FIZIOLOGIA
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Fiziologia - Physiology • 2016.26.2(90)FIZIOLOGIA

physiology
CONTENTS
1. Talking about oxytocin – physiological characteristics of the oxytocin system ................................................................................4
Liana Kobylinska, Ioana Mocanu, Cristina Gianina Anghel, Iuliana Dobrescu
2. New Marker that Correlates Epidemiological Indicators with Noninvasive Markers in Chronic Hepatitis C .................................14
Claudiu Popescu, Ioan Romoșan, Vasile Mălăiuș, Voichița Stoicoiu
3. Complications of Chemotherapy in Multiple Myeloma ........................................................................................................................19
Cristina Sorica-Romanescu, Ovidiu Potre, Adina Bucur, Ioana Ionita, Monica Pescaru, Claudiu Ionita, Mihai Ionita,
Hortensia Ionita
4. In Vitro Testing the Cytotoxic Effect of Titanium-Hydroxyapatite Biomaterial ..................................................................................23
Voisan R, Cristea M, Tanasie G, Bojin F, Nistor D, Anghel S, Ionac M
5. Neurophysiological and Neuropsychological Analysis of Cognition in New Hypothyroid Females ..............................................29
Pravriti Mishra, Preethi BL, Pramila Kalra
6. Evaluation of the In Vitro Secretory Profile of Mesenchymal Stem Cells in Contact With a Titanium-Hydroxyapatite
Biomaterial .............................................................................................................................................................................................33
Voisan R, Tanasie G, Cristea M, Marusciac L, Georgescu M, Tatu C, Ionac M
7. Predictors of experiencing trouble with the police in young drivers aged 18-20 years, alcohol and drugs consumers
residing in Timis County, Romania .....................................................................................................................................................39
Pantea C, Preda M.A ,Tuță Sas I, Băcean Miloicov C, Bagiu R, Popa M, Fira Mladinescu C, Petrescu C, Putnoky S, Suciu O, Vlaicu B
8. Acute posterior multifocal placoid pigment epitheliopathy - 18 months follow-up ..........................................................................44
Preda M.A, Ciobotea D., Vlaicu B., Boruga O., Munteanu M.

CUPRINS
1. Să vorbim despre oxitocină – caracteristicile fiziologice ale sistemului oxitocinergic ......................................................................4
Liana Kobylinska, Ioana Mocanu, Cristina Gianina Anghel, Iuliana Dobrescu
2. Un nou marker care coreleaza indicatorii epidemiologici cu markerii noninvazivi in hepatita cronica C ......................................14
Claudiu Popescu, Ioan Romoșan, Vasile Mălăiuș, Voichița Stoicoiu
3. Complicatii ale chimioterapiei in mielomul multiplu ............................................................................................................................19
Cristina Sorica-Romanescu, Ovidiu Potre, Adina Bucur, Ioana Ionita, Monica Pescaru, Claudiu Ionita, Mihai Ionita,
Hortensia Ionita
4. Investigarea efectului citotoxic in vitro a unui biomaterial de titan combinat cu hidroxiapatită .......................................................23
Voisan R, Cristea M, Tanasie G, Bojin F, Nistor D, Anghel S, Ionac M
5. Analiza neurofiziologica și neuropsihologică a cogniției la cazuri noi de hipotiroidie la femei ........................................................29
Pravriti Mishra, Preethi BL, Pramila Kalra
6. Evaluarea in vitro a modificărilor induse asupra profilului secretor al celulelor stem mezenchimale în contact
cu un biomaterial de titan-hidroxiapatită ............................................................................................................................................33
Voisan R, Tanasie G, Cristea M, Marusciac L, Georgescu M, Tatu C, Ionac M
7. Predictori ai experimentării de necazuri cu poliția la tineri de 18-20 ani cu permis de conducere, consumatori de alcoolși droguri din județul Timiș, România ....................................................................................................................................................39
Pantea C, Preda M.A , Tuță Sas I, Băcean Miloicov C, Bagiu R, Popa M, Fira Mladinescu C, Petrescu C, Putnoky S, Suciu O, Vlaicu B
8. Epiteliopatie pigmentară placoidă acută multifocală – 18 luni de urmărire clinică ...........................................................................44
Preda M.A, Ciobotea D., Vlaicu B., Boruga O., Munteanu M.

