metastasis [27, 28]. Podoplanine study
in tumor lesions was centered more on expression in
myofibroblasts type stromal cells which were
demonstrated to have an increase in local invasionleading to a decrease in survival [29].
Podoplaninhas become a therapeutic target in studies ontumor tissues using antibodies antipodoplanine and there
are reportsshowing significant inhibitory effects on
reducing tumor growth without harming normal tissues[30]. Podoplanin concordant expression in tumor cells inesophageal carcinomas reported it suggested that
6

positive interaction between stromal cells and tumor cells
is an independent prognostic factor in tumor biology ofthis type [31]. This support once again as a potential
therapeutic target podoplanine use. For this reason anddue to lack of data on podoplanin expression in tumor
cells in rectal cancer we consider that its quantification
should be correlated with that of VEGF.

CONCLUSION
The biological behaviour of rectal cancers is tailored bymany factors, most of them activating different pathwaysof vascular growth. Reasearch should focus on therelation between these factors and finding new ways oftargeting multiple factors during chemotherapy in order torestrict tumor blood supply by slowing the angiogenesis.

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19. Jannuzzi AT, Özhan G, Yanar HT, Alpertunga B. VEGF
gene polymorphisms and susceptibility to colorectal cancer.
Genet Test Mol Biomarkers, 2015;19(3):133-7
20. Jeon YJ, Kim JW, Park HM, Jang HG, Kim JO, Oh J,
Chong SY, Kwon SW, Kim EJ, Oh D1, Kim NK. Interplay
between 3'-UTR polymorphisms in the vascular endothelial
growth factor (VEGF) gene and metabolic syndrome indetermining the risk of colorectal cancer in Koreans. BMC
Cancer, 2014; 14:881.
21. Xu L, Duda DG, di Tomaso E, Ancukiewicz M, Chung DC,
Lauwers GY, Samuel R, Shellito P, Czito BG, Lin PC, Poleski M,

Fiziologia - Physiology • 2015.25.1(85)Bentley R, Clark JW, Willett CG, Jain RK. Direct evidence that
bevacizumab, an anti-VEGF antibody, up-regulates SDF1alpha,
CXCR4, CXCL6, and neuropilin 1 in tumors from patients withrectal cancer.Cancer Res, 2009;69(20):7905-10.
22. Karamanolis G, Delladetsima I, Kouloulias V, Papaxoinis
K, Panayiotides I, Haldeopoulos D, Triantafyllou K, Kelekis N,
Ladas SD. Increased expression of VEGF and CD31 in
postradiation rectal tissue: implications for radiation proctitis.
Mediators Inflamm, 2013;2013:515048.
23. Tsai WS, Changchien CR, Yeh CY, Chen JS, Tang R,
Chiang JM, Hsieh PS, Fan CW, Wang JY. Preoperative plasma
vascular endothelial growth factor but not nitrite is a usefulcomplementary tumor marker in patients with colorectal cancer.
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24. Kwon KA, Kim SH, Oh SY, Lee S, Han JY, Kim KH, Goh
RY, Choi HJ, Park KJ, Roh MS, Kim HJ, Kwon HC, Lee JH.
Clinical significance of preoperative serum vascular endothelial
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25. Wang Y, Yao X, Ge J, Hu F, Zhao Y.Can vascularendothelial growth factor and microvessel density be used as
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26. Ochoa-Alvarez JA, Krishnan H, Pastorino JG, Nevel E,
Kephart D, Lee JJ, Retzbach EP, Shen Y, Fatahzadeh M,
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7

MARKERI AI ANGIOGENEZEI ÎN CANCERELE RECTALEREZUMAT
Chirurgia a ajuns la unpunct critic in tratarea cancerelor rectale. Chimioterapia prezinta numeroase efecte
adverse, iar rata de supravietuire a atins un prag stabil, fara modificari in ultimele decenii. Pentru preventia
cancerelor colorectale sunt depuse eforturi in doua directii, prin testari screening pe grup epopulationale mari siprin cercetarea mecanismelor de progresie a cancerelor rectale si identificarea unor noi markeri pentru o maibuna tintire a noilor agenti anti-tumorali. In acest studiu ne propunem sa subliniem rolul markerilor angiogenici lanivelul actual al cunoasterii, pentru a stabili noi obiective ale cercetarii. Am identificat trei posibile cai de evaluare
a angiogenezei in cancerele rectale, care sunt utile cercetarii desfasurate prin acest studiu: densitatea
microvaselor cu antificata prin expresia CD34 si CD105, analiza morfometrica a vaselor intratumorale si
peritumorale, marcate cu CD34 siCD105, pentru evaluarea modelelor de progresie a cancerelor rectalesi analiza
combinata a VEGF si podoplaninei. Comportamentul biologic al cancerelor rectale este influentat de factori
multipli, majoritatea activand diferite cai implicate in dezvoltarea vasculara. Cercetarile ar trebui directionate spre
relatia dintre acesti factori si spre identificarea unor noi modalitati de tintire multifactoriala cu ajutorulchimioterapiei, pentru a restrictiona aportul sanguin la nivel tumoral, prin incetinirea procesului de angiogeneza.
Cuvintecheie: cancer rectal, markeri angiogenici, CD34, CD105, microvacularizație

8

Fiziologia - Physiology • 2015.25.1(85)BACTERIOLOGICAL PROFILE OF NOSOCOMIAL INFECTIONSIDENTIFIED IN THE ORTHOPEDIC WARD OF COUNTY CLINIC
EMERGENCY HOSPITAL OF BRAŞOV. CORRELATIONS WITH
THE MICROORGANISMS FROM HOSPITAL ENVIRONMENT
C. RAUTIA1, 2, C. NEMET2, M. IDOMIR1, 2, D. GODRI2
1Transilvania University of Brasov, Faculty of Medicine
2County Clinic Emergency Hospital of Braşov

ABSTRACTThe purpose of this paper is to implement in the orthopaedic ward of County Clinical Emergency Hospital of Brasov various
activities and means leading to decreased incidence of nosocomial infections. The target population is the inpatients on theorthopedic ward, in 2011-2013, a total of 7551 patients. We aimed a longitudinal, analytic cohort and retrospective study
through which to highlight the possible correlations between circulating flora from the orthopedic ward with the one
identified in reported nosocomial infections or in wound infections detected passively. The measure association was shownby the size of the correlation coefficient. Germs circulating in the ward and with potential of nosocomial infections wereidentified; the first is Staphylococcus aureus, which is found in the etiology of nosocomial infections from the ward, with
Acinetobacter, followed by Escherichia coli, Klebsiella and Acinetobacter with Enterococcus.
Key-words: nosocomial infections, circulating flora, microbial resistence

INTRODUCTION
Over time, nosocomial infections have been establishedin an important chapter of infectious pathology that hasconstantly developed along with the diversity of medical
procedures provided to healthy and ill people [2, 4, 5].
They appear due to changes occurring in the humanbody as a result of abusive use of antibiotics, the upward
trend for the prevalence of immunocompromised hosts,
the disturbance of relations between different categories
of pathogens, the decrease of general resistance ofimportant population groups, socio-economic justified,and due to the use of risk practices [1, 3, 6, 7].
The purpose of this paper is to implement, within theward of orthopedics, different activities and means thatlead to decreased incidence of nosocomial infections. As
objectives we mention highlight the associations of mostcommon germs from the orthopedic ward and the studyof their degree of resistance.

MATERIAL AND METHODSThe target population is the inpatients on the orthopedic
ward of County Clinical Emergency Hospital of Brasov, in
2011-2013, a total of 7551 patients. In the first stage of
research we have established the study group asthreported nosocomial infection cases that are confirmed
clinical, laboratory and epidemiological according to casedefinitions agreed at European level. The data from ourstudy were collected from the observation charts of
inpatients. Additionally, the records of surveillance ofwound nosocomial ininfection from the department ofprevention and control of the hospital were analyzed.
We consulted the laboratory data on the etiology ofwound infection detected and the degree of microbial
resistance to isolated germs and the data regarding thecirculating flora from the ward within the surveillancebacteriological activities of hospital environment. All data
were recorded in the registry books of the hospitallaboratory and stored by the program WHO / Net.
We aimed a longitudinal, analytic cohort andretrospective study to highlight the possible correlations
between circulating flora on hospital environment fromthe orthopedic ward with the one identified in reported
nosocomial infections or in wound infections detectedpassively, in order to control and reduce the nosocomial
infections in this ward. The measure association wasshown by the size of the correlation coefficient (r). The r
values of 0.3-0.4, a weak correlation is estimated at
0.5-0.7 values showed an average correlation and avalue of over 0.7 shows a correlation of high significance.

thReceived 10 of February 2015. Accepted 10 of March 2015. Address for correspondence: Codruta Nemet, MD, Emergency
Clinical County Hospital of Brasov, Faget Street No. 28, Brasov, Brasov County, RO-500035; phone: +40372 676100;
fax: +40372676351; e-mail: codruta_nemet@yahoo.com

Fiziologia - Physiology • 2015.25.1(85)9RESULTS
7 cases of nosocomial infection have been reported in2011, 5 of them were surgical wound infection, one
septicemia and one urinary infection. In 2012 and 2013
all infections were wound infections. During 2011-2013,
from the analysis of unreported cases of nosocomial
infections, identified from the medical records on
epidemiological, clinical and laboratory criteria - 101
cases, we found a similar distribution as the location of
the reported cases (Table I).
Table I. The types of nosocomial infection on the orthopedic
ward
Nosocomial infection
diagnosis Reported (R) or
unreported (UR)bronchopneumonia
Clostridium difficile
infection
deep surgical wound
infection
superficial surgicalwound infection
septicemia
urinary infection
Total

Frequency
2013
R
U

Frequency
2012
R
U

Frequency
2011
R
U

4

3

22

3

28

1

8

2

14

30

1
1
7

42

4

29

29

4

We mention that of the 116 possible cases of nosocomial
infections only 15 (10%) were reported by physicians, theremaining 101 were lost for various reasons, fear of
sanction or indifference to thick criteria of hospital
infections (clinical, epidemiological or laboratory microbiological).
In 2011, in the orthopaedic ward from the circulating flora
the largest share has MRSA, 45% of all strains followedby the Escherichia coli (15%), Klebsiella (9%),
Enterococcus (9%), Acinetobacter (9%), Enterobacter(4%), Proteus (4%), Pseudomonas (3%), Streptococcus
spp (1%) and Citrobacter (1%). For 2012, the share ofstaph remains unchanged (45%); Escherichia coli,
Enterococcus and Klebsiela also have a stationary
tendency 15% and 9% respectively. Frequency for
Acinetobacter has a slightly growing trend to 11%. In
2013, the circulating flora in the orthopaedic ward has aslightly upward trend for MRSA at 48%, the frequency
drops to 11% for Escherichia coli, Acinetobacter andEnterococcus remain at 10% and the share for Klebsiellatype strains drops to 8%.
In 2011, 72% of nosocomial infections are due toGram-positive flora: Staphylococcus, Enterococcus,
Clostridium dificille) circulated by medical personnel orpatients colonized and disseminated by nosocomial risk
medical practices. 28% is due to Gram negative flora(Proteus, Escherichia coli, Acinetobacter, Klebsiella, and10

Pseudomonas) present in the ward due to moderately
effective cleaning and disinfection (Figure 1).

Fig. 1. The etiology of nosocomial infections in the orthopedic
ward in 2011

In 2012 the situation on the etiology of nosocomial
infections in orthopedic ward radical changes,
incriminated germs being Gram negative, probably due
to transmission through contaminated objects (Figure 2).
In 2013 the etiology of nosocomial infections is 50%
Gram negative and 50% Gram positive, which is due
both staff and patient colonization and possibly tocontaminated objects (Figure 3).

Fig. 2. The etiology of nosocomial infections in the orthopedic
ward in 2012

Fig. 3. The etiology of nosocomial infections in the orthopedic
ward in 2013

In our study we tried to measure the association between
circulating germs and nosocomial infections both in thehospital and in the orthopedic ward. For the year theprocessed data showed the size of the correlation
coefficient between the germs isolated from hospital andthose from the nosocomial infections of the entire unit
0.158. This demonstrates a lack of correlation; theinfections acquired in our hospital as a result of medical
Fiziologia - Physiology • 2015.25.1(85)maneuvers are not due to environmental contaminationand to the lack of cleaning or disinfection, but rather areassociated with endogenous factors of the patient.
If we measure the same combination in orthopedic ward
we already observed an average correlation of 0.5326
between circulating germs and those involved in
nosocomial infections.

CONCLUSIONS
There were identified germs circulating in the orthopedic
ward and with potential for nosocomial infections, in firstare Staphylococcus aureus, which is found in the etiology
of nosocomial infections from the ward with Acinetobacter,followed by Escherichia coli, then Klebsiella andAcinetobacter with Enterococcus.
On the orthopedic ward we observe an averagecorrelation of 0.5326, between circulating germs andthose involved in nosocomial infections.

REFERENCES
1. Angelescu M, Alexandru C. Infecţiile nosocomiale- etiologie,
profilaxie, tratament. Viaţa Medicală 2000; 8: 1-3.
2. Buiuc D, Neguţ M. Tratat de Microbiologie Clinică, București,Editura Medicală, 2009.
3. Collee JG, Miles RS, Watt B. Tests for the identification ofbacteria. Mackie and McCartney. Practical Medical
Microbiolog. London: Ed. Churchill Livingstone, 1996.
4. Dashtbozorg A, Mahdinasab A, Latif M, et al. Prevalence of
infection after surgery of FX fixation in patients hospitalized in
the orthopedic ward of Ahvaz Razi hospital. Jundishaour Med J,
2008; 56: 45-51.
5. Deac L. Infecțiile nosocomiale, o permanentă realitate
medical. Cluj-Napoca: Ed. Dacia, 1997.
6. Harbarth S, Fankhauser C, Schrenzel J, et al. Universal
screening for methicillin-resistant Staphylococcus aureus at
hospital admission and nosocomial infection in surgical patients.
JAMA 2008; 299(10): 1149-1157.
7. Rioux C, Grandbastien B, Astagneau P. Impact of a six-year
control programme on surgical site infections in France: resultsof the INCISO surveillance. J Hosp Infect, 2007; 66: 217-223.

PROFILUL BACTERIOLOGIC AL INFECTIILOR NOSOCOMIALE DEPISTATEIN SECTIA DE ORTOPEDIE A SPITALULUI CLINIC JUDETEAN DE URGENTA
BRASOV. CORELATII CU FLORA CIRCULANTA DIN MEDIUL DE SPITALREZUMAT
Scopul acestei lucrări este de a implementa, în cadrul secţiei de ortopedie a Spitalului Clinic Judeţean deUrgenţă Braşov diferite activităţi şi mijloace ce duc la scăderea incidenţei infecţiilor nosocomiale. Populaţia ţintă
este reprezentată de bolnavii internaţi pe secţia clinică de ortopedie, în perioada 2011-2013, in total 7551 pacienţi.
Ne-am propus un studiu longitudinal, analitic, de cohortă, cu caracter retrospectiv, prin care să evidentiem
corelatiile posibile intre flora circulanta din sectia de ortopedie cu cea identificata in infectiile nosocomialedeclarate sau in infectiile de plaga depistate pasiv. Măsura asocierii a fost redată prin mărimea coeficientului decorelaţie. S-au identificat germenii circulanţi pe secţie şi cu potenţial de nosocomialitate, pe primul loc se situează
Staphylococcus aureus, care se regăsește și în etiologia în infecțiile nosocomiale de pe secție împreună cu
Acinetobacter, urmat de Escherichia coli, Klebsiella și Acinetobacter cu Enterococ.
Cuvinte cheie: infectii nosocomiale, germeni circulanți, rezistență microbiană.

Fiziologia - Physiology • 2015.25.1(85)11TEMPORAL ASPECTS OF FULL-FIELD ERG IN PATIENTSWITH DIABETES WITHOUT DIABETIC RETINOPATHY
DRAGOŞ OVIDIU ALEXANDRU1, ANDREEA CORNELIA CORÎCI2, OANA MARIACORÎCI2, TEODOR NICUŞOR SAS3, ALIN ŞTEFĂNESCU-DIMA4, MARIA IANCĂU2
1University of Medicine and Pharmacy of Craiova, Faculty of Medicine, Department of Medical Informatics and Biostatistics
2University of Medicine and Pharmacy of Craiova, Faculty of Medicine, Department of Physiology
3University of Medicine and Pharmacy of Craiova, Faculty of Medicine, Department of Radiology
4University of Medicine and Pharmacy of Craiova, Faculty of Medicine, Department of Ophthalmology

ABSTRACT
Aim: To evaluate implicit time changes of full-field ERG in patients with type 2 diabetes without diabetic retinopathy.
Material and Method: The prospective study included 11 diabetic patients, aged between 50 and 80 years old, without
diabetic retinopathy and 14 aged-matched controls. All of the participants underwent full-field ERG and ophthalmologic
examination to exclude any ophthalmologic pathology. The ERGs were recorded with Metrovision MonPackOne system,
which has the same stimulus parameters as the ISCEV standard. The implicit times were analyzed for “a” and “b” waves indark-adapted 0.01 ERG, dark-adapted 3 ERG, dark-adapted oscillatory potentials, light-adapted 3.0 ERG, and 30Hz flicker
ERG, and compared between diabetic patients and healthy subjects. Results: The significantly delayed responses,
between diabetic patients and healthy subjects older than 50 years, were the dark-adapted oscillatory potentials N2 (21.91± 0.85ms versus 22.45 ± 1.02ms, p=0.044) and P2 (25.49±1.01ms versus 26.13±0.94ms, p=0.027), dark-adapted,
scotopic 3 “b” wave (42.89±2.74ms versus 44.99±3.16, p=0.015) and light adapted, photopic 3 “b” wave (31.62±1.62ms
versus 32.72±1.36ms, p=0.014). Conclusion: The electrophysiological findings from the present study showed that, apartfrom oscillatory potentials changes, reported in previous studies, there was a significant delay in the cone system “b” wave,
which indicates that the functional integrity of the inner retina is compromised before the appearance of clinical changes.
Keywords: ERG, Diabetes Mellitus, ERG implicit time

INTRODUCTION
Diabetes mellitus is one of the most serious global healthproblems and is associated with increased levels ofvisual impairment after adjusting for the effects of age,sex, race, educational level, blood pressure, smoking
and body mass index [1]. The incidence of type 2
diabetes is increasing worldwide and hence the risk ofdeveloping complications [2]. The prevalence of visual
impairment in adults with and without diabetes vary from
3.8 to 13% and from 1.4 to 2%, respectively, and therelative risk of blindness is 5.2 times higher in patientswith diabetes than in those without diabetes [3].
Diabetic retinopathy (DR), the most common complication indiabetes, is the fifth leading cause of blindness worldwide
and the leading cause of visual impairment in adults ofworking age in industrialized countries [4]. It is notuncommon in clinical practice that DR is already present at
the first visit after diagnosis of type 2 diabetes [5].
Classically, DR is described as a microangiopathy,
affecting the perycites and endothelial cells [6], causedth

by the metabolic effects of hyperglycemia. In recent
years, more attention has been focused on theneurodegenerative aspects of DR [2]. Barber was thefirst who observed that one month after inducing diabetes
in rats, using streptozotocin there was a high rate of
apoptosis in the neuroretina without a significant
apoptosis in endothelial cells [7]. Glial cell activation isanother phenomenon that occurs in diabetic retina, apartfrom apoptosis in the neuroretina. In other words, anearly event in the pathogenesis of DR which participatesin the microcirculatory abnormalities is the retinal
neurodegeneration [7].
Since DR constitutes a major cause of visual
impairment and blindness in the world, is extremely
important to assess the retinal function in patients withdiabetes (X). Color vision defects, reduced contrast
sensitivity and visual field alterations have beenreported in diabetic individuals with minimal or no
retinopathy. All these are subjective tests. Thus, it isnecessary to evaluate the retinal function in diabetes inan objective way, using electrophysiological tests [5].

thReceived 10 of February 2015. Accepted 8 of March 2015. Address for correspondence: Andreea C. Corici, M.D., Universityof Medicine and Pharmacy of Craiova, Petru Rares St., No.2, Craiova-200349, Romania; phone/fax: +40251 426688; e-mail:corici.andreea@gmail.com

12

Fiziologia - Physiology • 2015.25.1(85)Although, clinical exams focus on the visualization ofretinal lesions in diabetic patients, electrophysiologicalchanges have been shown to occur before the onset ofclinically visible retinopathy. Hence, an electrophysiologicalassessment of retinal function could be a valuable
monitoring method for retinal health in diabetic patients[8].
Standard full-field ERG is an objective method whichreflects the function of the whole retina. In recent years, a
big number of studies showed the ERG changes inindividuals with diabetic retinopathy, but these studies
mainly focused on patients with long duration diabetes [2].
It is already known, that the oscillatory potentials are
reduced in amplitude and delayed in diabetic patients
without retinopathy, but it is not clearly shown what othercomponents of full-field ERG are modified in this group ofpatients [8]. As we mentioned above, there can be
retinopathic changes in the retina in non-diabetic
subjects, especially due to aging. The aim of our study
was to evaluate the ERG implicit time changes, inpatients with type 2 diabetes, without visible diabetic
retinopathy and to eliminate the changes that may be
due to aging by comparing them to an aged-match
healthy group of subjects.

MATERIAL AND METHOD
Subjects
Full-field ERGs were recorded for 28 eyes from 14
healthy subjects aged 50 to 80 years old (63.00 ± 11.62)
who had no history of ophthalmologic disease, normal
findings on eye examination, normal visual acuity, clear
optic media, no history of diabetes mellitus or othervascular or neurologic disease which could influence
the ERG. The ERGs were also recorded for 22 eyes of
11 diabetic patients without diabetic retinopathy, aged
between 50 and 80 years old (63.18 ± 7.09). All thesepatients were seen by an ophthalmologist and recruitedfrom the Diabetic Eye Department. The inclusion criteria
for these patients were: clinical diagnosis of type 2
diabetes mellitus for less than ten years, no othervascular or neurologic diseases, no history ofophthalmological disease, normal findings on eye
examination without any lesion of diabetic retinopathyon photo fundus, normal visual acuity, clear optic
media.
The study was performed according to the tenets of the
Declaration of Helsinki and was approved by the
University Ethics Committee. All the subjects were fully
informed about the possible consequences of theresearch protocol, and they sign their approval forparticipation.
Recording protocol
The ERGs were recorded at Ophthalmological ResearchCentre “Ocularius”, Craiova, under an agreement with
Fiziologia - Physiology • 2015.25.1(85)the University of Medicine and Pharmacy of Craiova. We
used the MonPackOne System (Metrovision, Perenchies,
France), which has stimulus parametres according to the
ISCEV standards. Recording electrodes were HK loop
type ("Hawlina- Konec loop") and the reference andground ones were Ag-AgCl cup type.
The research methodology consisted, for each subject,
in:
A. Usual eye examination: visual acuity, biomicroscopy,
intraocular pressure, fundus examination, refraction,color vision,
B. ERG recording according to ISCEV protocol:
1. fully dilatated pupils using 1% tropicamide and2.5% phenylephrine eye drops;
2. skin cleansing with an abrasive paste andmedicinal alcohol;
3. 4% xiline in the lower conjunctival bag;
4. electrods’ placement: the active electrodes
were placed on the free edge of the lower
eyelid for each eye; the reference electrodes
were placed near each orbital rim, with theground electrode placed on the vertex, using a
conductive paste;
5. dark adaptation for 20 minutes;
6. dark adapted ERG recording: rod response: the stimulus is a dim blue
flash of 0.01 cd.s.m-2 , with an interval of 2
seconds between flashes; combined rod-cone response: the stimulus
is a white flash of 3.0 cd.s.m-2 , with aninterval of 10 seconds between flashes; oscillatory potentials: the stimulus is a white
flash of 2.0 cd.s.m-2 , with an interval of 165
seconds between flashes.
7. light adaptation for 10 minutes;
8. light-adapted ERG recording: single flash cone response: a 3.0 cd.s.m-2
stimulus, with an interval of 0.5 seconds
between flashes and a backgroundluminance of 30cd.m-2
 30 Hz flicker: 30 stimuli of 3.0 cd.s.m -2 persecond.
Statistical analysis
The analysis of the results of ERG recordings was
performed using Microsoft Excel (Microsoft Corp.,
Redmond, WA, USA), together with the XLSTAT add-on
for MS Excel (Addinsoft SARL, Paris, France). We used
the Anderson-Darling test to assess data normality.
Because most of the data of diabetic patients did not
follow a Gauss distribution, we had to use nonparametrictests (i.e. Mann-Whitney test) to compare data between
the two groups.

13

RESULTS

thedark-adapted
oscillatory
potentials
N2(21.91±0.85ms versus 22.45±1.02ms, p=0.044, Figure 2)
and P2 (25.49±1.01ms versus 26.13±0.94ms, p=0.027,
Figure 3), dark-adapted, scotopic 3 “b” wave
(42.89±2.74ms versus 44.99±3.16, p=0.015, Figure 4)
and light adapted, photopic 3 “b” wave (31.62±1.62ms
versus 32.72±1.36ms, p=0.014, Figure 5). Other ERG
parameters showed a delay for DM+ patients, but thestatistical significance was slightly above 0.05, so further
studies on larger patients group should be conducted.

Fig. 1. Sample ERG recording for subjects without Diabetes
Mellitus (DM-) and subjects with Diabetes Mellitus (DM+)

We computed mean values and standard deviations forthe implicit times of all parameters described for the ERG
responses, recorded according to the ISCEV standard
procedure (Figure 1).
Table I. Comparison of mean implicit times between subjects
without Diabetes Mellitus (DM-) and subjects with Diabetes
Mellitus (DM+)
p
Parameter

DM-

DM+

Mann-Whitney

Significance29.62 ± 2.29

30.31 ± 2.57

0.320

- NS

61.27 ± 2.99

62.90 ± 2.99

0.061

- NS

21.03 ± 2.43

21.40 ± 2.12

0.573

- NS

42.89 ± 2.74

44.99 ± 3.16

0.015

-S

15.87 ± 1.10

15.82 ± 0.46

0.833

- NS

31.62 ± 1.62

32.72 ± 1.36

0.014

-S

18.38 ± 2.07

18.70 ± 2.04

0.586

- NS

30.69 ± 2.57

31.37 ± 2.18

0.324

- NS

21.91 ± 0.85

22.45 ± 1.02

0.044

-S

25.49 ± 1.01

26.13 ± 0.94

0.027

-S

29.01 ± 1.37

29.64 ± 1.04

0.081

- NS

32.72 ± 1.78

33.32 ± 1.26

0.189

- NS

Scotopic
0.01 ERG a

Fig. 2. Comparison of mean values of implicit times fordark-adapted oscillatory potentials N2 wave between DM- andDM+ subjects

Scotopic
0.01 ERG b
Scotopic 3.0
ERG a
Scotopic 3.0
ERG b
Photopic 3.0
ERG a
Photopic 3.0
ERG b
Photopic 3.0
Flicker a
Photopic 3.0
Flicker b
Oscillatory
potentials N2
Oscillatory
potentials P2
Oscillatory
potentials N3
Oscillatory
potentials P3

Fig. 3. Comparison of mean values of implicit times fordark-adapted oscillatory potentials P2 wave between DM- andDM+ subjects

The significantly delayed responses, between diabetic
patients and healthy subjects older than 50 years, were
14

Fiziologia - Physiology • 2015.25.1(85)Fig. 4. Comparison of mean values of implicit times fordark-adapted (scotopic) 3 “b” wave between DM- and DM+
subjects

The full-field ERG is an objective method which reflects
the electrical answer from the whole retina [5] and thus a
specific tool for assessing the functional response of thistissue. As symptoms do not appear in the first stages ofdiabetic retinopathy, early diagnosis is a challenging stepin the prevention of complications.
Oscillatory potentials deterioration was one of the earliest
functional retinal abnormalities reported in the literature[1, 9] in patients with diabetes but without diabetic
retinopathy. Our study is in consent with these reports,
showing a delayed OP2, which probably indicates earlydamage of the neuronal synaptic activity of the amacrineand horizontal cells.
The “a” wave of both rod and cone systems had a slightly
higher, but statistically insignificant implicit time (p>0.05),
compared to age-matched controls. This finding is not inconsent with a previous study that showed a delayed “a”
wave [2], reflecting that the photoreceptors are more
affected by age than diabetes, at least at the onset of thedisease as yet there are no changes of diabetic
retinopathy.
In our study, the averaged “b” wave implicit time wasdelayed for both rod and cone systems in diabetic
patients compared to the aged-matched controls. Thesechanges in the “b” wave implicit time are consistent withdamage in the inner retinal layers in the early stages of
disease. Holopigian et al showed in their study [10], thatapart from oscillatory potentials, “b” wave is also asensitive indicator of retinal damage in patients withdiabetes and no visible diabetic retinopathy.

CONCLUSIONS

Fig. 5. Comparison of mean values of implicit times forlight-adapted (photopic) 3 “b” wave between DM- and DM+
subjects

DISCUSSIONSAs we stated before, diabetes is a disease with bothneurologic and vascular components. Recent studies
showed that neurodegeneration is an early event in thepathophysiology of diabetic retinopathy and is alreadypresent before any vascular abnormalities can be seen in
ophthalmoscopic examination [7]. The results of thepresent study strengthen these findings, showing ERG
abnormalities in patients with diabetes and normal
ophthalmologic examination.
Fiziologia - Physiology • 2015.25.1(85)By eliminating age influence on ERG responses, because
the most affected parameters in the diabetic patients without
diabetic retinopathy are the scotopic oscillatory potentials,“b” wave in scotopic 3.0 and photopic 3.0 ERG, we canconclude that the retinal dysfunction in Diabetes Mellitus
appears first in the inner retinal layers. Therefore, theaforementioned parameters could be used to assess theevolution in the initial stages of DM, before clinicalophthalmological symptoms appear.
The electrophysiological findings from the present study
showed that, apart from oscillatory potentials changes,
reported in previous studies, there was a significant delayin the cone system b wave, which indicates that thefunctional integrity of the inner retina is compromised
before the appearance of clinical changes.
ACKNOWLEDGMENT
Dragoş O. Alexandru received financial support for thispaper under the frame of European Social Fund, Human
Resources Development Operational Programme
2007–2013, project no. POSDRU/159/1.5/S/133377.
Andreea C. Corici received financial support for thispaper under the frame of European Social Fund, Human
15

Resources Development Operational Programme
2007–2013, project no. POSDRU/159/1.5/S/136893.
REFERENCES
1. Zhang X, Gregg EW, Cheng YJ, Thompson TJ, Geiss LS,
Duenas MR, Saaddine JB. Diabetes mellitus and visual
impairment: national health and nutrition examination survey,
1999-2004. Arch Ophthalmol. 2008; 126(10): 1421-7.
2. Tyrberg M, Lindblad U, Melander A, Lövestam-Adrian M,
Ponjavic V, Andréasson S. Electrophysiological studies in newly
onset type 2 diabetes without visible vascular retinopathy. Doc
Ophthalmol. 2011; 123(3): 193-8.
3. Trautner C, Icks A, Haastert B, Plum F, Berger M. Incidenceof blindness in relation to diabetes. A population-based study.
Diabetes Care. 1997; 20(7): 1147-53.
4. Luu CD, Szental JA, Lee SY, Lavanya R, Wong TY,
Correlation between retinal oscillatory potentials and retinal
vascular caliber in type 2 diabetes. Invest Ophthalmol Vis Sci.
2010; 51(1): 482-6.
5. Tyrberg M, Ponjavic V, Lövestam-Adrian M. Multifocal
electroretinography (mfERG) in insulin dependent diabetics withand without clinically apparent retinopathy. Doc Ophthalmol.
2005; 110(2-3): 137-43.
6. Lasta M, Pemp B, Schmidl D, Boltz A, Kaya S, Palkovits S,
Werkmeister R, Howorka K, Popa-Cherecheanu A, Garhöfer G,
Schmetterer L. Neurovascular dysfunction precedes neuraldysfunction in the retina of patients with type 1 diabetes. Invest
Ophthalmol Vis Sci. 2013; 54(1): 842-7.
7. Villarroel M, Ciudin A, Hernández C, Simó R.
Neurodegeneration: An early event of diabetic retinopathy.
World J Diabetes. 2010; 1(2): 57-64.
8. Hancock HA, Kraft TW. Oscillatory potential analysis and
ERGs of normal and diabetic rats. Invest Ophthalmol Vis Sci.
2004; 45(3): 1002-8.
9. Lecleire-Collet A, Audo I, Aout M, Girmens JF, Sofroni R,
Erginay A, Le Gargasson JF, Mohand-Saïd S, Meas T,
Guillausseau PJ, Vicaut E, Paques M, Massin P. Evaluation ofretinal function and flicker light-induced retinal vascularresponse in normotensive patients with diabetes without
retinopathy. Invest Ophthalmol Vis Sci. 2011; 52(6): 2861-7.
10. Holopigian K, Seiple W, Lorenzo M, Carr R. A comparison ofphotopic and scotopic electroretinographic changes in early
diabetic retinopathy. Invest Ophthalmol Vis Sci. 1992; 33(10):2773-80.