Fiziologia - Physiology • 2016.26.2(90)3TALKING ABOUT OXYTOCIN – PHYSIOLOGICAL
CHARACTERISTICS OF THE OXYTOCIN SYSTEM
Liana Kobylinska1,2, Ioana Mocanu2, Cristina Gianina Anghel2, Iuliana Dobrescu2
1. Department of Physiology and Fundamental Neuroscience, “Carol Davila” University of Medicine and Pharmacy, Bucharest,
Romania
2. Child and Adolescent Psychiatry Department, “Prof. Dr. Al. Obregia” Clinical Psychiatry Hospital, Bucharest, Romania

Abstract: Oxytocin has been highly researched in the past two decades, particularly for its involvement in social behaviors.
From the initial identified roles in breast-feeding and uterine contractions during birth, it is now known that it is the key factor inpair bonding, attachment behavior and social functioning. In the context of an alarming increase in the incidence of psychiatricdisorders involving deficits of social functioning, the physiological mechanisms of action of oxytocin have come into focus.
Moreover, it has been shown that oxytocin circuits are deregulated in several psychiatric pathologies, with depression, autism
spectrum disorders and anxiety being the most prevalent. However, obsessive-compulsive disorder, schizophrenia andborderline personality disorder also associate modifications in oxytocin secretion or response. This review aims to summarize
the current findings in the field, emphasizing oxytocins effects at both central and peripheral levels.
Key-words: oxytocin, vasopressin, peripheral and central actions

BACKGROUND
Oytocin is the first artificially synthesized hormone andit is used by clinicians for induction and maintenance oflabor. Since the beginning of 1990s when oxytocin was
linked with specific attachment behaviors, numerous
studies have been completed in order to investigate thepotential benefits of oxytocin as adjuvant medication in
psychiatric diseases with social deficits, like schizophrenia
spectrum disorders, autism spectrum disorder andimpulse-control disorders.
In Greek, the term “oxytocic” means `quick birth`. This
name was given to the hypothalamic nonapeptide in 1906
when Sir Henry Hallet Dale discovered its uterine contracting
properties [1]. Several years later, in 1910 and 1911, Ott andScott [2] then Schafer and Mackenzie [3] described themilk-ejection property of oxytocin through contraction of thesmooth muscle layer of the galactophore ducts. The nineaminoacid sequence was discovered in 1953 [4] and oxytocin
became the first artificial synthesized human hormone [5].
Oxytocin is produced in the hypothalamicmagnocellular neurosecretory cells of the supraoptic andparaventricular nuclei, and it is stored in the posterior
pituitary gland in Heering bodies. The axons that store
oxytocin probably have connections with oxytocin
receptors in the accumbens [6] and medullar and corticalspecific areas [7]. Depending on the species, oxytocin
receptors can be located in the amygdala or in the stria
terminalis. In the pituitary gland, oxytocin is stored in
vesicles bound to neurophysin I.

Non-neural sources of oxytocin have been identified:
corpus luteum [8,9], interstitial cells of Leydig [10], theretina [11], the adrenal medulla [12], the thymus [13] andthe pancreas [14]. The possible actions on these tissues
are not fully known. In women, oxytocin released by the
corpus luteum and estrogen help synthesize prostaglandin
E2 involved in the involution of the corpus luteum. In men,oxytocin secretion in interstitial Leydig cells is correlatedwith a high intake of vitamin C [15].
In mammals, the majority of oxytocin effects are due topituitary secretion. Some of the most known actions are:lactation, uterine contractions, social behavior, injury
recovery [16], sexual behavior [17,18].
Due to its role in sexual behavior like orgasm andcouples attachment [19] oxytocin is also known as the 'love
hormone'.