ASPECTE TEMPORALE ALE ERG FULL-FIELD LA PACIENTII CU DIABET FĂRĂRETINOPATIE DIABETICA
REZUMAT
Scop: Evaluarea modificărilor latentei undelor ERG full-field la pacienții cu diabet zaharat tip 2, fără retinopatie
diabetica. Material și metodă: Studiul prospectiv a inclus 11 pacienți cu diabet zaharat, cu vârste cuprinse între 50
și 80 de ani, fără retinopatie diabetica si 14 subiecți cu vârste similar, folosiți ca lot martor. Tuturor participanților li
s-a efectuat ERG full-field și examinare oftalmologică pentru a exclude orice patologie oftalmologică asociată.
Electroretinogramele au fost înregistrate cu sistemul Metrovision MonPackOne, care are parametrii de stimulare
in conformitate cu standardul ISCEV. A fost analizata latenta pentru undele "a" și "b", pentru ERG 0,01 cu
adaptare la întuneric, ERG 3 cu adaptare la întuneric, potențialele oscilatorii cu adaptare la întuneric, și ERG
3.0, 30Hz flicker cu adaptare la lumină, care au fost comparaţi între pacienții diabetici și subiecții sănătoși.
Rezultate: Răspunsurile întârziate semnificativ, la compararea lotului de pacienți diabetici cu lotulde subiecți
sănătoși cu vârstă peste 50 de ani, au fost potențiale oscilatorii cu adaptare la întuneric N2 (21.91 ± 0.85ms
versus 22.45 ± 1.02ms, p = 0,044) și P2 (25.49 ± 1.01ms față 26.13 ± 0.94ms, p = 0,027), unda "b" pentru ERG
3, cu adaptare la întuneric (42.89 ± 2.74ms versus 44.99 ± 3.16, p = 0,015) și unda "b" pentru ERG 3, cu
adaptare la lumina (31,62 ± 1.62ms versus 32.72 ± 1.36ms, p = 0,014). Concluzie: Rezultatele electrofiziologice
din prezentul studiu au arătat că, în afară de modificările potențialelor oscilatorii, raportate în studiile anterioare, aexistat o întârziere semnificativă pentru unda "b", ceea ce indică faptul că integritatea funcțională a starturilorinterne ale retinei este compromisă înainte apariția modificărilor clinice.
Cuvinte cheie: ERG, diabet zaharat, latenta

16

Fiziologia - Physiology • 2015.25.1(85)ANTIBIOTIC RESISTANCE OF ENTEROCOCCUS SPP .
STRAINS ISOLATED FROM OUTPATIENTS WITH URINARY
TRACT INFECTIONSLIA PEPELEA, LAURA SIMON, OANA SERBAN, MONICA JUNIE
Deparment of Microbiology, University of Medicine and Pharmacy ”Iuliu Hațieganu”, Cluj-Napoca

ABSTRACT
Objective: The main aim of our study was to isolate and identify enterococci from cases of urinary tract infection, toknow antibiotic susceptibility pattern of the isolates and also to establish the resistance phenotypes.
Material and Methods: The present study, was conducted in the Microbiology Department of Intermed Laboratory,
Cluj-Napoca, from january 2010 to december 2012. Identification, isolation and testing of the germs were done by thestandard conventional methods.The testing of antibiotic resistance was performed through Kirby-Bauer disk-diffusion
method, according to CLSI standards.
Results: A total of 62 strains of enterococci were isolated from a total of 5261 urine samples.Out of 62 strains, 58 (94%)
were Enterococcus faecalis, 4(6%) were E. faecium. The antimicrobial resistance profile for Enterococcus faecalis was:
24.13% to ampicillin, penicillin, streptomicin, gentamicin, 15.51% to nitrofurantoin, 13.79% to ciprofloxacin, levofloxacin,
ciprofloxacin, 6.89% to tetracycline. Most of the four Enterococcus faecium strains were sensitive to antibiotics, only
one from these strains presents resistance to fluoroquinolones
Conclusion: The role of the microbiology laboratory, essential for a correct anti-infective therapeutic approach,consists of performing an antibiogram, of establishing and interpreting the resistance phenotypes, thus providing
information about the resistance development mechanisms and the way of spreading of these phenotypes.
Keywords: Enterococcus; Enterococcus faecalis; Enterococcus faecium; urinary tract infections; urine culture; antibiotics

INTRODUCTION
It is a known fact that enterococci cause a large variety of
infections. Most frequently, they infect the urinary tract,
the abdomen, the blood flow, the endocardium, the biliary
tract, lesions caused by burns, and permanent catheters[1]. 80 to 90% of these infections are caused by
E.faecalis and just 10% by E.faecium [2].
The data provided by the literature indicate thatenterococci rank 4th among the causes for infections that
require hospitalization, and 3rd among the causes forbacteremia in the United States [3]. The statistics forthese cases/the degree of fatality for enterococcal
bacteremia show values ranging from 12 to 68%, withdeaths caused by septicaemia in 4 to 50% of cases [4].
Urinary tract infections are among the most frequent
human infections [5].
The aetiology of urinary infections is varied: the species
isolated most frequently is E.coli, which produces
approximately 90% of these infections, but lately,Enterococcus species have also been frequently isolated
and have become important etiological agents of these
infections [6].

The ideal treatment for urinary infections requires knowing
the bacteria involved in the aetiology of these infections andtheir sensitivity to antibiotics. The increase in the number ofantibiotics as well as their empirical and abusive use
favoured the occurrence of certain strains that are
multiresistant to antibiotics. Antibiotic resistance represents
one of the difficult issues that medical practice is facing, butalso one of the priority research themes in microbiology [7].

MATERIAL AND METHODS
A total of 5261 urine sample recovered from outpacients,
were processed. Samples from outpatients from whomEnterococcus species were isolated were collectedduring the period from january 2010 to december 2012.
Midstream urine sample in early morning was collected inwide mouth sterile container. The urine samles werecollected in sterile condition and all of them were cultured
on sheep blood agar and lactose medium forenterobacteria and incubated at 37°C.
After 48 hours, each of the enterococcal positive cultures
over than 105 CFU/ml was identified as UTI [8].

Received 15th of March 2015. Accepted 20th of April 2015. Address for correspondence: Lia Pepelea, Microbiology
Department, UMF ’’Iuliu Haţieganu” Cluj-Napoca, 6th Pasteur Street, RO-400349, Cluj-Napoca, Romania; phone:+40750774432; fax: +40264597257, e-mail: liapepelea@yahoo.com / microb@umfcluj.ro

Fiziologia - Physiology • 2015.25.1(85)17Identification of the isolates to the genus level was
performed according to: reaction on Gram staining;cellular morphology; growth and blackening of thebile-esculine agar; growth on CPS medium (bioMérieux)
and Trypticase soy agar - 5% sheep blood (bioMérieux);absence of catalase.
Antibiotic susceptibility studies were performed by discdiffusion method (Kirby-Bauer) according to the Clinicaland Laboratory Standard Institute (CLSI 2007) [9].
High level aminoglycoside resistance was determined byusing discs with 120µg/mL of gentamicin and 300µg/mLof streptomycin. Isolates were interpreted as susceptibleor resistant according to the sensitivity zones of theparticular antimicrobial as recommended by CLSI.
Comparing antibiotic susceptibility was performed for thefollowing antibiotics: Ampicillin (AM), Penicillin (P),Gentamicin (GM120), Streptomycin (STR 300),
Tetracycline (TE), Linezolid (LZD), Ciprofloxacin (CIP),
Levofloxacin (LEV), Rifampicin (RIF),Norfloxacin (NOR),
Nitrofurantoin (FT), Vancomycin (VA).
To confirm the secretion of beta lacatamase we realized
nitrocefin test (Nitrocef Matchbook stick-Hardy Diagnostics).

Enterococcus faecium (1%), Staphylococcus aureus (1%)
in that descending order (Table I). Enterococci wereisolated in pure cultures in clinically significant numbersin 62 (6%) specimens.
Table II. Different Enterococcus spp. isolated
Enterococccus spp.
Enterococcus faecalisEnterococcus faecium
TOTAL

No. of isolates
58
462

Percentage
94
6
100

Only two species of enterococci were isolated. Out ofthese two, E. faecalis (94%) was the predominant
species followed by E. faecium (6%)(Table II).
Table III. Susceptibility of the Enterococcus faecalis isolates todifferent antimicrobials
Antimicrobial

Susceptible

Intermediate

Resistant

disc

Num
ber

Percen
tage

Numb
er

Percen
tage

Num
ber

Percentage

Penicillin

44

75.86

-

-

14

24.13

Ampicillin

44

75.86

-

-

14

24.13

Levofloxacin

50

86.2

-

-

8

13.79

RESULTS

Ciprofloxacin

48

82.75

2

3.44

8

13.79

Norfloxacin

48

82.75

2

3.44

8

13.79

A total of 5261 urine samples were studied from patientswith suspected signs and symptoms of urinary tract
infection, out of which 976 specimens were positive inculture.

Gentamicin

44

75.86

-

-

14

24.13

Streptomicin

44

75.86

-

-

14

24.13

Rifampicin

58

100

-

-

-

-

Tetracyclin

54

93.1

-

-

4

6.89

Linezolid

58

100

-

-

-

-

Table I. Frequency distribution of microbial pathogens in
midstream urine of outpatients studied

Vancomycin

58

100

-

-

-

-

Nitrofurantoin

49

84.48

-

-

9

15.51

Microbial Isolates / Strains

No. of strains

Frequency%

E.coli703

72%

Proteus mirabilis

43

4%

Klebsiella pneumoniae

495%Enterobacter cloacae

1Enterococcus faecium

41%Enterococcus faecalis

58

5%Streptococcus agalactiae

55

6%Staphylococcus

24

3%

Staphylococcus epidermidis

saprophyticus

222%

Staphylococcus aureus

91%Serratia odorifera

2Serratia marcescens

3

Pseudomonas fluorescens

1

Pseudomonas aeruginosa

2

TOTAL

976

100 %

The frequency of occurrence of the isolates and theirstrains therefore, were as follows: E.Coli (72%),
Streptococcus agalactiae (6%), Enterococcus faecalis(5%), Klebsiella pneumoniae (5%), Staphylococcus
saprophyticus (3%), Staphylococcus epidermidis (2%),
18

E. faecalis isolates were resistant to ampicillin, penicillin,
streptomicin, gentamicin (24.13%) and sensitive tovancomicin, linezolid and rifampicin (100%).
Most of the four Enterococcus faecium strains weresensitive to antibiotics, only one from these strainspresents resistance to fluoroquinolones.

Fig. 1. The distribution of resistance phenotypes of E.
faecalis strains. Antibiotic resistance of Enterococcus spp.
strains isolated from outpatients with urinary tract infections

Fiziologia - Physiology • 2015.25.1(85)DISCUSSIONSCONCLUSIONSOver the past years, there has been an increasing
interest in enterococci, not only because of their capacity
to induce severe infections, but also due to theirincreased resistance to a number of antimicrobial agents[10].
In the present study, E. coli was the bacterium
predominantly involved in urinary tract infections (72%),
which is in accordance with the data obtained in otherstudies [11].
Miskeen et al. [12] detected enterococci in 7.4% of
patients with urinary tract infections.
Cornia et al. [13] identified enterococci as etiologicalagents of bacteriuria among elderly male inpatients andoutpatients in 22.5% of the cases.
In our study, from 976 positive urocultures, 58 E. faecalis
strains (94%) and 4 E. faecium strains (6%) were isolated.
These 2 identified species are known to be significantly
associated with severe clinical infections, their isolation
being a serious reason for concern [14].
These results are comparable to those obtained by otherauthors [15], who mention the fact that E. faecalis has
been isolated in the majority of the urinary tract infection
cases (63.89%), being followed by E. faecium (11.37%).
From the point of view of resistance, enterococci have an
intrinsic resistance to oxacillin and cephalosporins. E.
faecalis is naturally resistant to clindamycin.
For enterococci isolated from urinary tract infections, the
CLSI excludes erythromycin and clindamycin from thetest panel, so that no data on macrolide and lincosamide
resistance are available.
Our study showed that the isolated enterococci strains had a
24.13% resistance to penicillin, ampicillin, gentamicin andstreptomycin, and only a 6.89% resistance to tetracycline.
In a study including 27 European countries performed by
Schouten et al. [16], the resistance of enterococci togentamicin varies between 1%-49%.
Recent literature data indicate a dramatic increase in theresistance levels of enterococci to penicillin of up to
100% [17], and to ampicillin of up to 62% [18].
Data obtained in our study evidence a low resistance(15.51%) of enterococci strains to nitrofurantoin, similarly
to results obtained in other studies [19].
Doctors should be encouraged to use nitrofurantoin for
the treatment of urinary tract infections due to the highefficiency and low cost of this antibiotic.
All enterococci strains identified in our study had a 100%
sensitivity to vancomycin, which is in agreement withdata published in other recent studies [20].
Antimicrobial resistance surveillance in Europe 2012 [21]shows a vancomycin resistance lower than 1% for Romania.
Resistance to glycopeptides (vancomycin and teicoplanin)
is variable, ranging between 1%- 39% for countries using
glycopeptide derivatives (avoparcin) as growth factors inanimals [13].

1. The aim of this paper was to study the antibiotic
resistance of Enterococcus spp. strains isolated fromoutpatients, as well as to establish the resistancephenotypes of these strains in order to select themost efficient antibiotic and to prevent the selection ofmultiresistant bacterial strains.
2. The study was performed on 62 Enterococcus spp.
strains isolated from 5261 outpatient urocultures.
3. Two species of enterococci were identified: E. faecalis(94%) and E. faecium (6%).
4. From the point of view of resistance, our study
showed that only one E. faecium strain had anisolated resistance to fluoroquinolones, while theisolated E. faecalis strains had a 24.13% resistance toampicillin, penicillin, gentamicin, streptomycin and a
100% sensitivity to vancomycin.
5. The monitoring of antibiotic prescription is required,because empirical treatment in the absence of an
antibiogram induces an increase in the resistance ofEnterococcus spp. strains.
6. The role of the microbiology laboratory, essential for acorrect anti-infective therapeutic approach, consists ofperforming an antibiogram, of establishing andinterpreting the resistance phenotypes, thus providing
information about the resistance development
mechanisms and the way of spreading of these
phenotypes.

Fiziologia - Physiology • 2015.25.1(85)REFERENCES
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2. Moellering RC Jr. Emergence of Enterococcus as a
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Infections, Including the Role of the Microbiology Laboratory.
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4. Landry SL, Kaiser DL, Wenzel RP. Hospital Stay andMortality Attributed to Nosocomial Enterococcal Bacteremia:
A Controlled Study. Am. J. Infect. Control 1989; 17: 323-329.
5. Kallen AJ, Welch HG, Sirovich BE. Current Antibiotic
Therapy for Isolated Urinary Tract Infections in Women.
Arch Intern Med, 2006; 166: 635-639.
6. Fritsche TR, Sader HS, Jones RN. Epidemiology of
Antimicrobial Resistance: Species Prevalence, Susceptibility
Profiles, and Resistance Trends. In: Victor L Antibiotics inLaboratory Medicine. 5th Edition, Lippincott Williams &Wilkins, 2005: 815-835.
7. Heiberg IP, Schor N. Abordagem diagnostica e
terapeutica na infeccao do trato urinario, ITU. Rev. Assoc.
Med. Bras. 2003; 49: 4-15.
8. Forbes BA, Sahm DF, Weissfeld AS, Trevino E. Bailey and
Scott's Diagnostic Microbiology. Mosby Company, 1990, USA.

19

9. CLSI - Clinical and Laboratory Standards Institute.
Performance Standards for Antimicrobial Susceptibility Testing;
Sixteenth Informational Supplement 2007; M100S-17.
10. Mendiratta DK, Kaur H, Deotale V, Thamke DC, Narang R,
Narang P. Status of high level aminoglycoside resistantEnterococcus faecium and Enterococcus faecalis in a rural
hospital of central India. Indian J Med Microbiol 2008; 26:36971.
11. Astal ZY, Sharif FA. Relationship between demographic
characteristics and community-acquired urinary tract infection.
East Mediterr Health J 2002; 8: 164-71.
12. Miskeen PA, Deodhar L. Antimicrobial susceptibility
pattern of Enterococcus species from urinary tract infections.
J Assoc Physicians India 2002; 50: 37881.
13. Cornia PB, Takahashi TA, Lipsky BA. The microbiology ofbacteriuria in men: A 5year study at a Veterans' Affairs
hospital. Diagn Microbiol Infect Dis 2006; 56: 2530.
14. Murray BE. The life and times of the Enterococcus. Clin
Microbiol Rev 1990; 3: 4665.
15. Miskeen PA, Deodhar L. Antimicrobial susceptibility
pattern of Enterococcus species from urinary tract infections.
J Assoc Physicians India 2002; 50: 37881.

16. Schouten MA, Voss A, HoogkampKorstanje JA.
Antimicrobial susceptibility patterns of enterococci causing
infections in Europe. The European VRE Study Group.
Antimicrob Agents Chemother 1999; 43: 25426.
17. Jain S, Kumar A, Kashyap B, Kaur RI. The
clinico-epidemiological profile and the high-level
aminoglycoside resistance in enterococcal septicemia at a
tertiary care hospital in east Delhi. Int J App Basic Med Res.
2011; 1(2): 80-83.
18. Mulla S, Patel KG, Panwala T, Rewadiwala S. The
prevalence of Enterococci with a higher resistance level in a
tertiary care hospital: A matter of concern. National J Med Res.
2012; 2(1):25-27.
19. Butt T, Leghari MJ, Mahmood A. In vitro activity ofnitrofurantoin in enterococcus urinary tract infection. J Pak
Med Assoc 2004; 54: 466-9.
20. Prakash VP, Rao SR, Parija SC. Emergence of unusual
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Infectious Diseases. 2005; 5(14): 106-110.
21. Antimicrobial resistance suveillance in Europe 2012

REZISTENTA LA ANTIBIOTICE A TULPINILOR DE ENTEROCOCCUS SPP.
IZOLATE DIN INFECTII URINARE LA PACIENTII DIN AMBULATORREZUMAT
Scop: Studiul urmareste izolarea, identificarea si rezistenta la antibiotice a tulpinilor de Enterococcus spp. izolate
din infectii urinare la pacientii din ambulator, precum si stabilirea fenotipurilor de rezistenta in care se incadreaza
aceste tulpini.
Material si metoda: In perioada ianuarie 2010 - decembrie 2012 s-au luat in studiu 5261de uroculturi provenite
de la pacientii laboratorului de analize medicale Intermed Cluj-Napoca. Izolarea, identificarea si testarea
sensibilitatii germenilor izolati s-a realizat in laboratorul mai sus mentionat. Testarea rezistentei la antibiotice s-a
realizat prin metoda difuzimetrica conform standardelor CLSI.
Rezultate: Din cele 5261 uroculturi efectuate la pacientii laboratorului Intermed, 976 au fost pozitive, izolandu-se
58 de tulpini de E. faecalis si 4 de E.faecium. Tulpinile de E. faecalis izolate au prezentat o rezistenta de: 24,13%
la ampicilina, penicilina, gentamicina si streptomicina, 15,51% la nitrofurantoin, si de doar 6,89% la tetraciclina.
Din cele 4 tulpini de E. faecium numai una a prezentat rezistenta izolata la fluoroquinolone.
Concluzii: In elaborarea unei terapii antiinfectioase corecte, rolul laboratorului de microbiologie consta inefectuarea antibiogramei, precum si stabilirea si interpretarea fenotipurilor de rezistenta, oferind astfel informatii
asupra mecanismului de instalare a rezistentei si asupra modalitatilor de raspandire a acestor fenotipuri.
Cuvinte cheie: Enterococcus; Enterococcus faecalis; Enterococcus faecium; infectiile tractului urinar; urocultura;
antibiotice

20

Fiziologia - Physiology • 2015.25.1(85)AUTOFLUORESCENCE IMAGING VIDEO ENDOSCOPYSYSTEM: PRINCIPLES, TECHNIQUE, COLOUR PATTERNSOF EARLY LARYNGEAL CANCERMARIOARA DOBRE, CAIUS ION DOROS, NICOLAE CONSTANTIN BALICA,MARIOARA POENARU
Department of ENT, "Victor Babeș" University of Medicine and Pharmacy, Timisoara, Romania