Oxytocin release increases during birth and lactation,and during orgasm (in men and women), during caresses
and pleasant social interactions. In women, there is a cyclic
elevation of plasma levels during ovulation.
Oxytocin effects on the central nervous system are due
to specific receptors distributed in the pituitary gland,
lateral septal nucleus, periaqueductal grey matter, Broca's
area, Meynert nucleus, locus coeruleus, the vagus nerve,
the trigeminal nerve and the reticular formation [20-22].
Experimental results suggest that oxytocin has animportant role in promoting social interactions. Animalstudies using knockout models, oxytocin agonists andantagonists showed that oxytocin facilitates:

Address for correspondence: e-mail: vnedescu@yahoo.com;
Child and Adolescent Psychiatry Department, “Prof. Dr. Al. Obregia” 10 Berceni Road, Sector 4, RO - 041914 Bucharest,
Romania

4

Fiziologia - Physiology • 2016.26.2(90)social closeness or mood for interacting with ananimal from the same species‐ social memory or the ability to remember a peer fromthe same species with whom they previouslyinteracted;‐ forming stable bounds between couples members.
This effect was highlighted through the discovery of ahigh number of oxytocin receptors in prairie voles
that are monogamous, unlike other vole species that
do not form stable couples [23].
Recent studies on young healthy adults suggest thatthe social effects of oxytocin observed in animals can begeneralized in humans. It looks like oxytocin can promote
positive social behavior towards strangers through:decreasing amygdala activation and the stress response
associated with the perception of social threatening [24-27];
increasing accuracy in interpretation of other people's
emotions [28]; improvement in eye contact [29]; increasing
the level of trust and cooperating behavior [30,31].
Also, oxytocin increases the ability to remember faces[32,33] and the positive assessment of a unknown person:
after oxytocin administration, strangers where consideredmore attractive and trustworthy [34] and the negative
experiences with a stranger where minimized [35,36].
Considering these results, the scientific community
focused on the potential effect of oxytocin administration intreatment of some psychiatric disorders. It was suggested
that oxytocin may help disorders that involve or lead toabnormal approach of other individuals. Currently, there
are studies on human subjects that assess the potential ofoxytocin as adjuvant treatment in autism spectrum
disorders, schizophrenia, social conduct disorder, social
phobia, anorexia, depressive disorders (especially
postpartum depression), mother-child attachment disorders,with promising results.
In order to exert its effects on the central nervous
system, exogenous oxytocin needs a suitable way ofadministration, such as intranasal. Research shows thatmelanochortin, vasopressin and insulin can cross the
blood-brain barrier when they are administered intranasal.
It has been highlighted that oxytocin crosses the
blood-brain barrier through intranasal administration, by
measuring oxytocin plasma levels after 10 and 30 minutes
since administration [37]. Other research groups provedthat intranasal administration skips the blood-brain barrier
through distribution in the subarachnoid space [38].
Furthermore, this way of administration had no adverse
reactions for doses of 18-40 IU in short-term experimental
conditions in voluntary adults [39]. One International Unit ofoxytocin is equivalent to 2 micrograms of pure oxytocin.
The aim of this review is to summarize oxytocin’s main
physiological mechanisms of action at the level of different
organs and systems throughout the body. In this sense, an
extensive pub-med and gene-cards search was conductedin March-April, 2016. The conclusions on the subject aresummarized in the following pages.
‐

Fiziologia - Physiology • 2016.26.2(90)I. Oxytocin-vasopressin structural analogy
Vasopressin, also known as arginine vasopressin (AVP)
is a neurohormone produced by magnocellular neurons in
the paraventricular and supraoptic nuclei. The neuron
projections reach the posterior pituitary gland, where
vasopressin is released into the bloodstream [40,41]. At the
peripheral level, vasopressin acts as a hormone and is
involved blood pressure regulation and water reabsorption
in the distal convoluted tubes [42]. AVP is also produced byparvocellular neurons located in the paraventricular
nucleus. Their projections reach the anterior pituitary gland[43]. At this level, AVP stimulates the secretion of ACTH,
which means it is directly involved in the body's reaction tostressors, modulating hypothalamic-pituitary-adrenal axis
activity [44].