ABSTRACT
We conducted a prospective study to evaluate visualization of laryngeal lesions using AFE videoendoscopy, which is a
new illumination system that allows for real-time WLE, but also makes it possible to switch to AFE endoscopy by thepress of a single button. In total, 78 patients (53 men and 25 women) with signs or symptoms suggesting laryngeal
dysfunction were included in this analysis. Histopathology revealed 30 cases (24%) of benign lesions such as
granullomas, polyps, chronic laryngitis and papillomas. Mild dysplasia occurred in 35 cases (28%), moderate dysplasia
in 18 cases (14.4%), severe dysplasia or carcinoma in situ in 32 cases (25.6%) and early invasive carcinoma in 10
cases (8%). Our results similar to other studies, have demonstrated a sensitivity by white light laryngoscopy of 88%
and specificity of 77%. The sensitivity by autofluorescence laryngoscopy was 94% and specificity to 87%. The
diagnosis accuracy by white light laryngoscopy was 73% (n=57) and that of autofluorescence 84% (n=66). Interest influorescence diagnostics in vivo has increased considerably in the last few years, as a result of several factors: theavailability, decreasing cost and clinical suitability of the required technologies, development of new exogenous
fluorophores and more detailed understanding of autofluorescence.
Key words: photodiagnosis, autofluorescence endoscopy, colour laryngeal patterns

INTRODUCTION
1. Fluorescence imaging: historical background
Early detection and proper treatment monitoring of
neoplastic disease with the main function of preserving
organs are some of the most challenging problems in thefield of ENT oncology [1, 2]. In recent years there has
been a great interest in fluorescence-based techniqueapplied to clinical oncology especially in the ENT field.
These techniques such as: laser-induced fluorescence(LIF), fluorescence correlation spectroscopy (FCS),
photodynamic diagnosis (PDD) by autofluorescence (AF)or by exogenous fluorescence (induced fluorescence orIF) have a significant impact on the development ofphotodynamic therapy (PDT) of solid tumors.
The first study in cancer photodetection or tissue
characterization using AFE (autofluorescence endoscopy)
was reported by Policard in 1924 concerning thefluorescence of tumours under illumination with UV/violet
light [3]. In 1942 Auler and Banzer [4] and Figge [5]observed red fluorescence of animal tumours afteradministration of exogenous porphyrins. The first use offluorescein, to improve the detection and identification ofthbrain tumours in vivo, was reported by Moore et al. in1948 [6]. The groups of Profio [7], Alfonso [8], Lohmann[9] and Yang [10] did pioneering in vitro and in vivo
studies of human and animal tumour autofluorescence.
Recently, Zhang et al. [11] reported differences influorescence excitation and emission spectra between
slowly and rapidly growing cells.
After exposing of instruments and tissue fluorescenceprinciples we will discuss the clinical applications of invivo fluorescence endoscopy for early cancer detection.
Secondly we will classify the colour patterns of earlylaryngeal lesions in AFE (autofluorescence endoscopy)
images and then we will investigate the correlation
between the patterns and histopathological findings.
2. Physical Principles and Instrumentation
When the mucosal surface is illuminated by light, the light
can be reflected, back-scattered, absorbed or induce
cellular autofluorescence. During malignant transformation
morphological alterations of premalignant and normal
tissues produce AF spectral changes.
Autofluorescence endoscopy is based on the excitationof tissue-specific fluorescence of the mucosa bythReceived 15 of February 2015. Accepted 20 of March 2015. Address for correspondence: Marioara Dobre, PhD Student in
Otorhinolaryngology, MD, Department of ENT, "Victor Babeș" University of Medicine and Pharmacy, 2 Eftimie Murgu Square,
300041, Timisoara, Romania, Phone: +40786330040, e-mail: mary2dobre@yahoo.com.

Fiziologia - Physiology • 2015.25.1(85)21short-wave blue light of the visible spectrum (375-440nm).
The appearance and degree of autofluorescence depend
especially on tissue structure’s fluorophores content.
Fluorophores involved in cellular metabolism include
reduced nicotinamide adenine dinucleotide (NADH),
flavins, aromatic amino-acids (e.g. tryptophan, tyrosine,
phenylalanine), carious porphyrins and lipopigments (e.g.
ceroids, lipofuscin) that are the end-products of lipid
metabolism. In addition, red porphyrin fluorescence due
to bacteria may be significant in certain body sites and/or
lesions.
Characteristics that are important to consider in
optimizing and interpreting clinical studies include thefollowing: a) each florophore has a distinct excitation andemission spectrum. b) the fluorophores are not uniformly
distributed in tissue. c) any given tissue contains a
mixture of many fluorophores of different concentration.
First AF endoscopy proved to be of great value in thedetection of early lung cancer using the LIFE (Laser
Induced Fluorescence Endoscopy) System (Xillix
Technology Vancouver) [12, 13]. Then multiple
publications were reported on its clinical applicability inthe diagnosis of head and neck malignancies, mainly inlaryngeal localizations [14, 15].
Recently the Storz Company, using the xenon lightsource with a band of excitation wavelength from 375 to
440 nm and producing AF imaging in a green spectral
range, has developed a less expensive device, theD-light AF System [16].
3. Clinical applications
In urology, the detection of early-stage urothelial lesions
with fluorescence illumination has pushed forwards thedevelopment of PDD technique [17]. Also in gynaecology
PDD is used to diagnose precursors of malignant tissue
that are not visible under white light [18]. Fluorescencebronhoscopy seems to be successful in the detection ofearly lung cancer [19] and in otolaryngology,
fluorescence imaging of PP IX seems to be helpful in thedetection of neoplastic lesions in the oral cavity [20]. In
dermatology, efforts have been made to diagnose anddemarcate basal cell carcinoma, for example [21].
Neurosurgeons se PP IX fluorescence for intraoperative
detection of tumours and tumour rests after resection ofmalignant gliomas [22].

METHODSAFE videoendoscope system
We conducted a prospective study to evaluate
visualization of laryngeal lesions using AFE
videoendoscopy, which is a new illumination system thatallows for real-time WLE, but also makes it possible toswitch to AFE endoscopy by the press of a single button.
The blue light source was provided by a xenon short arc
lamp with an excitation wavelength ranging from 375 to
22

440nm (D-Light C/AF Light System, 20133620), while an
integrated optic filter block out the remitted excitation
light up to 440nm. The camera system was a target integrating high-resolution colour charge device(CCD-camera, Tricam PDD, 20221037; Karl Storz Co.).
This camera was connected to a 70° rigid-angled
endoscope (Karl Storz, Tuttlingen, Germany).
Patients: In total, 78 patients (53 men and 25 women)with signs or symptoms suggesting laryngeal dysfunction
were included in this analysis. Main symptoms were:
hoarseness, foreign body sensation in the throat, throat
pain and fatigue of voice. The ages of the patients
ranged from 34 to 79 years (mean 55 years).
Endoscopic examinations (procedure): First, routine
endoscopic examinations were carried out using the
WLE mode of the AFE videoendoscope system toidentify abnormal mucosal areas in the larynx. We thenexamined the larynx by switching to the AFE mode with apress of a foot switch. Pictures made of abnormal
mucosal areas were then assembled of both WLE and
AFE and examined. In each case tissue sample weretaken from the areas that were suspected. According to
the size and extension of a laryngeal lesion
microlaryngoscopy with excisional biopsy or laser CO2
cordectomy was performed in all cases to confirm thediagnosis histologically. Diagnostic accuracy, sensitivity
and specificity were evaluated by correlating
histopathologic diagnosis with visual impression.

RESULTSA total of 78 patients with a total of 125 benign andmalignant lesions of the vocal folds were examined.
Histopathology revealed 30 cases (24%) of benign
lesions such as granullomas, polyps, chronic laryngitis
and papillomas. Mild dysplasia occurred in 35 cases(28%), moderate dysplasia in 18 cases (14.4%), severe
dysplasia or carcinoma in situ in 32 cases (25.6%) andearly invasive carcinoma in 10 cases (8%).
In the larynx all lesions were localized in the glottis region.
Under AF videoendoscopy, the healthy mucosal areas of
the head and neck regions displayed a typical brightgreen colour. The AF appearance of the premalignant
lesions was found to be variable. If marked
hyperkeratosis was present, it appeared as a light green
or white colour, depending on its thickness.
The bright green colour in marked keratosis can be
explained by the intensive fluorescence of keratin in the
superficial epithelial layer. However, the intensity of thesuperficial layer may hide a subjacent precancerous
lesion rendering impossible a complete evaluation.
Therefore, the result should be regarded critically instrongly keratinizing lesions. Mucosal alteration,
histologically defined as moderate dysplasia, showed aclearly recognizable altered green fluorescence with a
Fiziologia - Physiology • 2015.25.1(85)shift to reddish-blue, violet fluorescence. In situ andinfiltrative squamous cell carcinomas all displayed amarkedly altered green fluorescence, presenting
reddish-violet colour. The AF image of the cancer wasless variable, in most of the cases being recognized as aviolet or reddish-blue colour (Table I).
Table I. The recorded AFE images and the colour patterns wereclassified into the following types depending on the epithelial
thickness, inflammation or vascularization grade
Laryngeal lesion

AFE (LAF)

Remarks

Hyperkeratosis

Green toDepending on thewhite(0)thickness

Green(0)Better contrast ofNormal mucosa,
benign lesions

Fig. 1. Invasive Carcinoma (pT1a). White light endoscopy (a)demonstrates an irregular thickening of the right vocal fold;
Autofluorescence endoscopy (b, c) shows a marked loss ofautofluorescence, whereas induced fluorescence endoscopy.

blood vessels

Simple hyperplasia,

Green toDepending on

mild dysplasia

violet(0-1)

inflammation grade

Chronic laryngitis,

Violet to

Depending on

moderate dysplasia

red-violet(1-2)

inflammation grade

Teleangiectatic

Violet to

Depending on

polyp, granulomas

red-violet(1-2)

vascularization

Papillomas

Red-violet(2)Fig. 2. Moderate differentiated squamous cell carcinoma of theright vocal fold. White light endoscopy (a); Yellow light endoscopy(b); Autofluorescence endoscopy (c) shows a marked loss ofautofluorescence, whereas induced fluorescence endoscopy.

grade
Depending on
vascularization
grade
Severe dysplasia

Red-violet(2)No differentiationfrom CIS/ICA

Carcinoma in situ

Red-violet(2)No differentiationfrom DIII/ICA

Invasive cancer

Red-violet(2)Yellow-orange inulcerated tumors

Abbreviations: AFE autofluorescence endoscopy; LAF loss ofautofluorescence(0=none, 1=mild,2= marked); CIS carcinoma
in situ; ICA invasive cancer; DIII severe dysplasia.

Moreover, smaller, flat superficial epithelial lesions were
found to be better outlined by AF photodiagnosis thenlarge, ulcero-infiltrative tumours. The demarcation ofthese lesions provided a sharp, good image quality, but
often showed a greater size than we expected from thewhite light examination, indicating the structural changes
in the dysplastic mucosa around the infiltrating tumour.
Some benign lesions, such as granulation tissue,
papilloma and vocal fold polyp, also displayed a loss ofgreen fluorescence. This may be the results that the hem
molecule in dilated subepithelial vessels absorbs theincident light and therefore contributes to a further loss ofautofluorescence. On the ulcerated necrotic surface ofthe tumours bacterial colonization was observed,
reflecting as an orange or yellow bright fluorescence
under the excitation light.

Fiziologia - Physiology • 2015.25.1(85)Fig. 3. Pyogenic granuloma on the right vocal fold: White lightendoscopy (a); Autofluorescence endoscopy (b, c) shows amarked loss of autofluorescence, whereas induced
fluorescence endoscopy.

Our results similar to other studies, have demonstrated
sensitivity by white light laryngoscopy of 88% andspecificity of 77%. The sensitivity by autofluorescencelaryngoscopy was 94% and specificity to 87%. The
diagnosis accuracy by white light laryngoscopy was 73%(n=57) and that of autofluorescence 84% (n=66).

DISCUSSION AND FUTURE PERSPECTIVESInterest in fluorescence diagnostics in vivo has increased
considerably in the last few years, as a result of several
factors: the availability, decreasing cost and clinicalsuitability of the required technologies, development of
new exogenous fluorophores and more detailed
understanding of autofluorescence.
The detection of premalignant lesions or early cancer
using autofluorescence depends on changes in one ormore of:a) the fluorophore concentration or spatial distribution;b) the tissue architecture, such as mucosal thickening orloss of layered structure;23c) the biochemical/biophysical microenvironment of thetissue;d) the metabolic status: NADH is fluorescent only in its
reduced form;e) the wavelength-dependent light attenuation due totheconcentration
anddistributionof(non-fluorescent)chromophores,
particularly
haemoglobin.
But, the large number of false positive results with aconsequent low positive predictive value implies a
potential benefit from adjunct methods such as
microendoscopy-narrow band imaging (ME-NBI), opticalcoherence tomography (OCT) or confocal laser
endomicroscopy (CLE) [23] which would provide greater
detection specificity. The use of trimodal imaging
endoscopy that includes WLE, AFI and ME-NBI
incorporated in one endoscopy system might improve
diagnostic accuracy for high-grade dysplasia and earlycancer [24]. ME-NBI is currently considered thetechnique of choice for improvement of diagnostic
accuracy because it reduces the high rate offalse-positive results associated with WLE and AFI.
Consequently, the development of infrared technique
may provide greater tissue penetration, obtaining images
with greater contrast between lesions and theirsurrounding regions, and allowing the visualization ofvascularization in deeper lesions, including the
submucosa. Another possible strategy could be thesupplementation of the AF technique with contact
endoscopy (CE) [16, 25] or the combination of the AF
method with protoporphyrin IX induced fluorescenceendoscopy [26]. Although our series includes only a small
number of patients treated by endoscopic laser surgery,this clinical application of the D-Light AF System inindirect laryngoscopy seems to be a very promising
method in the control of the surgical margins.
If the goal of early detection of primary or recurrent
tumours can be achieved, this may increase thelikelihood of successful radical treatment and reducecomplications. Additional potential applications of thesetechniques are to provide guidance in locationg the
optimum sites for biopsy [27] to define the surgicalmargins for tumour resection [28, 29] and to optimize
and monitor PDT treatments [30].

CONCLUSIONSThe potential clinical advantages of fluorescence
techniques include: the high sensitivity; flexibility in the
anatomical sites that can be investigated, especiallyusing small-diameter optical fiber probes; reduction in theuse of random tissue biopsies and the ease of use by theclinician. The potential for reduced health-care costs as aconsequence for the minimally invasive nature andspeed of the techniques and the improved patient
outcome may be significant.
24

ACKNOWLEDGMENT
The study was financed by the University of Medicine
and Pharmacy "Victor Babeș", Timisoara, Romania;
Doctoral Grant No:978/31.01.2013.

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8. Alfonso RR, Tata DB, Cordero J, et al. Laser induced
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lesions: ramdomized study. JNCI, 2001; 93: 1385-1391.
13. Lam S, MacAulay C, Hung J. Detection of dysplasia
and carcinoma in situ using a lung imaging fluorescenceendoscope (LIFE) device. J Thorac Cardiovasc Surg, 1993;
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14. Delank W, Khanavkar B, Nakhosteen JA, Stoll W. A
pilot study of autofluorescence endoscopy for the in vivo
detection of laryngeal cancer. Laringoscope, 2000; 110:368-373.
15. Zargi M, Fajdiga I, Smid L. Autofluorescence imaging
in the diagnosis of laryngeal cancer. Eur Arch
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16. Arens C, Malzahn K, Dias O, et al. Endoscopic imaging
techniques in the diagnosis of laryngeal cancer and its
precursor lesions. Laryngol Rhinol Otol, 1999; 78: 685-691.
17. Zaak D, Wagner S, Stepp H, et al.
Fluoreszzenzquantifierung bei der 5-Aminolevulinsäure
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20. Leunig A, Betz CS, Mehlmann M, et al. Detection ofsquamous cell carcinoma of the oral cavity by imaging
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IX
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SISTEMUL VIDEO DE ENDOSCOPIE CU AUTOFLUORESCENTA: PRINCIPII,TEHNICA, MODELE DE CULOARE PENTRU CANCERUL LARINGIAN INCIPIENTREZUMATAm efectuat un studiu prospectiv pentru a evalua modelele de culoare ale leziunile laringiene utilizand
videoendoscopia cu autofluorescenta, un sistem nou de iluminare ce permite in timp real vizualizarea atat cu
lumina alba, cat si posibilitatea de a schimba in modul autofluorescent numai prin apasarea unui singur buton. In
studiu au fost inclusi un numar de 78 pacienti (53 barbati si 25 femei) cu semne si simptome sugestive pentru o
disfunctie laringiana. Rezultatele histopatologice au evidentiat 30 cazuri (24%) de leziuni benigne ce includ
granuloame, polipi, laringita cronica si papiloame. Displazia slaba a putut fi decelata in 35 de cazuri (28%),
displazia moderata in 18 cazuri (14,4%), displazia severa sau carcinomul in situ in 32 de cazuri (25,6%) si
carcinomul invaziv incipient in 10 cazuri (8%). Rezultatele noastre, similare cu alte studii, au demonstrat osensibilitate pentru endoscopia cu lumina alba de 88% si o specificitate de 77%. Sensibilitatea in cazul
endoscopiei cu autofluorescenta a fost de 94% si specificitatea de 87%. Acuratetea diagnostica pentru
endoscopia cu lumina alba a fost de 73% (n=57), iar pentru endoscopia cu autofluorescenta de 84% (n=66).
Interesul pentru diagnosticul fluorescent in vivo a crescut considerabil in ultimii ani ca si rezultat al catorva factori:
disponibilitatea, costurile mai scazute, adaptarea clinica a noilor tehnologii cerute, dezvoltarea unor noi fluorofori
exogeni si nevoia de intelegere mai amanuntita a autofluorescentei.
Cuvinte cheie: fotodiagnosticul, endoscopia cu autofluorescenta, modele de culoare laringiana.