As well as for oxytocin, it is thought that vasopressin
has been evolutionary preserved, the two nanopeptides
differing by only two aminoacids, both being involved inmodulating certain social behaviors, like couple formation,social recognition and social cognition in humans and otheranimal species [45,46]. It seems that some of oxytocin
effects are mediated through vasopressin 1a receptors[47].
The two aminoacids that differ are located in position 3
and 8 (isoleucine and leucine in oxytocin are replaced byphenylalanine and arginine in vasopressin).
Allvertebrates produce structural analogs for oxytocin andvasopressin, with similar functions. It is thought that theseevolved from common precursors. The genes that code thevasopressin precursor and the oxytocin precursor are
located on chromosome 20 [48]. The two loci are reverse
transcribed and it is thought that evolutionary it is the samegene duplicated and reversed. Estimates made on
nucleotide levels of the genes that code oxytocin andvasopressin suggest that the ancestral gene for the
precursor should be more than 500 million years old. Given
that structural preservation of the two nanopeptides it isvery likely that it was a simultaneous development andpreservation phenomenon of substances, enzymes andspecific receptors. Moreover, vasotocin is present in themost primitive forms of cyclostomes identified until now; itis thought that the ancestral gene duplication witch lead theformation of the two nanopeptides was produced bycyclostomes iradiation [20].
Both nanopeptides are produced in the parvocellular
neurons located in the hyptohalamus, and their projections
reach the anterior pituitary gland and other cortical andsubcarotical areas. Beside the classic secretion from axons,
oxytocin and vasopressin are secreted by dendrites ofmagnocellular neurons in the hypothalamus. Bothsubstances diffuse in the extrecellular space and have aneuromodulating effect [7].
Peptides similar in structure and function asvasopressin and oxytocin are found in almost all vetebrates.
Fish with bone skeleton, terrestrial vertebrae ancestors,5have izotocin and vasotocin. Vasotocin is also found in
cyclostomes, as previously mentioned. Fish withcartilaginous skeleton present numerous peptides similar in
structure with oxytocin, with important functions inmaintaining ion homeostasis. Some of them even have thesame oxytocin found in mammals. The majority ofvertebrate secrete mesotocin. Except two Nord American
marsupials, Isoodon macrourus and Didelphis virginiana
who secrete oxytocin, all other marsupials exclusively
secrete mesotocin. The most primitive species from whoma peptide similar to oxytocin was isolated (anetocin) is
earthworm Eisenia foetida. Injecting anetocin in earthworm
or leech induces eggs deposit [20].

II. Oxytocin and vasopressin receptor system
Oxytocin receptors
Until now, only one type of oxytocin receptor has been
identified -OTR [44]. The OTR belongs to the G-protein
coupled receptor family. It is encoded by a single gene,located on chromosome 3 [48]. Various polymorphisms ofa single nucleotide are associated with autism spectrum
disorders, anxiety and depression [49-51]. It is thought that
metilation of oxytocin receptor gene is involved in the
etiology of postpartum depression [52]. In borderline
personality disorder, specific changes in the oxytocin
receptor gene structure can predict resilience to theadverse family environment [53].
Oxytocin receptor's affinity for oxytocin is ten times
higher than for vasopressin, both of them being able tobound to OTR [48].
Although the distribution of oxytocin and vasopressin
receptors in the central nervous system of various animalmodels is accurately known, studying these systems in thecentral nerous system in humans is less accessible. In
2013 Boccia et al published a study concerning thelocation of oxytocin receptors in the human cortex. The
method of identification used was immunohistochemistry.