Fiziologia - Physiology • 2015.25.1(85)25PLACENTAL STEM CELLS MODULATE TUMORFORMATION IN MOUSE MODEL OF UTERINE SARCOMAANASTASIU MD1, CEAN A2, BOJIN F2, GAVRILIUC O2, GLUHOVSCHI A1,
ANASTASIU D1, CRAINA M1, TATU C2, TANASIE G2, PANAITESCU C2, PAUNESCU V2
1Department of Obstetrics and Gynecology
2Department of Functional Sciences“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACT
Background: The adult mesenchymal stem cells (MSCs) are one of the most studied populations of adult multipotent
stem cells, being isolated from bone marrow, peripheral blood, or other solid tissues. The fetal MSCs are isolated fromplacenta, amniotic fluid, umbilical cord and umbilical cord blood. Placental stem cells (PSC) are at the crossroads ofadult stem cells and embryonic stem cells (ESCs). Although they do not have the same proliferative and differentiation
potentials of ESCs, the placenta-derived multipotent cells may be superior to adult stem cells, so that their increased
potential should be explored in more detail for further therapeutic strategies.
Materials and methods: Placental stem cells (PSCs) were isolated from 20 term-delivered placentas using enzymatic
digestion (Collagenase I A). Further cultivation and expansion of single-cell suspension was performed in standard
medium containing alpha-MEM, 10% FCS, and antibiotic solution, placed in 6-well culture dishes at 37o C and 5% CO2.
Morphological characterization showed fibroblast-shape cellular appearance, adherent to plastic surface, and reachingconfluence in spindle-like colonies. For mouse model (CD1 Nu/Nu) of uterine sarcoma we used an immortalized cellline commercially available (MES-SA), admixed with Matrigel scaffold and we injected 1x106 tumor cells in theexperimental animal group (n = 10). Tumor formation was completed in 4 weeks. PSCs were transfected with GFP byelectroporation method, and 1x106 GFP-expressing PSCs were injected at tumor site for every mouse. For comparative
analysis we used bone marrow-derived MSCs stably expressing GFP, and we visualized cellular migration, homing andeffects by serial pictures taken every 3 days for 2 weeks using in vivo imaging system Hamamatsu Aequoria.
Results: PSCs obtained using enzymatic digestion were similar to bone marrow-derived mesenchymal stem cells inmorphological and functional characteristics. Tracking of in vivo migration of GFP-PSCs comparative with MSCs-GFP
expressing showed an increased ability of PSCs to decrease the size of initial tumor, evidenced by measurement oftumor diameter every 3 days for 2 weeks.
Conclusion: The present study shows that PSCs have an increased ability to modulate tumor growth compared totheir counterparts, the bone marrow-derived MSCs, which makes them more suitable for therapeutic approaches incancer treatment.
Key words: mesenchymal stem cells, placental stem cells, tumor growth

INTRODUCTION
The adult mesenchymal stem cells (MSCs) are one of
the most studied populations of adult multipotent stem
cells, being isolated from bone marrow, peripheral blood,or other solid tissues. The fetal MSCs are isolated fromplacenta, amniotic fluid, umbilical cord and umbilical cord
blood. Placental stem cells (PSCs) are at the crossroadsof adult stem cells and embryonic stem cells (ESCs).
Although they do not have the same proliferative anddifferentiation potentials of ESCs, the placenta-derived
multipotent cells may be superior to adult stem cells, soththat their increased potential should be explored in moredetail for further therapeutic strategies.
Despite recent advances in understanding of the molecularabnormalities associated with uterine cancer, these results
have not been translated into better therapy for patients[1-3]. Uterine cancer is the most common cancer of thefemale genital tract. These cancers include carcinomas,
sarcomas, and mixed epithelial-mesenchymal tumors. For
all subtypes, the only available treatment is hysterectomy.
There is no effective therapy once these tumors havespread outside the uterus; adjuvant therapy for metastatic
disease does not enhance survival. Thus, further study isthReceived 14 of January 2015. Accepted 24 of February 2015. Adress for correspondence: Diana Anastasiu, MD, PhD
student, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square N o. 2A, RO-3000041, Timisoara,phone/fax: +40256220479; e-mail: diana_anastasiu@yahoo.com

26

Fiziologia - Physiology • 2015.25.1(85)needed, not only to identify chemotherapeutic agents totreat uterine cancer after it has developed, but also to
discover new strategies to prevent or delay diseaseprogression.
Mesenchymal stem cells can be broadly grouped into two
different subgroups adult MSCs and fetal MSCs. Adult
MSCs are isolated from bone marrow, peripheral blood.
Fetal MSCs are isolated from Placenta, amniotic fluid,
umbilical cord and umbilical cord blood [4]. Placenta
provides one of the most reliable and abundant source of
MSCs [5]. Term placental tissues are discarded after birth,
hence these tissues can be effectively utilized forresearch as well as clinical application without much
ethical concern.
The aim of the present study was to establish the role of
the PSCs in modulating tumor development. For thisreason we comparatively analyze the bonemarrow-derived MSCs and PSCs from morphological andphenotypical point of view. Further we induced tumor
formation in mouse model and infused both MSCs and
PSCs in order to establish their role in tumor development.

MATERIALS AND METHODS
Cell culture and expansion
Placental stem cells (PSCs) were isolated from 20
term-delivered placentas using enzymatic digestion(Collagenase I A) or explants method. Further cultivationand expansion of single-cell suspension was performed
in standard medium containing alpha-MEM, 10% FCS,
and antibiotic solution, placed in 6-well culture dishes at
37o C and 5% CO2. Morphological characterization
showed fibroblast-shape cellular appearance, adherent
to plastic surface, and reaching confluence in spindle-likecolonies. At passage 2, cells were submitted todifferentiation assays towards osteoblasts, adipocytes,
and chondrocytes, and were analyzed based on
flowcytometric markers (CD34, CD45, CD90, CD73, andCD105) (data not shown).
For mouse model (CD1 Nu/Nu) of uterine sarcoma weused an immortalized cell line commercially available(MES-SA, ATCC® CRL-1976™). This cell line was wasestablished from a surgical tumor specimen obtained at
the time of hysterectomy. Initially, the cells were grown insoft agar, and later they were transferred to multiwellplates. Using the MES-SA cell line, tumors developed
within 21 days at 100% frequency (5/5) in nude miceinoculated subcutaneously with 107 cells. Literature data
provides evidence that the tumor is a poorly differentiateduterine sarcoma. The nonepithelial origin of the cells was
supported by ultrastructural studies and the absence ofstaining for mucin. The cells are sensitive to a number ofchemotherapeutic agents including doxorubicin,
dactinomycin, mitomycin C, taxol and bleomycin. They are
resistant to vinblastine, dacarbazine, cisplatin, melphalan,vincristine, methotrexate and etoposide.
Fiziologia - Physiology • 2015.25.1(85)The base medium for this cell line is ATCC-formulated
McCoy's 5a Medium Modified (ATCC®, Manassas,
Virginia, USA) supplemented with fetal calf serum (FCS,ATCC®) to a final concentration of 10%.
Transfection procedure
The PSCs used in tumor models were transfected with
Green Fluorescent Protein (GFP) using Amaxa™
Nucleofector™ II Device (Amaxa, Lonza Group Ltd,Basel, Switzerland). GFP is derived from the copepod
Pontellina and can be used as a positive control tomonitor transfection efficiency in cells of interest (Figure1). The cells were passaged 2-3 days before
nucleofection and cells were nucleofected after
reaching 70 - 85% confluency (higher cell densities may
cause lower nucleofection efficiencies). We used
plasmid pmaxGFP (Amaxa) conc 0.5 ug/ul - 4
uL/reaction, and the procedure was as follows:
nucleofection with Amaxa Nucleofector II, program U-23,
for maximum eficiency. The kit used for transfection
was Lonza (Amaxa) MSC Nucleofector kit. We used 5 x
105-106 cells/reaction. After trypsinization, the cells
were centrifuged and then resuspended in 100 ul
Nucleofector MSC solution and 2 ug GFP-containing
plasmid. The cells are further electroporated, transferedin a new culture flask and expanded in mesenchymal
stem cells medium supplemented with 20% FCS.

Fig. 1. GFP containing plasmid

Tumor formation in mouse models
MES-SA cells admixed with Matrigel scaffold(BD Matrigel™ Basement Membrane Matrix, BD
Bioscience) and we injected 1x106 tumor cells in theexperimental animal group (n = 10). Tumor formation wascompleted in 4 weeks. PSCs were transfected with GFP
by nucleofection method, and 1x106 GFP-expressing
PSCs were injected at tumor site for every mouse. For
comparative analysis we used bone marrow-derived
MSCs stably expressing GFP, and we visualized cellular27

migration, homing and effects by serial pictures taken
every 3 days for 2 weeks using in vivo imaging system
Hamamatsu
Aequoria(Hamamatsu
Photonics
Deutschland GmbH, Germany).
The mice were anesthetized with Sevofluran – induction
and maintenance, while the dark box in which theanimals were placed was maintained at 37o C. Cellularmigration was tracked for 2 weeks, taking serial
photographs, which were interpreted with the dedicatedsoftware of the in vivo imaging system. All animal
experiments described herein comply with the European
Convention for the Protection of Vertebrate Animals used
for Experimental and Other Scientific Purposes (Directive
86/609, Strasbourg, 1986) and the experimental protocol
was reviewed and approved by the University of
Medicine and Pharmacy Timisoara Board for Animal
Experiments.

RESULTS AND DISCUSSION
Following infusion of 1x106 tumor cells (human uterine
sarcoma cell line MES-SA), subcutaneous tumor
formation occurred within 4 weeks (Figure 2, Panel A).
Peri-tumoral injection of 1x106 GFP-expressing MSCs
induced a decrease in size of mouse tumors after 1 weekand 2 weeks (Figure 2, Panel B). Prior to injection attumor site, 2x106 PSCs were transfected with GFP with atransfection rate of 50%. Then, 1x106 GFP-expressing
PSCs were injected in tumor mouse model and wenoticed a progressive decrease in tumor diameter at 1
week and 2 weeks (Figure 2, Panel C). Compared to
MSCs-treated tumors, the ratio between initial tumor sizeand final tumor size was favorable to tumors treated with
PSCs.

types, including chondrocytes, osteoblasts, adipocytes,
muscles, neurons, stromal cells, and other cell types [6].
Several studies have indicated that humanplacenta-derived mesenchymal stem cells (PSCs) aresimilar to stem cells from bone marrow with respect tocell characteristics and their potential for multilineage
differentiation [7-9]. As placental tissues originate during
the first stages of embryological development, thesetissues might contain cells that have retained theprosperities of early embryonic cells from which they
derive. Furthermore, the placenta is fundamental for
maintaining feto-maternal tolerance during pregnancy,suggesting that cells present in placenta tissue may haveimmonomodulatory characteristics. Meanwhile, recent
studies showed that mesenchymal isolated from placenta
tissue have the ability to specifically homing to multiple
tumor site.
These three key aspects make cell from placenta
extremely attractive candidate for possible use in celltherapy approaches in direct cancer therapy [10]. Somestudies have engineered MSCs to express interferon b(IFNb) in gliomas [11], metastatic melanoma [12], andbreast cancer models [13]. These studies showed that
MSCs can travel to the same homing destination as the
migrating cancer stem cell with unusual abilities tomigrate to oncogenetic site. Meanwhile, PSCs havebeen shown to be more advantageous in cellprocurement, storage, and transplantation than bonemarrow-derived MSCs. Moreover, mesenchymal
stromal cells from bone marrow have a risk of viral
infection [14] and their differentiation capacity
decreases significantly with donor age [15].
Furthermore, the placenta is generally discarded after
birth; as such, this tissue is available in large supply,
and the isolation of stem cell from this tissue does not
involve any invasive procedures for the donor andavoids ethical controversy. These key aspects makecells isolated from placenta good candidates forpossible use in cell therapy. The primary interest of thisstudy has been focused on whether PSCs could
specifically migrate to the tumor site and overcome
tumor progression in a human uterine cancer model.

CONCLUSION
The present study shows that PSCs have an increased
ability to modulate tumor growth compared to theircounterparts, the bone marrow-derived MSCs, whichmakes them more suitable for therapeutic approaches incancer treatment.
Fig. 2. Timeline of tumor development induced by MES-SA
uterine sarcoma cells in CD1 nu/nu mice, injected with
GFP-expressing MSCs or GFP-transfected PSCs

ACKNOWLEDGEMENT
This work was supported by CNCS-UEFISCSU,PNII-Parteneriate 120/2012 and PNII-IDEI 318/2011.

Mesenchymal stem cells (MSCs) are multipotent stem
cells with the ability to differentiate into a variety of cell
28

Fiziologia - Physiology • 2015.25.1(85)REFERENCES
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2. Ronnett BM, Zaino RJ, Ellenson LH, Kurman RJ.
Endometrial carcinoma. In: Kurman, RJ., editor. Blaustein's
Pathology of the Female Genital Tract. 5th ed..
Springer-Verlag; New York: 2002; 501-59.
3. Pautier P, Genestie C, Rey A, et al. Analysis ofclinicopathologic prognostic factors for 157 uterine sarcomas
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4. Hass R, Kasper C, Bohm S, Jacobs R. Different
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MSC. Cell Communication and Signaling, 2011; 9: 12.
5. Semenov OV, Koestenbauer S, Riegel M, et al.
Multipotent mesenchymal stem cells from human placenta:
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after isolation. American Journal of Obstetrics andGynecology, 2010; 202: 193.e1-193.e13.
6. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK,
Douglas R, et al. Multilineage potential of adult humanmesenchymal stem cells. Science 1999; 284: 143-147.
7. Kassem M, Kristiansen M, Abdallah BM. Mesenchymal
stem cells: cell biology and potential use in therapy. Basic
Clin Pharmacol Toxicol, 2004; 95: 209-214.
8.
Miao Z, Jin J, Chen L, Zhu J, Huang W, et al. Isolationof mesenchymal stem cells from human placenta:comparison with human bone marrow mesenchymal stem
cells. Cell Biol Int, 2006; 30: 681-687.
9.
Fukuchi Y, Nakajima H, Sugiyama D, Hirose I,
Kitamura T, et al. Human placenta-derived cells havemesenchymal stem/progenitor cell potential. Stem Cells,
2004; 22: 649-658.
10. Pereboeva L, Komarova S, Mikheeva G, Krasnykh V,
Curiel DT. Approaches to utilize mesenchymal progenitor
cells as cellular vehicles. Stem Cells, 2003; 21: 389-404.
11. Nakamizo A, Marini F, Amano T, Khan A, Studeny M, etal. Human bone marrow-derived mesenchymal stem cells in
the treatment of gliomas. Cancer Res, 2005; 65: 3307-3318.
12. Studeny M, Marini FC, Champlin RE, Zompetta C,
Fidler IJ, et al. Bone marrow-derived mesenchymal stem
cells as vehicles for interferon-beta delivery into tumors.
Cancer Res, 2002; 62: 3603-3608.
13. Komarova S, Kawakami Y, Stoff-Khalili MA, Curiel DT,
Pereboeva L. Mesenchymal progenitor cells as cellularvehicles for delivery of oncolytic adenoviruses. Mol Cancer
Ther, 2006; 5: 755–766.
14. Eichna DM, Brown KS, Breen A, Dean RB.
Mucormycosis: a rare but serious infection. Clin J Oncol
Nurs, 2008; 12: 108-112.
15. Mareschi K, Ferrero I, Rustichelli D, Aschero S,
Gammaitoni L, et al. Expansion of mesenchymal stem cells
isolated from pediatric and adult donor bone marrow. J Cell
Biochem, 2006; 97: 744-754.