Thus, the authors highlight OTR in the basolateral regionsof the amygdala, medial preoptic area, anterior andventromedial hypothalamus, olfactory bulb, anterior branchof Broca’s diagonal band, ventrolateral septum, the anterior
cingulate, hypoglossal nuclei and the solitary tract. Unlikein the rodents brain, in this study the authors did not find
OTR expression in the following structures in the humanbrain: hippocampus, parietal lobe, raphe nuclei,
ambigouous nucleus or pons [54]. In the beginning of the
90's, Loup et al published two studies [21,22] using
autoradiography of oxytocin and vasopressin in the central
nervous system. Beside previously listed locations, theyfound OTR expression in basal nuclei of Meynert andglobus pallidus. Their method identified pons andmesencephalic receptors.
Oxytocin and vasopressin neurons have oxytocin andvasopresin receptors thus modulating their secretion.
Activating the receptors doesn't necessary generate a6action potential in this case [55]. Studies involving murinae
models suggest that oxytocin secretion induced by pituitaryoxytocin neuron depolarization is facilitated by thetransmembrane glycoprotein CD38. Blocking its expression
alters maternal and social behavior in murinae models [56].
Variations in CD38 expression have been identified inpatients diagnosed with autism spectrum disorder [57].
Oxytocin binding to its receptor generates
conformational change and G protein cascade activationand further calcium release from intracellular deposits
resulting in phosphorylation of intracellular proteins,
activation of nitric oxide synthetase and vasodilatation,smooth muscle contraction, increasing gene transcription
and neuronal excitability [49]. OXTR and vasopressin
receptor type 2 have a rapid desensitization mechanism
through receptor internalization [20]. In 2001, Oakley et al
highlighted that oxytocin receptor internalization involves
binding with beta-arrestin in membrane pores coated withclathrin [58]. Around 85 percent of receptors return to thecell's surface in 4 hours since internalization [59]. This
process is important for chronic administration regarding
the frequency of administration, in order to obtain long-term
effects on behavior. In 2004, Huang et al evidenced that 2times per day chronic administration of oxytocin reducedsocial behavior in mice, while acute administration had
prosocial effects [60].
Magnesium, manganese and cholesterol increaseoxytocin's receptor affinity for its ligands and allosteicallyregulate the receptor's activity, while progesterone and its
analogs act as antagonists [20].
In 2001, Gimpl and Farenholtz published an extensive
review about the known locations of oxytocin receptors [20].
Specific, they are found in uterus, ovaries, testis, prostate,
the mammary gland, adipose tissue, pancreas, GI tract,kidneys, adrenal glands, cardio-vascular system, thymus
and some neoplastic tissue in the mammary gland.
Oxytocin’s actions at these levels will be further discussed.
Vasopressin Receptors
There are three subtypes of vasopressin receptors AVRP1a, AVRP1b (also known as V3) and V2 [63]. The
genes responsible for the synthesis are located on
chromosome 20 [20].
The AVRP1a receptor is located in the vascular smoothmuscle, skeletal muscle, liver, heart, red bone marrow,erythrocytes, thymus, lymph nodes, kidneys, adrenal
glands, bones, GI tract, thyroid gland, salivary glands,
lungs, pancreas, retina, spinal cord, skin, uterus, ovaries,
testis, prostate and visceral adipose cells. In the central
nervous system, AVPR1a is found in the hypothalamus,
hippocampus, thalamus, cerebellum, amygdala, striatum,
medulla, pons and cortex. Maximum incidence of AVPR1aexpression is found in the following areas: arteries, liver,kidneys, cortex, heart, adrenal gland and visceral adipose
cells. AVPR1a is a G protein-coupled receptor, activating
Fiziologia - Physiology • 2016.26.2(90)the phosphatidylinositol calcium second messanger
signaling pathway. Its function is contraction, cellproliferation, platelet aggregation, releasing coagulation
factors and glycogenolysis. Receptor activation modulates
blood pressure, aggressive maternal behavior andgenerates metabolic and macroergic precursors. AVPR1aabnormalities have been identified in ACTH-independent
macronodular adrenal hyperplasia, the syndrome of
inappropriate antidiuretic hormone secretion, small cell
carcinoma and autism spectrum disorder [64].