CELULELE STEM DERIVATE DIN PLACENTA MODULEAZA FORMAREATUMORILOR IN MODELUL ANIMAL DE SARCOM UTERINREZUMAT
Introducere: Celulele stem mezenchimale adulte (MSCs) sunt dintre cele mai studiate populatii de celule
stem multipotente adulte, fiind izolate din maduva osoasa hematogena, sange periferic sau alte tesuturi.
Celulele stem mezenchimale fetale sunt izolate din placenta, lichid amniotic, cordon ombilical si sange dincordonul ombilical. Celulele stem izolate din placenta (PSCs) se afla la limita dint re celule stem adulte si
celule stem embrionare (ESCs). Cu toate ca nu au acelasi potential proliferativ si de diferentiere ca al ESCs,
celulele stem multipotente izolate din placenta sunt superioare celulelor stem adulte, astfel incat potentialul lor
crescut ar trebui explorat in detaliu in vederea utilizarii acestora pentru diverse strategii terapeutice.
Materiale si Metode: Celulele stem placentare (PSCs) au fost izolate din 20 de placente la termen folosind
digestia enzimatica (Coagenaza I A). Cultivarea ulterioara si expandarea celulara au fost efectuate in placi decultura cu 6 godeuri, la 37o C si 5% CO 2, in mediul de cultura standard, care contine alpha-MEM, 10% ser fetal
bovin (FCS) si solutie de antibiotic. Caracterizarea morfologica a aratat celule alungite, fibroblast-like,aderente la suprafata de plastic, care ajung la confluenta prin formarea de colonii in vartejuri. Pentru modelul
animal (CD1 Nu/Nu) de sarcom uterin am folosit o linie celulara imortalizata disponibila comercial (MES -SA),care a fost amestecata cu matrice Matrigel si au fost injectate 1x10 6 celule tumorale/animal in grupul
animalelor folosite pentru acest experiment (n=10). Formarea tumorilor a fost completa in decurs de 4
saptamani de la injectare. PSCs au fost transfectare cu GFP prin metoda electroporarii si au fost injectate
1x106 PSCs fluorescente/animal la nivelul tumorilor formate. Pentru analiza comparativa am folosit celule
Fiziologia - Physiology • 2015.25.1(85)29stem mezenchimale izolate din maduva osoasa care exprima stabil GFP (MSC/GFP) si am urmarit migrarea
celulara, localizarea si efectul acestor celule prin imagini seriale efectuate la fiecare 3 zile timp de 2 saptamani
cu ajutorul sistemului de imagistica in vivo Hamamatsu Aequoria.
Rezultate: PSCs obtinute prin digestie enzimatica au fost similare celulelor stem mezenchimale derivate dinmaduva osoasa in ceea ce priveste caracteristicile morfologice si functionale. Urmarirea comparativa amigrarii in vivo a PSCs/GFP si MSCs/GFP a aratat ca celulele stem placentare prezinta o capacitate mai marede a induce regresia dimensiunii initiale a tumorilor, evidentiata prin masurarea diametrului tumoral timp de 2
saptamani, la fiecare 3 zile.
Concluzie: Studiul prezent arata ca PSCs au o capacitate crescuta de a modula dezvoltarea tumoralacomparativ cu celulele stem mezenchimale izolate din maduva osoasa, fiind mai potrivite pentru abordari
terapeutice in tratamentul cancerului.
Cuvinte cheie: celule stem mezenchimale, celule stem placentare, dezvoltare tumorala

30

Fiziologia - Physiology • 2015.25.1(85)EPITHELIAL MARKERS EXPRESSED BY HUMANMESENCHYMAL STEM CELLS UPON IN VITRO INDUCTION
RADU IVANESCU, RADU LAZAROVICZ, FLORINA BOJIN,CARMEN PANAITESCU, VIRGIL PAUNESCU, GABRIELA TANASIE
Department of Functional Sciences, ”Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT
Although the property of adult stem cells to regenerate and repair damaged tissues may be controversial, bone marrowderived cells are reported to have potential contribution to tissue repair in both physiological and pathological conditions,
including various types of epithelia. In this study we verify the effect of some chemical inductors to differentiate in vitro
bone marrow-derived adult mesenchymal stem cells (MSCs) into epithelial-like cells. After isolation and expansion,induction of MSCs towards the epithelial lineage was made using various cytokines and growth factors (epidermal
growth factor – EGF, hepatocyte growth factors – HGF, fibroblast growth factor - FGF), added in the culture alone or in
combination. Differentiated cells were analyzed using immunofluorescence for vimentin, cytokeratin and E-cadherin
markers. In all differentiated MSCs, some modifications of the cell morphology were noticed, as they become morepolygonal and had a tendency to form multilayered culture. Expression of vimentin was weaker in the epithelial-likecells, compared with MSCs, whereas expression of cytokeratin was stronger in epithelial-differentiated cells. The
results of RT-PCR showed increased expression of cytokeratin 19 and E-cadherin in epithelial cells, especially in cells
cultured in media without FGF, suggesting a possible role FGF in inhibition of mesenchymal to epithelial transition. The
experiments showed that MSCs differentiation toward the cells expressing epithelial markers is relatively easy to obtainusing a certain combination of inductors, without genetic manipulation of the cells.
Key words: mesenchymal stem cells, epithelial-like cells, differentiation media, immunocytochemistry

INTRODUCTION
The epithelium is a type of tissue that lines all the bodysurfaces and cavities including skin, ocular surface,gastrointestinal tract or uro-genital system, providing aprotective barrier [1,2]. Cell loss is continuously
counter-balanced by cell production via the paracrine
effects of various cytokines and growth factors including
Epidermal Growth Factor (EGF), Hepatocyte Growth
factor (HGF) or Fibroblast Growth Factor (FGF)
maintaining thus epithelial homeostasis [2]. The source ofcells involved in tissue regeneration is poorly defined andremains unknown. A possible source can be stem-likeprogenitor cells, found mainly in neural [5], vascular [6],hepatic [7], pancreatic [8] and epidermal tissue [9], orbone marrow derived stem cells, including hematopoietic
stem cells (HSCs), involved in blood cell production andmesenchymal stem cells (MSCs) [2]. Various approaches
have been followed in the field of stem cell engineering,in order to exploit the differentiation ability of in vitro
MSCs to not only the well-known osteogenic, adipogenic
and chondrogenic lineages [3], but also towards neuraland epithelial lineages [1,2].
nd

The aim of our study was to verify the effect of somechemical inductors to differentiate in vitro MSCs intoepithelial-like cells, without genetically manipulate thecells.

MATERIALS AND METHODSMSCs isolation and expansion
Normal human mesenchymal stem cells (MSCs) wereobtained from bone marrow of 10 healthy Orthopedics
patients undergoing hip replacement surgery.
Approximately 10 ml of bone marrow were placed inculture plates, and the fibroblastic-like, plastic adherent
fraction, was isolated following multiple passages andused in our experiments. The MSCs were further cultured
and expanded in alpha-minimum essential medium(MEM; Gibco BRL, Invitrogen, Carlsbad, CA, USA),supplemented with 10% fetal calf serum (FCS;
PromoCell,
Heidelberg,
Germany)
and2%
Penicillin/Streptomycin mixture (Pen/Strep, 10,000 IU/ml;
PromoCell), by incubation at 37oC in 5% CO2
atmosphere. Medium replacement was performed every
third day and when reaching 80-90% confluence, thethReceived 2 of February 2015. Accepted 5 of March 2015. Address for correspondence: Ivanescu Radu, Department of
Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041,
Timisoara, phone/fax: +40256 490507, e-mail: radu.ivanescu@yahoo.com

Fiziologia - Physiology • 2015.25.1(85)31cells were passed using 0.25% Trypsin-EDTA solution(Sigma-Aldrich Company, Ayrshire, UK) followed bycentrifugation (10 minutes, 300g) and replated in T75
culture flasks at a density of 10,000 cells/cm 2. Startingwith passage two, part of the cells were used for further
phonotypical analyses and differentiation assays, while
MSCs expanded to passages 2-5 were used in thesubsequent experiments.
All tissue samples were obtained after signing theinformed consent elaborated under an approved protocol,
according to the World Medical Association Declarationof Helsinki.
Epithelial differentiation of human MSCs
Human MSCs at passage three, obtained as describedabove, were plated on adherent uncoated culture flasks,
and the differentiation process was induced using a
protocol previously described [2]. Shortly, the induction
medium was constituted of basal DMEM low glucose (1g/l, Gibco BRL, Invitrogen, Carlsbad, CA, USA) with 10%
FCS (PromoCell, Heidelberg, Germany), supplemented
gradually with 10 ng/ml keratinocytes growth factor (KGF,
Peprotech Inc., Rocky Hill, NJ), 20 ng/ml epidermal
growth factor (EGF, Peprotech Inc., Rocky Hill, NJ), 10
ng/ml hepatocyte growth factor (HGF, Peprotech Inc.,
Rocky Hill, NJ) and 60 ng/ml insulin-like growth factor-2(IGF-2; Human Recombinant, Peprotech Inc., Rocky Hill,
NJ).

Fig. 1. Immunofluorescence staining for Vimentin, Cytokeratin,
E-cadherin, and DAPI. Cell were analyzed by fluorescencemicroscopy at 20x magnification, for undifferentiated hMSCs (A),HK2 epithelial line (B) and differentiated hMSCs (C).

Expression of MSCs characteristic markers assessed by
flowcytometry confirmed their definition criteria: positive
for CD90, CD73 and CD105, these cells being negative
for CD34 and CD45 (Figure 2).

Differentiation analysis
After 24 days of differentiation, cells morphology wasassessed using light and fluorescence microscopy.
Specific cell surface antigens, including stem markers:
CD29, CD44, CD54, CD73, CD90, CD95, CD105, CD106,
CD166, and epithelial markers - E-cadherin, Ep-CAM and
HER2, were measured by flow cytometry (FACScalibur,
BD
Biosciences,
San
Jose,
CA,
USA).
Immunofluorescence staining was also performed forcytokeratin, vimetin, and E-cadherin. All the analysis was
performed for the differentiated MSCs and for normal
undifferentiated MSCs as control.
RESULTS AND DISCUSSION
In all differentiated MSCs, some modifications of the cellmorphology were noticed, as they become morepolygonal and had a tendency to form multilayered
culture. Expression of vimentin was weaker in theepithelial-like cells, compared with MSCs, whereas
expression of cytokeratin was stronger inepithelial-differentiated cells (Figure 1).

32

Fig. 2. Undifferentiated MSCs expressing characteristic surface
markers

When the MSCs were induced towards epithelial lineage,
the cells were induced towards a different expression
pattern of surface markers (Figure 3).

Fiziologia - Physiology • 2015.25.1(85)for regeneration and repair of damaged tissues. Even
though there are controversies regarding stem cells
regeneration ability, there are solid evidence that stem
cells can induce tissue repair in both physiological andpathological conditions, in different non-hematopoietic
tissues, including here diverse epithelial types.
MSC homing and differentiation in vivo into type I
pneumocytes was demonstrated [12], as well as
acquirement of phenotypical characteristics of all mainlung cells [13], using an animal model of lung injury.
Moreover, MSCs were differentiated into pigmented
retinian cells [14,15], skin [16], ductal pithelial cells [17],and epithelial renal tubular cells [18,19]. In this study, wedemonstrated that MSCs can differentiate intoepithelial-like cells with keratinocyte phenotype, which
can be further used in cellular therapies.
Fig. 3. Epithelial-differentiated MSCs showing different patternsof expression for surface markers

Regarding expression of epithelial characteristic markers,
we demonstrated an increase of Ep-CAM, E-cadherin
and Her2 in epithelial-differentiated cells, compared toundifferentiated MSCs (Figure 4).

CONCLUSION
The experiments showed that MSCs differentiation
toward the cells expressing epithelial markers is relatively
easy to obtain using a certain combination of inductors,
without genetic manipulation of the cells.
ACKNOWLEDGEMENT
This work was supported by CNCSIS-UEFISCSU, project
number PNII-IDEI 318/2011 and 259/2011.

REFERENCES

Fig. 4. Flowcytometric analysis of surface markers for
undifferentiated MSCs (upper panel) and epithelial-like cells(lower panel)

There are few data regarding MSCs in vitro differentiation
towards epithelial-like cells, so that MSCs ability in thiscase is an unrevealed subject. This phenomenon isinteresting for evaluation of epithelial-mesenchymal(EMT) and mesenchymal epithelial transition (MET),
mechanisms which are involved in tumor development[10, 11]. Although the data from literature provides
information regarding the culture condition containing
trans retinoic acid for induction of differentiation, ourprotocol suggested the use of growth factors, such as
KGF (keratinocyte growth factor), EGF (epidermal growth
factor), HGF (hepatocyte growth factor), and IGF-2(insulin-like growth factor-2).
Several in vitro studies supported the idea of pluripotent
ability of mesenchymal stem cells, which could be used
Fiziologia - Physiology • 2015.25.1(85)1. Takafumi Takebayashi, Takashi Horii, Hiromi Denno, et al.
Human mesenchymal stem cells differentiate to epithelial
cells when cultured on thick collagen gel. Bio-Medical
Materials and Engineering, 2013; 23: 143-153
2. Paunescu V, Deak E, Herman D, Siska IR, Tanasie G,
Bunu C, et al. In vitro differentiation of human mesenchymal
stem cells to epithelial lineage. J Cell Mol Med, 2007; 11(3):502-508.
3. Baksh D, Song L, Tuan RS. Adult mesenchymal stem
cells: Characterization, differentiation, and application in celland gene therapy. J Cell Mol Med, 2004; 8: 301-316.
4. Horiuchi T, Miyamoto K, Miyamoto S, et al. Image
analysis of remesothelialization following chemical woundingof cultured human peritoneal mesothelial cells: The role ofhyaluronan synthesis. Kidney Int, 2003; 64: 2280-90.
5. Gage HH. Mammalian neural stem cells. Science, 2000;
287: 1433-38.
6. Kuznetsov SA, Mankani MH, Gronthos S, Satomura K,
Bianco P, Robey PG. Circulating skeletal stem cells. J Cell
Biol. 2001; 153: 1133-39.
7. Alison MR, Poulsom R, Forbes SJ. Update on hepatic
stem cells. Liver. 2001; 21: 367-73.
8. Bonner-Weir S, Sharma A. Pancreatic stem cells. J
Pathol. 2002; 197: 519-26.
9. Janes SM, Lowell S, Hutter C. Epidermal stem cells. J
Pathol. 2002; 197: 479-91.
33

10. Song L, Tuan RS. Transdifferentiation potential of
human mesenchymal stem cells derived from bone marrow.
FASEB J. 2005; 18: 980-982.
11. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD.
Human mesenchymal stem cells differentiate to a
cardiomyocyte phenotype in the adult murine heart.
Circulation. 2005; 105: 93-98.
12. Kotton DN, Ma By, Cardoso WV et al. Bonemarrow-derived cells as progenitors of lung alveolar
epithelium. Development 2001; 128: 5181-88.
13. Rojas M, Xu J, Woods CR et al. Bone marrow-derived
mesenchymal stem cells in repair of the injured lung. Am J
Respir Cell Mol Biol 2005; 33: 145-152.
14. Arnhnold S, Heiduschka P, Klein H et al. Adenovirallz
transduced bone marrow stromal cells differentiate into
pigment epithelial cells and induce rescue effects in RCS
rats. Invest Ophthalmol Vis Sci 2006; 47: 4121-29.
15. Arnhold S, Absenger Y, Klein H et al. Transplantation ofbone marrow-derived mesenchymal stem cells rescue

photoreceptor cells in the dystrophic retina of the rhodopsin
knockout mouse. Graefes Arch Clin Exp Ophthalmol 2007;
245: 1414-22.
16. Nakagawa H, Akita S, Fukui M et al. Human
mesenchymal
stem
cells
successfully
improveskin-substitute wound healing. Br J Dermatol 2005; 153:
29-36.
17. Fu X, Fang L, Li X et al. Enhanced wound-healing
quality with bone marrow mesenchymal stem cells
autografting after skin injury. Wound Repair Regen 2006; 14:414-22.
18. Morigi M, Imberti B, Zoja C et al. Mesenchymal stem
cells are renotropic, helping to repair the kidnez and improvefunction in acute renal failure. J Am Soc Nephrol 2004; 15:
1794-1804.
19. Herrera MB, Bussolati B, Bruno S et al. Mesencymal
stem cells contribute to the renal repair of acute tubularepithelial injury. Int J Mol Med 2004; 14: 1035-41.

MARKERI EPITELIALI EXPRIMATI DE CELULELE STEMMEZENCHIMALE UMANE IN CONDITII DE DIFERENTIERE IN VITROREZUMAT
Cu toate că abilitatea celulelor stem adulte de a regenera și repara țesuturile lezate rămâne controversată,celulele derivate din măduva hematogenă sunt raportate ca având o contribuție însemnată proceselor dereparare tisulară, atât în condiții fiziologice cât și patologice, incluzând de asemenea și numeroase tipuri deepitelii. În acest studiu, verificăm efectul anumitor substanțe biochimice inductoare pentru diferențierea in vitro acelulelor stem mezenchimale adulte (MSCs) derivate din măduva hematogenă, către celule epithelial-like. Dupăizolare și expandare, inducția MSCs spre linie epitelială s-a realizat utilizând diferite citokine și factori de creștere
(EGF – Factorul de creștere epidermală; HGF – Factorul de creștere al hepatocitelor; FGF – Factorul de creștere
al fibroblastelor), adăugati individual în cultură sau în combinație. Celulele diferențiate au fost analizate prin
imunohistochimie pentru vimentină și citokeratină, iar expresia genică pentru citokeratină 19 și E-caderină, prin
RT-PCR. În toate MSC-urile diferențiate, s-au observat modificări morfologice, ele devenind mai poligonale cu
tendință de a forma culturi pluristratificate. Expresia de vimentină a fost scăzută în celulele epithelial-like, în
comparație cu MSC, pe când expresia de citokeratină a fost mai intensă în celulele diferențiate. Experimentele
au demonstrat că diferențierea MSC spre celule care exprimă markeri epiteliali este relativ ușoară, princombinația diferiților factori inductori, fără modificări genetice ale celulelor.
Cuvinte cheie: celule stem mezenchimale, celule epithelial-like, mediu de diferențiere epitelială, RT-PCR,
imunohistochimie

34

Fiziologia - Physiology • 2015.25.1(85)EVALUATION OF LARGE AND GIANT INCIZIONALHERNIA REPAIR: A 5-YEARS RETROSPECTIVEOBSERVATIONAL STUDYNODITI GEORGE1, LAZAR CAIUS1, NODITI GHEORGHE2, LAZAR FULGER3
1Surgery Clinics II, Emergency Clinical County Hospital Timisoara
2Clinics of Plastic and Reconstructive Surgery - Austria House, “Victor Babes” University of Medicine and Pharmacy
Timisoara
3Surgery Clinics II, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT
Large and giant incisional hernia defect is challenging in order to improve thepostoperative results. We conducted a 5
years retrospective observational study on large and giant incisional hernia repair in our Clinics in order to evaluate thesurgical outcomes in relation with the patient’s demographic characteristics, comorbidities, and the local surgicalconditions. We have established the target group of 124 patients with large and giant ventral herniaout of a total
number of 409, using inclusion and exclusion criteria. We collected data about demographics, comorbidities, hernia
characteristics, operative details, and follow-up data (length of hospital stay, incidence of early complications). Data arepresented as mean and range.Sex distribution shown a ratio of F: M= 3.34: 1. The peaks of incidence for female
patients was the decade 60-69 y and 50-59 y, and for males the decade 70-79 y and 80-89 y. Prior hernia onset
gynecology surgical procedures (39.5%) and classic gallbladder surgery (33.8%), have been noticed. 19% of thepatients were admitted for a recurrence or multi recurrence. High blood pressure, obesity, diabetes, cardiac andrespiratory diseases were the most obvious comorbidities. Alloplastic substitution with polypropylene, polyester andDacron mesh has been used in all cases. The most preferred mesh position was intraperitoneal (78%), thenretromuscular (15%) and preperitoneal (7%). Immediate postoperative complications occurred in 10% (12 cases) of thepatients. 58.3% of them have been postoperative wound infection, then hemorrhagic shock, acute renal insufficiency,
parietal abscess. Length of hospital stay varied widely according to the presence of complications, between 7 days andmaximum 62 days. In large and giant incisional hernia defect size, location, depth of involvement, contamination, andcomorbidity influence the repair outcomes. Pre, intra and postoperative management focused on patient’s status andcharacteristics has to be done in a multidisciplinary team of clinicians, anesthesiologist, general surgeons and plastic
surgeons.
Key words: large hernia, giant hernia, comorbidities, alloplastic substitution, mesh position, hospital stay

INTRODUCTION
Incisional hernias whose real incidence is difficult todetermine are considered one of the most common
complications after abdominal surgery with high morbidity
and important socioeconomic costs. Sanders si Kingsnorthin a review on scientific papers from 1970-2012 havefound big differences on repored incidence values [1]. The
reported incidence after a midline laparotomy ranges from
3% to 20% and is doubled if the index operation iscomplicated by wound infection [2]. About 50% ofincisional hernias are detected within one year of surgery,but they can occur several years afterwards, with asubsequent risk of 2% a year [2, 3]. Apparently incisionalthhernias can occur after any type of laparotomy incision but
the most common are after midline (especially upper
midline) and transverse incisions [3].
Since now risk factors such as patient’s multiple
comorbid conditions (age >65, male, obesity, diabetus,
renal failure, smoking, others) have been reported inmany studies, but the relative importance of many of the
proposed risk factors is poorly understood yet [1, 4, 5].
Surgical factors that can influence the surgical outcomes
have been mentioned in several studies. Type oftechnique as well as type of suture are to be mentioned.
Reported recurrence rates remain high. For instance, therecurrence rate after open suture repair can be up to
54%, and up to 36% for open mesh repair. In general,thReceived December 10 2014. Accepted February 5 2015. Address for corresponding: Noditi George, MD, Surgery Clinics II,
Emergency Clinical County Hospital Timisoara; 22 Zimnicea Steet, RO-300317, Timisoara; phone: +40755636258; e-mail:georgenoditi@gmail.com

Fiziologia - Physiology • 2015.25.1(85)35recurrence rates are slightly lower, with a mean of about
15% [6]. A systematic review found that in the case ofhernia repair without prosthetic mesh the recurrence rates
is 12-54%, whereas in the case of hernia repair with mesh
the recurrence rates range between 2-36% [6, 7].
In the case of large incisional hernia defect size, location,
depth of involvement, contamination, and comorbidity
influence the management of abdominal wall defects.
We conducted a 5 years retrospective observational
study on large and giant incizional hernia repair in our
Clinics in order to evaluate the patients demographic
characteristics, comorbidities, and the surgical conditons(defect size, infection, bowel resection, mesh type, drainuse, length of hospital) and surgical outcomes.

MATERIAL AND METHODS
We have conducted a retrospective observational study
on big and large hernia repair with alloplastic substitution
performed in the Surgery Clinics II of Emergency County
Hospital Timisoara. Electronic medical of all 409
consecutive patients who had a ventral hernia repair from2008 until 2012 have been revised. We have establish
the target group of 124 patients with large and giant
ventral hernia for whom we have revised also theoperative reports, clinic notes, imaging reports. Hernia
size of 10-15 cm was defined as large and more than 15
cm as giant. Data were collected using a standard
Microsoft excel worksheet. We included in the study
primary ventral hernia repairs and recurrent hernia
repairs. Every recurrent hernia repair during the time
period of the study was counted as a separately. We
excluded ventral hernia repairs that were performed as apart of other procedures from our study.
The data that was obtained included demographics,
comorbidities, and hernia characteristics such as statusof natural history (reduced versus incarcerated).
Operative details included the hernia management:
defect size, mesh type, bowel resection type (colon vssmall bowel), mesh positioning. Followup data consistedof length of hospital stay, the incidence of earlycomplications. Data are presented as mean and range.

Fig. 1. Age groups and sex distribution of the cases.

Legend: blue - females; red - males.

According to the type of laparotomy incision prior hernia
onset we noticed: 39.5% gynecology surgical procedures(49 cases), 33.8% clasic gallbladder surgery (42 cases),
11.3%colon cancer surgery (14 cases), followed by otherpathology conditions: gastric perforated ulcer, umbilicalhernia, hidatic cyst, intestinal infarction, apendicectomy,
pancreas cancer, ilio-femural bypass (Figure 2).

Fig. 2. Distribution of type of laparotomy incision prior hernia onset

In the group of 124 patients, 81% presented norecurrence. The rest of 19% have been distributed asfollowing: 15% (18 patients) have been recorded withrecurrence afer direct suture of the initial defect and 45 (5patients) with multi recurrence (Figure 3).

RESULTS
During 2008-2012, 409 patients have been operated in
our Clinics for ventral hernia. Out of them 124 patients(23%) presented large and giant hernia. Most of them(87%) had a hernia defect between 10-15cm and the rest(13%) presented a giant hernia.
Distribution according to sex has shown a ratio of F:M =3.34 : 1.
The age limits of the group were between 30-89 y. The
peaks of incidence for female patients was the decade60-69 years and 50-59 years, and for males the decade70-79 years and 40-49 years (Figure 1).
36

Fig. 3. Percentage distribution of recurrences.

Comorbidities associated with our group consisted inorder in high blood pressure, obesity, diabetes, cardiac
and respiratory diseases.
Fiziologia - Physiology • 2015.25.1(85)Considering the localisation of hernia the most frequant wasat midline (78%), then xifo-umbilical, sub-umbilical andsupra-umbilical. Lateral hernia heve been encoutered in
17% of cases, and transversal hernia in 5% (Table I).
Table I. Localisation of hernia
MedianLateralTransvers

xifo-ombilical39

subcostal

10

supra-ombilical28

paramedian8

sub-ombilical

30

Right iliac fosse

3

TOTAL

97(78%)TOTAL

21(17%)

sub-ombilical

6

TOTAL

6(5%)

In 17 cases out of 124, the intervention was an emergency(irreducible or incarcerated ventral hernia and strangulated
ventral hernia). All the patients were operated on undergeneral anesthesia. For alloplastic substitution the mostused were from polypropilene, polyester and Dacron. As formesh position the most preferred was intraperitoneal (97cases), retromuscular (19 cases) and preperitoneal (8cases). Figure 4 is illustrating the precentage distribution ofmesh positioning.

Fig. 4. Distribution of mesh positioning

Immediate postoperative complications have been notedin 10% (12 cases) of the patients: wound infection (7cases), hemorrhagic shock, acute renal insufficiency,
parietal abscess.
Length of hospital stay varied widly according to thepresense of complications, between 7 days andmaximum 62 days.

DISCUSSION
The correction of abdominal hernias remains one of themost common surgical procedures since it occurs inabout 11% of laparotomies [8].
In our study we noticed as types of laparotomy incisionprior hernia onset gynecology surgical procedures (39.5%),
clasic gallbladder surgery (33.8%), colon cancer surgery(11.3%), and other pathology conditions. 19% of the group
have been prezented for a recurrence or multi recurrence.
In 17 cases out of 124, the intervention was an emergency
for irreducible or incarcerated ventral hernia andstrangulated ventral hernia. Considering the localisation ofhernia the most frequant was at midline (78%).
Better appreciation of patient characteristics may help to
support which type of procedure or mesh use will likely
Fiziologia - Physiology • 2015.25.1(85)succeed. Risk factors for the development of abdominal
wall incisional hernias may include the following:
overweight, smoking, age greater than 60 years, wound
infection,
re-laparotomy,
postoperative
wound
dehiscence, postoperative wound infection chronic
medical conditions (such as cirrhosis or cardiopulmonary
disease, diabetes), and chronic steroid use [9, 10, 11].
In our study the risk factors were age greater than 60
years, female sex, and associated comorbidities
consisted in order in high blood pressure, obesity,
diabetes, cardiac and respiratory diseases.
In terms of hernia repair there are three options withrespect to the location of the prosthesis: premuscleaponeurotic (onlay), retro-muscleaponeurotic(underlay or sublayer) or retro-muscular, andintraperitoneal (inlay) [8]. It seems to be no statisticallysignificant the difference regarding recurrence between
prostheses
placedinretro-muscularor
pre-muscleaponeurotic position [12]. The placement of
the prosthesis intraperitoneally in the open operation, is
an indication to the large incisional hernias, to themulti-recurrent, when associated with intra-abdominal
injury, in obese patients and when the laparoscopic
approach is contraindicated [8].
In our study, alloplastic substitution with polypropilene,
polyester and Dacron mesh have been used in all cases. As
for mesh position the most preferred was intraperitoneal(78%), retromuscular (15%) and preperitoneal (7%).
Suture repair is likely to produce results twice as bad asmesh repair and the current techniques that surgeons areusing to repair incisional hernias with prosthetic mesh
continue to yield recurrence rates of greater than 20% [13].
Major complications which can occur in repair of large
incisional hernias include mesh infection andenterocutaneous fistula which may result in prolonged
morbidity and require re-operation [14].
Our followup data have shown immediate postoperativecomplications in 10% (12 cases) of the patients. Most ofthem (58.3%) confrunted with postoperative wound
infection (7 cases), then hemorrhagic shock, acute renal
insufficiency, parietal abscess. Lenght of hospital stayvaried widly according to the presense of complications,
between 7 days and maximum 62 days.
Before performing a surgical approach for large or giant
hernia repair it is important that the patient is advised
about the possibility that their expectations regarding theoutcome, both aesthetic and functional, could notto bealways achieved.
Langer and colleagues in a comparative, retrospective studyof over 400 incisional hernia operations over a 25-year
period, estimated that the most important prognostic factor isthe surgeon's experience [15]. Actually pre, intra andpostoperative management of hernia repair focused on
patient’s status and charateristics should be done in a
multidisciplinairy team of clinicians, anethesiologits, generalsurgeons and plastic surgeons.
37

REFERENCES
1. David L Sanders, Andrew N Kingsnorth The modern
management of incisional hernias. BMJ, 2012; 344.
2. Mudge M, Hughes LE. Incisional hernia: a 10 year
prospective study of incidence and attitudes. Br J Surg,
1985; 72: 70-71.
3. Bucknall TE, Cox PJ, Ellis H. Burst abdomen andincisional hernia: a prospective study of 1129 major
laparotomies. BMJ, 1982; 284: 931-3.
4. Rutkow IM. Demographic and socioeconomic aspects ofhernia repair in the United States in 2003. Surg Clin NorthAm, 2003; 83:1045-51.
5. Sanders RJ, DiClementi D. Principles of abdominal
wound closure. II. Prevention of wound dehiscence. ArchSurg, 1977; 112: 1188-91.
6. Girotto JA, Malaisrie SC, Bulkely G, Manson PN.
Recurrent ventral herniation in Ehlers-Danlos syndrome.
Plast Reconstr Surg, 2000; 106: 1520-6.
7. Ceydeli A, Rucinski J, Wise L. Finding the best
abdominal closure: an evidence-based review of theliterature. Curr Surg, 2005; 62: 220-5.
8. Speranzini MB, Deutsch CR. Giant incisional hernias.
Arq Bras Cir Dig, 2010; 23(4): 280-286.

9. Vilallonga R, Fort JM, Gonalez O, Baena JA, Lecube A,
Armengol M. Review Article Management of Patients with
Hernia or Incisional Hernia Undergoing Surgery for Morbid
Obesity. J Obes, 2011; 2011: 860942.
10. Wong SY, Kingsnorth AN. Prevention and surgicalmanagement of incisional hernias. Int J Surg Invest., 2001;
3: 407-14.
11. van't Riet M, deVos van Steenwijk PJ, Bonjer HJ,
Steyerberg EW, Jeekel J. Incisional hernia after repair ofwound dehiscence: incidence and risk factors. Ann Surg.,
2004; 70:281-6.
12. Vidović D1, Jurisić D, Franjić BD, Glavan E, Ledinsky
M, Bekavac-Beslin M. Factors affecting recurrence after
incisional hernia repair. Hernia, 2006; 10: 322-325.
13. Luijendijk RW, Hop WCJ, van den Tol MP, de Lange
DC, Braaksma MM, Jermans N, et al. A comparison of
suture repair with mesh repair for incisional hernia. N Engl J
Med, 2000; 343:392-8.
14. Kingsnorth A. The Management of Incisional Hernia.
Ann R Coll Surg Engl., 2006; 88(3): 252-260.
15. Langer C, Schaper A, Liersch T, Kulle B, Flosman M,
Füzesi L, et al. Prognosis factors in incisional hernia surgery:25 years' of experience. Hernia, 2005; 9:16-21.

EVALUAREA REPARĂRII HERNIILOR INCIZIONALE MARI ȘI GIGANTE:STUDIU RETROSPECTIV OBSERVAȚIONAL PE 5 ANIREZUMAT
Repararea defectului cauzat de hernia incizională mare și gigantă este o provocare pentru îmbunătățirea rezultatului
postoperator. Am efectuat unstudiu retrospectiv observațional pe 5 ani privind repararea herniei mari și gigante în
Clinica noastră, pentru a evalua rezultatele chirurgicalein relație cu caracteristicile demografice si comorbiditațile
pacientului si condițiile chirurgicale locale. A fost selectat un grup țintă de 124 pacienți cu hernia incizională mare și
gigantă din cei 409, folosind criterii de acceptare și respingere. Au fost colectate date demografice, de comorbiditate,caracteristici ale decfectului parietal, detalii de intervenție chirurgicală si date de supraveghere (durata de spitalizare,incidenta complicatiilor imediate). Datele sunt prezentate ca medii si distributii.Repartiția pe sexe a arătat o ratăF:M= 3,34: 1. Vârfurile de incidență pentru femei au fost decadele 60-69 ani și 50-59 de ani, iar pentru bărbați
decadele 70-79 si 80-89 de ani. Intervențiile chirurgicale din sfera ginecologică (39,5%) și chirurgia vezicii biliare(33,8%) au fost cele mai frecvente laparotomii înainte de apariția herniei. 19% dintre pacienți s-au internat pentru
recidivă sau multi recidivă.Cele mai frecvente comorbidități semnalate au fost hipertensiunea arterială, obezitatea,
diabetul zaharat, afecțiuni cardiace și respiratorii. In toate cazurile s-a folosit substituția alloplastică cu meșe dinpolipropilenă, poliester sau Dacron. Poziționarea meșei s-a făcut cel mai des intraperitoneal (78%), apoi
retromuscular (15%) și preperitoneal (7%). Complicațiile postoperatorii imediate au apărut la 10% dintre pacienți (12cazuri). 58,3% dintre ele au fost reprezentate de infectarea plăgii postoperatorii, urmate de șoc hemoragic,
insuficiență renală acută, abces parietal. Durata de spitalizare a fost in funcție de prezența
complicatiilorposoperatorii, respectiv între 7 zile și maxim 62 zile. In cazul defectelor parietale mari și gigante,mărimea defectului, localizarea, structurile implicate, prezența contaminarii și comorbiditățile pot influența rezultatele
postoperatorii. Managementul pre, intra și postoperator focusat pe statusul și caracteristicile pacientului este depreferat să se facă în echipă multididciplinară de medici interniști, anesteziști, chirugi generalițti si chirurgi plasticieni.
Cuvinte cheie: hernie mare, hernie gigantă, comorbidități, substituție alloplastică, poziționarea meșei, durata despitalizare

38

Fiziologia - Physiology • 2015.25.1(85)POLYURETHANE NANOSTRUCTURES INCORPORATINGURSOLIC AND OLEANOLIC ACIDS: IN VITROANTIPROLIFERATIVE EVALUATIONCAMELIA OPREAN1, FLORIN BORCAN2, MIRABELA CRISTEA3,FLORINA BOJIN4, ALEXANDRA IVAN4, MARIUS MIOC*1,
CRISTINA TRANDAFIRESCU1, CODRUTA SOICA1, VIRGIL PAUNESCU4
1Department of Pharmaceutical Chemistry, “Victor Babes” University of Medicine and Pharmacy Timisoara
2Department of Analitical Chemistry, “Victor Babes” University of Medicine and Pharmacy Timisoara
3 “Pius Brinzeu” Timișoara County Emergency Clinical Hospital, Timişoara
4Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACTIn the last years, the frequency of melanoma is increasing while melanoma death-related causes currently represent
more than 80% of all skin cancers. Oleanolic and ursolic acids are pentacyclic triterpenes that were proven as efficient
antitumor agents in several cancers such as colon, liver, breast and prostate. The aim of this study was synthesis of a
new delivery system for ursolic and oleanolic acids, as a solution for their poor water solubility that presumably causes
low bioavailability. Using the interfacial polycondensation technique combined with spontaneous emulsification,
nanoparticles with diameter between 30 and 68 nm were obtained.In vitroantriproliferative assay was conducted on
several melanoma cell lines using the free pure compounds as blank A significant antiproliferative effect of ursolic acidon human and murine melanoma cells was observed, while the same doses of oleanolic acid exhibited only low
potency on all tested cell lines. When incorporated into polyurethane nanoparticles, the active compounds lack theirantiproliferative effect, probably due to a weak release from the final nanostructures.
Keywords: antiproliferative, melanoma, oleanolic acid, polyurethane nanoparticles, ursolic acid

INTRODUCTION
In the last years, the frequency of melanoma isincreasing [1] while melanoma death-related causes
currently represent more than 80% of all skin cancers [2].
The highest incidence occurs between age 20 and 45,
with a mortality rate higher in men than in women;
however, the incidence is higher among women, due tothe fact that,usually, melanoma occurs on areas hard to
be noticed on men, while on women it occurs on lower
legs [3, reviewed in 4].
New
treatment
strategies
aregeneratedbyincreasedresearch in the field, natural compounds
being a significant part of the currently studiedcompounds. Oleanolic and ursolic acids are pentacyclic
triterpenes that were proven as efficient antitumor agentsin several cancers such as colon, liver, breast andprostate [5-7]. Also, several studies have reported theirefficacy against melanoma [8, 9].
The aim of this study was synthesis of a new delivery
system for ursolic and oleanolic acids, as a solution forththeir poor water solubility thatpresumably causes low
bioavailability. The delivery system is based onpolyurethane nanoparticles which were physicochemicallyanalyzed in terms of particle size; in order to evaluate theirefficacy, an in vitro antriproliferative assay was conducted
on several melanoma cell lines using the free purecompounds as blank.