AVRP1b/V3 has the same location as AVPR1a, withover expression in the posterior pituitary gland modulating
ACTH release. AVPR1b is a G protein-coupled receptor,
activating the phosphatidylinositol calcium second
messanger signaling pathway. Expression or structuralabnormalities of AVPR1b have been identified in
ACTH-independent macronodular adrenal hyperplasia,
melancholy/anhedonia-avolition, Asperger syndrome(reclassified in 2013 as social-communication disorder,
according to DSM-V), Cushing syndrome and
ACTH-secreting pituitary adenoma. A important paralog is
Neuropeptide S receptor 1 involved in asthma etiology andother bronchopathies [65,66].
AVPR2 is part of the G protein-coupled receptor superfamily, bound to a Gs protein which activates adenylyl
cyclase. V2 receptor is expressed in the glomeruli,
predominantly in the renal collecting ducts and distal
convoluted tubes. The primary role at this level isregulating aquaporin expression and water reabsorbtion,
thus maintaining internal homeostasis. Structural orfunctional abnormalities of AVPR2 generate nephrogenic
diabetes insipidus. AVPR2 is also found in the lungs, where
it is involved in releasing clotting factors. The exact
mechanism of this pathway is partially understood, as
primary nephrogenic diabetes insipidus does not associatesecondary coagulopathies. Another location is inpulmonary tumors. AVPR2 generally have the same
distribution as AVPR1a and AVPR1b, with an overexpression in kidneys, testis (Leydig cells), heart, thyroid
and parathyroid glands [67].
In the central nervous system, vasopressin receptors
have been identified predominantly in the hypothalamus
and limbic system [68]. Areas with a high affinity for AVP
and no affinity for oxytocin can be found in the lateral
septal nucleus and some thalamic nuclei [21]. Areas with alower affinity for AVP are the hippocampus, parts of basal
ganglia, solitary tract nucleus and spinal trigeminal nucleus.
A study from 2014 involving titi monkey brains highlighted
that these primates have a circumscribed location ofoxytocin receptors, and vasopressin receptors have a
diffused localization. The reason why these monkeys
where studied is that they are monogamous with a welldefined social structure [69].
It has been previously shown that type 1a vasopressin
receptors, dopamine and oxytocin receptors from themedial prefrontal cortex contribute to monogamous
Fiziologia - Physiology • 2016.26.2(90)behavior, and in voles to polygamous behavior [70]. Also
modifying AVPR1a expression in the ventral globus
pallidus in prairie voles loses the monogamous behavior[71]. Oxytocin’s action on type 1a vasopressin receptors
from the lateral septum alters monogamous behavior infemale prairie voles [72]. This data suggests thatvasopressin and oxytocin receptors in the brain can
modulate diverse behaviors, sometimes opposed
according to different neural networks involved and thetargeted cerebral areas.

III. Oxytocin and vasopressin systems activation effects and modulators
Central nervous system effects
Both oxytocin and vasopressin are responsible forvarious behavior and somatic effects. For example, at
cardiovascular level, both oxytocin and vasopressin
influence blood pressure through vascular caliber, dieresisand hydric input, especially through vasopressin receptors[73]. Patients with autism spectrum disorder haveautonomic dysfunctions associated with alteredcardiovascular reactivity that can be explained by atypicalfunctioning of the oxytocin-vasopressin system [74].
Oxytocin neuron projections to the limbic system andsomatosensory cortex are observed only in adult animals[75]. Dendrite secretion seems to play an important role inanimal models for social anxiety [76], while axonal
secretion modulates fear reaction in mice [77].
Considering that oxytocin receptor expression exceeds
areas of neuron networks involved in decision-making andsocialization, and that there are great differences inregional oxytocin receptor distribution, numerous species
dependent behaviors have been described. One of thecurrently accepted hypotheses regarding the way oxytocin
modulates social interactions is focusing attention on
external stimuli associated with social interactions byselectively ignoring irrelevant stimuli [78].