MATERIALS AND METHODS
Substances
Ursolic and oleanolic acids of analytical purity werepurchased from Fluka (Sigma Aldrich, Steinheim,
Germany). The reagents were purchased as follows:
Merck (Germany) provided isophoronediisocyanate(IPDI), Polyethylene glycol M = 200 (PEG), acetone,
Span®85 and Tween®20. 1,4-butanediol (BD) was
purchased from Carl Roth GmbH (Germany) andethylene glycol (EG) from Lach-Ners.r.o. (Czech R.).

th

Received 10 of February 2015. Accepted 12 of March 2015. Address for correspondence: Marius Mioc, Department ofPharmaceutical Chemistry, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A,
RO-300041, phone/fax: +40256490507, e-mail: marius.mioc@umft.roFiziologia - Physiology • 2015.25.1(85)39Synthesis of polyurethane drug delivery system
The method described in literature by Bouchemalet al.
(10) was used for the polyurethane particles synthesis.
The method consists in a multi-step procedure: interfacial
polycondensation
techniqueandspontaneous
emulsification. An organic phase (mixture of 1.5 ml IPDI,
1.5 ml Span®85, and 15 ml acetone heated at 30oC) was
injected into an aqueous phase (0.8 ml EG, 0.8 ml BD,
0.3 ml PEG, and 1.5 ml Tween®20 mixed with 15 ml
distilled water and heated at 30o C) under magnetic
stirring (500 rpm). The final mixture was heated at 40oC
for four hours in order to ensure complete chemical
reactions. The nanoparticles obtained from the proceduredescribed above were maintained as layers of 3 mm
thickness in Petri dishes at 80o C for 12 hours, for waterand acetone removal, and then the products were
washed with a mixture (water-acetone 1:1 v/v). The twotriterpenic acids were added in concentration of 57 µM in
two experiments and empty polymeric nanoparticles
were used as a control.

the concentrations of 25, 50, 75 and 100 μM. The twohighest concentrations (75 and 100 μM) of theempty
polyurethane nanoparticles were tested. After the
exposure time, 15 μL of the Alamar Blue solution wasadded and the cells were incubated for at least 4 h at 37°C. A microplate reader was used tospectrophotometrically analyze the samples at 570 nm
and 600 nm respectively. Untreated cells were used ascontrols. Since the tested substances were dissolved indimethyl sulfoxide (DMSO; Sigma-Aldrich, Ayrshire, UK)and stored as stock solutions, the highest concentrationof DMSO in the final medium (0.1%) was tested and did
not have any significant effect on cell proliferation. For all
experiments, final concentrations of the tested
compounds were prepared by serial dilutions of the stock
solution (10 mM) with the DMEM and EMEM medium.
Cell viability was calculated using the formula:

Nanoparticles’ size measurements
A particle size analyzer (Vasco from Cordouan Tech.,France) was used in order to measure the size of theobtained polyurethane nanoparticles. Solutions of 1:100(w/w) in ethanol were prepared. The parameters used
were: 190 channels, 25o C temperature, 80% laser power,
18 µs time interval, continuous acquisition mode andLog-normal dispersion. The average value of threeconsecutive measurements was considered.

untreated positive growth control]} × 100

Alamar Blue in Vitro Analysis
A375 human melanoma cells (Sigma-Aldrich, Bucharest,
Romania) were cultured in DMEM (Sigma-Aldrich,
Bucharest, Romania) containing 15% FCS (fetal calf
serum, PromoCell, Heidelberg, Germany) and 1%
penicillin-streptomycin (Pen/Strep, 10,000 IU/mL;
PromoCell). SK-mel 2 human melanoma cells (ATCC,
Germany) were cultured in EMEM (ATCC, Germany)
containing 10 % FBS (fetal bovine serum, ATCC,
Germany) and 1% penicillin-streptomycin (Pen/Strep,
10,000 IU/mL; PromoCell). B16 4A5 murine melanoma
cells were cultured in DMEM (Sigma-Aldrich, Bucharest,
Romania) containing 10% FCS (fetal calf serum,
PromoCell,
Heidelberg,
Germany),1%
penicillin-streptomycin (Pen/Strep, 10,000 IU/mL;
PromoCell) and 1% glutamine (PromoCell). Cells weremaintained at an atmosphere of 5% CO 2 at 37°C.
The cell lines were seeded onto a 96-well microplate(5,000 cells/plate) and attached to the bottom of the wellfor 24 h. The next day, 150μL of new medium containing
the tested substances were added and cells wereincubated for 48 h.ursolic acid (UA), polyurethane
nanoparticles incorporating ursolic acids (UA_nano),oleanolic acid (OA) and polyurethane nanoparticles
incorporating oleanolic acid (OA_nano) were added in
40{[(εOX)λ2 Aλ1 − (εOX)λ1 Aλ2 of test agent
dilution]/[(εOX)λ2 A°λ1 − (εOX)λ1 A°λ2 ofWhere,εOX= molar extinction coefficient of alamar Blue oxidized
form (BLUE);
A = absorbance of test wells;
A° = absorbance of positive growth control well (cells
without tested compounds);
λ1= 570 nm and λ2 = 600 nm
All in vitro experiments were performed on microplateswith at least four parallel wells. The results are arepresented as mean ± standard deviation. One way Anova
test was used to determine the statistical difference
between various experimental groups;*, ** and ***indicate p<0.05, p<0.01 and p<0.001.

RESULTS
Nanoparticle’s diameter
Polyurethane nanoparticles with the two tested acids
were prepared. Figure 1 exhibits the results of particle
measurements for the polyurethane nanostructures witholeanolic acid (a), polyurethane nanoparticles with ursolic
acid (b) and empty polyurethane nanoparticles (c). One
can notice that nanoparticles with diameter between 30
and 68 nm were obtained, as follows: 30.91 nm forpolyurethane nanoparticles with oleanolic acid, 58.90 nm
for polyurethane nanoparticles containing ursolic acidand 67.63 nm for empty polyurethane samples. Only one
population was obtained for all the samples, thus
indicating their homogeneous nature.

Fiziologia - Physiology • 2015.25.1(85)Fig. 1. Diameter of polyurethane structures for: (a)polyurethane nanoparticles with oleanolic acid, (b)polyurethane nanoparticles with ursolic acid and (c) empty
polyurethane nanoparticles. Parameters used were:temperature (25o C), 190 channels, laser power (80%), timeinterval (18 µs), acquisition mode (continuous) and analysis
mode (Pade-Laplace).

Cell proliferation assay
Figure 2 present the in vitroantiproliferative effects ofursolic and oleanolic acids compared to the encapsulated
compounds on A375 human melanoma cell line.
Exposure to ursolic acid for 48 h caused thedecrease ofA375 cells’ viability in a dose-dependent manner. The
viability values were as follows: 96.60 ± 1.63 %, 76.25 ±4.60 %, 52.61 ± 9.74 % and 33.23 ± 3.20 % for 25, 50,
75 and 100 μM, respectively. Encapsulation of ursolic
acid inside polyurethane nanoparticles led to thefollowing results: 97.98 ± 2.46 %, 85.74 ± 8.15 %,100.44 ± 7.37 %, and 79.49 ± 4.54 %, for the same
concentrations, respectively. In case of oleanolic acid, theviability values were: 77.94 ± 7.92 %, 75.70 ± 2.88 %,75.15 ± 6.90 %, 82.54 ± 0.20 % for 25, 50, 75 and100 μM, respectively. The same concentrations of thepolyurethane nanoparticles incorporating ursolic acid led
to the results: 86.68 ± 2.48 %, 88.03 ± 7.83 %, 88.59 ±6.37 %, 85.58 ± 5.15 %, respectively. Also, the highest
concentrations (75 and 100 μM) of empty polyurethane
nanoparticles were tested and the viability values after 48
h of exposure were: 94.72 ± 6.69 % and 86.33 ± 2.20 %,respectively.

Fiziologia - Physiology • 2015.25.1(85)Fig. 2. In vitro viability of A375 human melanoma cells after
48 h of exposure to: (a) ursolic acid (UA), polyurethane
nanoparticles incorporating ursolic acid (UA_nano), empty
polyurethane nanoparticle (empty_nano) and (b) oleanolicacid (OA), polyurethane nanoparticles incorporating
oleanolic acid (OA_nano), empty polyurethane nanoparticle(empty_nano) as compared to the untreated cells (control).

Fig. 3. In vitro viability of SK-Mel 2 human melanoma cells
after 48 h of exposure to: (a) ursolic acid (UA), polyurethane
nanoparticles incorporating ursolic acid (UA_nano), empty
polyurethane nanoparticle (empty_nano) and (b) oleanolicacid (OA), polyurethane nanoparticles incorporating
oleanolic acid (OA_nano), empty polyurethane nanoparticle(empty_nano) as compared to the untreated cells (control).

41

As figure 3 shows, the viability values for SK-Mel 2 cells
treated with ursolic and oleanolic acids as well as theirnanoparticles are: 95.40 ± 1.89 %, 67.90 ± 4.77 %,50.12 ± 2.16 %, 11.24 ± 2.19 % for 25, 50, 75 and 100
μM of ursolic acid, respectively. Treatment with
nanoparticles incorporating ursolic acid resulted in thefollowing values: 101.34 ± 4.77 %, 101.76 ± 2.22 %,102.91 ± 1.35 % and 100.00 ± 2.41 % for 25, 50, 75 and100 μM, respectively. Oleanolic acid’s treatment led to
the following results: 99.25 ± 2.19 %, 92.30 ± 4.23 %,90.83 ± 3.16 % and 85.73 ± 4.38 % for 25, 50, 75 and100 μM, respectively. For the same concentration ofpolyurethane nanoparticles incorporationgoleanolic acidthe viability values were: 101.23 ± 2.31 %, 106.51 ±0.48 %, 113.07 ± 2.00 % and 102.21 ± 3.28 %,respectively. The empty nanoparticles led to 90.20±
2.37 % for 75 μMand 89.34 ± 3.26 % for 100 μM.

Fig. 4. In vitro viability of B16 4A5 murine melanoma cells
after 48 h of exposure to: (a) ursolic acid (UA), polyurethane
nanoparticles incorporating ursolic acid (UA_nano), empty
polyurethane nanoparticle (empty_nano) and (b) oleanolicacid (OA), polyurethane nanoparticles incorporating
oleanolic acid (OA_nano), empty polyurethane nanoparticle(empty_nano) as compared to the untreated cells (control).

B16 4A6 murine melanoma cells viability values after
treatment with the tested compounds are presented in
Figure 4. Ursolic acid’s treatment produced decrease incell viability as follows: 70.35 ± 9.23 %, 36.04 ± 2.32 %,25.85 ± 7.80 % and 12.70 ± 4.11 % for 25, 50, 75 and100 μM, respectively. For the same concentrations of
nanoparticles with ursolic acid the results were: 76.08 ±3.31 %, 73.68 ± 4.10 %, 68.43 ± 3.53 % and 64.77 ±0.68 %, respectively. Treatment with oleanolic acidresulted in the following values: 81.23 ± 4.23 %, 73.00 ±42

6.49 %, 77.41 ± 6.57 % and 77.31 ± 5.18 % for 25, 50,
75 and 100 μM, respectively. At the same concentrations,the results for nanoparticles with oleanolic acid were:
86.68 ± 2.48 %, 85.04 ± 8.53 %, 88.59 ± 6.37 % and85.58 ± 5.15 %, respectively.

DISCUSSION
The role of pentacyclictritepenes in cancer treatment is the
subject of many reports. Ursolic and oleanolic acidextracted from various plants were proven to act as
antiproliferative agents in many cancer types. Menget al.
[11] reported the ursolic acid’s activity on DU 145, PC-3
and LNCap prostate cancer lines with IC50values ranging
between 22 and 36 μM. Also, they reported ursolic acid’s
antitumor effect on HeLa (cervix) and RPMI 8226
(myeloma) cells [11]. Shan et al. [12] reported the in vitro
antitumor activity of ursolic acid on HL60 and K562
(leukemia) cells as well on HL60 and H562 – Adriamycin
resistant leukemia cells. Other studies also reported theinvivo anticancer activity of ursolic acid in HCT11 coloncancer xenograft [13] as well as in DU145 prostate cancer
cells implanted in athymicBalb/c nude male mice [14].
In terms of oleanolic acid’s anticancer activity, in vitro
effects were reported for BGC-823 (gastric) and A549
(lung) cancer cells [15]. Kartiniet al. reported positive
results of oleanolic acid against SiHa (cervix) cells [16].
Cheng et al. reported the oleanolic acid antitumor activity
on MCF-7 (breast) cancer cells; Cheng’s results arecontradictory to Hsu et al. that reported a weak
anticancer activity of oleanolic acid on MCF-7 and
MDA-MB-231 cells [17].
Our data revealed the in vitroantiproliferative activity ofursolic acid on all tested melanoma cell lines. The viability
decrease manifested in a dose-dependent manner for all
three cell lines, however with different potencies. The
IC50values ranged between 75 and 100 μM for A375
(human) melanoma cells, between 25 and 50 μM for B16
4A5 (murine) melanoma cells and 75 μM for SK-Mel 2(human) melanoma cells. The same concentration ofoleanolic acid seems to produce a weak response as
antiproliferativeagent on the tested cells. For all cell lines,
the IC50 values were not found in the tested range ofconcentration, the lower viability being recorded for B16
4A5 (73 %). SK-Mel 2 proved to be the most resistant tooleanolic acid. Our results are consistent with previous
data, suggesting that ursolic acid is more effective than theoleanolic acid [18, 19] in terms of antiproliferative activity.
In recent years, nano- and micro-particles have raised
medical interest, being used as drug delivery systems [20].
Some of the new strategies of drug delivery in
nanotechnology
arepolymericnanoparticles,
microemulsions, liquid cristals systems, solid lipid
nanoparticles and liposomes [21]. Along with otheradvantages, it was reported that nanostructures could
improve the solubility and stability of the active substances
Fiziologia - Physiology • 2015.25.1(85)andalsoassociatecompounds
withdifferent
hydrophilicity/lipophilicity degrees [22, 23]. Theseadvantages could lead to the reduction of the therapeutic
dose and the improvement of pharmacological activity [21].
The present synthesis of polyurethane nanoparticles was
based on the method previously described by
Bouchemalet al. [10], who obtained nanocapsules as drug
carriers for α-tocopherol, a good antioxidant with a highsensitivity on light, heat and oxygen. Based on thisprocedure, Borcanet al. also obtained microparticles usingisophorondiisocyanate, with nontoxic in vitro activity on
mesenchymal stem cells and reducedin vivo noxiousness
on CD1Nu/Nu mice [20].
As the present study revealed, the encapsulation ofursolic and oleanolic acid into poly(ether) urethane
nanoparticles did not improve the in vitro activity of theactive compounds, leading to a high viability of the threecancer cell lines, as compared to the antiproliferative
activity of the free substances. A possible explanation
may consist in the strong entrapment of the active agent
inside the polymeric nanostructures which presumablycauses an impossibility to exert its pharmacologicalactivity. Further research should be focused on the invivo study of the polyurethane nanoparticles loaded withthe active compounds.

CONCLUSIONSThe present study reportsa significantantiproliferative
effect of ursolic acid on human and murine melanoma
cells while the same doses of oleanolic acid exhibited
only low potency on all tested cell lines. When
incorporated into polyurethane nanoparticles, the active
compounds lack their antiproliferative effect, probably
due to a weak release from the final nanostructures.
ACKNOWLEDGEMENTS
This paper was published under the frame of European
Social Found, Human Resources Development
Operational Programme 2007-2013, project no.
POSDRU/159/1.5/S/136893.

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NANOSTRUCTURI POLIURETANICE INCORPORÂND ACIZII URSOLIC ȘI
OLEANOLIC: EVALUAREA EFECTELOR ANTIPROLIFERATIVE IN VITROREZUMATÎn ultimii ani, frecvența melanomului este în creștere, iar mortalitatea produsă de melanom reprezintă mai mult de
80% din cancerele pielii. Acizii oleanolic și ursolic sunt triterpene pentaciclice care s-au dovedit a fi agenți
antitumorali eficienți în câteva tipuri de cancere precum colon, ficat, sân și prostată. Scopul studiului a fostsinteza unui noi sistem de livrare a acidului ursolic și oleanolic, ca o soluție pentru slaba solubilitate în apă, careposibil să fie cauza unei biodisponibilități scăzute. Utilizând tehnica de policondensare interfacială combinată cu
emulsificarea spontană, au fost obținute nanoparticule cu diametru intre 30 și 68 nm. S-au realizat teste
antiproliferative in vitro, pe câteva linii celulare de melanom, utilizând compuși puri liberi ca martori. A fostobservat un efect antiproliferativ semnificativ al acidul ursolic pe liniile celulare melanomice umane și murinice, întimp ce aceleași concentrații de acid oleanolic au avut o potență redusă pe toate liniile celulare testare. Dupăincorporarea în nanoparticule poliuretanice, compușii activi și-au pierdut efectul antiproliferativ, probabil datorită
unei eliberări scăzute din nanostructurile finale.
Cuvinte cheie: antiproliferativ, melanom, acid oleanolic, nanoparticule poliuretanice, acid ursolic

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