In songbirds, for example, OXTR are found in neuralnetworks involved in regulation of social behavior (pallium
avium) and motor nuclei of several cranial nerves
suggesting a role in vocal efferent pathways [79]. The
presence of oxytocin receptors in motor nuclei has also
been highlighted in monkeys (vagus motor nucleus andtrigeminal nucleus)[80] and in rodents [81] suggesting thatoxytocin is capable of rapid and direct influence in motor
and autonomous response to social stimuli. In rodents,
OXTR are also found in high amount in the olfactory cortex,
as smell is the primary way of interaction and social
exploration in these animals. In primates, oxytocin
receptors have been identified in cortical regions
responsible for visual attention, a crucial element for
primate interaction [78].
Another important aspect generated by activation/
inhibition of oxytocin-vasopressin system is its relationshipwith the monoaminergic system [49].
7

Serotonin appears to contribute to oxytocin andvasopressin effects in social functioning through regulationof peptide secretion or modulation of fear and anxiety
responses [49]. Oxytocin and vasopressin stress response
is serotonin mediated, and oxytocin growth in stressconditions is serotonin 1A, 2A, and 2C receptor activation
dependent [82]. Also serotonin blocks aggressive behavior
induced by AVP administration in murine models [83]. In
2012, a study highlighted a inverse correlation between
oxytocin and serotonin plasma levels in patients with
autism spectrum disorder [84], while in adult patients with
autism spectrum disorder the binding property of theserotonin transporter was reduced overall [85]. Anotherstudy measured serotonin and vasopressin levels in CSF
of patients with personality disorders and found direct
correlation between AVP and level of aggressiveness, andan indirect correlation between serotonin andaggressiveness [86]. The results of animal studies in thefield suggest that activation of some serotonin receptors
can trigger and may be needed for oxytocin andvasopressin secretion, which, in turn, stimulate serotoninrelease [49]. Another assumption of oxytocin andvasopressin behavioral effects relates to their potential inmodulating hypothalamic-pituitary-adrenal axis stressresponse [87].
Oxytocin and vasopressin activity seems to depend onsex steroids, with significant changes in OXTR, AVPR inneutered animals [49]. A study from 2003 underlined thatknocking-out the oxytocin gene or the estrogen alpha/beta
receptor gene altered considerably social abilities in mice[88]. Autism spectrum disorder incidence is higher in males;
however, the exact sex steroids-oxytocin-vasopressin
connections are not yet known [89].
Correlations between dopaminergic system and oxytocin
and vasopressin social effects seem to be related to reward
pathways. Oxytocin’s action could mediate satisfaction felt insocial contexts. In praire voles, maintaining preferences inelecting a partner requires simultaneous activation of
dopaminergic system and oxytocin and vasopressin action[70]. On the other hand, as in humans oxytocin receptors
could not be identified in the nucleus accumbens, probably
the action is collaterally mediated through the basal region of
the telencephalon [90].
During the last years, scientists studied the effects ofneuropeptides in rapid activation of interneurons.
Interneurons adjust the firing rate of main neurons, throughinhibiton using GABA [91]. Recently the role of oxytocin
has been shown to increase stimulus/ noise ratio throughits effect on interneurons. Specifically, oxytocin exposure of
the hippocampic interneurons raised their inhibition ability,
decreasing reflected signal from the main neuron.
Consecutively, the fidelity and intensity of fierings from demain neuron increased, with consequent augmentation of
the signal through the entire neural network [92]. Nakajima
et al (2014) showed that sexual and social behavior in miceare modulated by interneurons in the frontal cortex that8express oxytocin receptors [93]. It is possible thatvasopressin acts through a similar mechanism, because it
has been proven to increase neurotransmission in
hippocampic neurons [94]. Administration of AVP on tissue
sections from the hippocampus increases the frequency of
inhibitory signals. AVPR1a mediates this process, byincreasing GABA discharge. Simultaneously, AVP has an
excitatory effect on pyramidal main neurons [95]. Oxytocin
and AVPR1a receptors also mediate mutually inhibitory
effects through GABA interneurons in different regions of
the amygdala in rats [27]. GABA interneurons are alsopresent in primates’ telencephalon [96].
New optogenetics methods allowed modulation ofoxytocin receptor activation, highlighting increased firing of
GABA interneurons in the amygdala, and consecutive
reduction of 'freeze' behavior in preconditioned rats [77].
Another recent optogenetic study showed oxytocin secretionfrom hypothalamic projection in the pyriform cortex isnecessary for positive and negative social learning [97],meanwhile oytocin projections from autonomic nuclei in thebrainstem moderate cardiac rhytm variability [98].
Excitation/Inhibition imbalance also represents a theoryof autism spectrum disorder ethiology [99,100]. A potential
action mechanism of oxytocin in GABA neurotransmissionis through modulation of chlorine channel activity.
Increasing intracellular chlorine quantity in GABA neurons
from immature rats brains, therefore inducing membrane
hyperpolarization and lowering neural excitability seems to
be a protection mechanism in anoxic perinatal brain injury
in rats [101].
Somatic effects
Except the numerous actions in the central nervous
system listed above, both oxytocin and vasopressin havemultiple effects on various organs and tissues in the
periphery expressing the same receptors.
The uterus with its segments and layers represents
one of the primary oxytocin targets. An increase in uterine
secretion of oxytocin during pregnancy and peripartum
period has not been found, but the number of uterine
receptors increases significantly during this period.
Oxytocin is one of the most powerful uterotonic agents. Its
action at this level allows the expulsion of the fetus andplacenta with closing underlying placental vessels, and thearrest of peripartum hemorrhage [20].
In many species, ovarian oxytocin amount is higher thanin the blood, suggesting that the ovaries might also secrete
oxytocin [102]. It is possible for oxytocin to participate in
fertilization and early embryonic development [103], especiallysince oxytocin increases sperm motility in cultures [104].
At the same time, the secretion of oxytocin varies
depending on the stage of the menstrual cycle, in the luteal
phase is significantly low, and a correlation between
plasma levels of oxytocin and the degree of vaginal
moistening, which supports the involvement in female
Fiziologia - Physiology • 2016.26.2(90)sexual dynamics[105]. Moreover, ejaculation is correlatedwith a sudden increase in plasma oxytocin, probably due toa hypothalamic pulse of secretion. It acts both in the
periphery, by stimulating the smooth muscle in the seminal
ducts, as well as centrally, being involved in the behaviors
related to the sexual act. Two main functions of oxytocin
have been described at the testicular level – the tuning of
the seminal ducts motility and modulating steroidogenesys.
The effects on smooth musche contractility at this level are
mediated through AVPR1a, whose testicular density isseven times higher than that of oxytocin receptors [106].
Oxytocin increases the activity of 5 alpha-reductase in thetesticles and epididimus [107]. In the male reproductive
tract, oxytocin is also found in high amounts in the prostate
gland, where it increases the basal tone of the gland [20],which suggests its involvement in prostate contraction andseminal liquid ejection [106]. Due to its action on 5 alphareductase, oxytocin may be a paracrine factor in prostate
cell growth, with a possible involvement in benign prostate
hypertrophy [108].
As previously mentioned, one of the main roles ofoxytocin is in the mammary gland. Here it triggers milk
ejection. This is mediated through tactile receptors whose
neurons project in the hypothalamus. However, as central
oxytocin release is crucial for this reflex, it is triggered not
only through tactile stimuli, but also through reflex
responses to the baby’s behavior, such as to its crying[109]. Some studies even suggest a protective role ofoxytocin against breast tumors in women [110].
Another target organ for the neurohypophysal
hormones is the kidney. Here, they govern water andelectrolytes
excretion,bothhypovolemia
andhyperosmolarity being triggers for oxytocin andvasopressin release. At plasma sodium concentrations
above 130mM, both hormones levels increase
exponentially [111]. In dogs, oxytocin decreases sodium
reabsorbtion in the distal and collecting ducts, but thiseffect has not been found in humans [112].
At the level of the cardiovascular system, oxytocin
decreases mean arterial pressure in murine models, theeffect being apparent even in congenital hypertensive
animals, but only in males, probably due to the vascularresponse being modulated through sexosteroids [20].
As CD8+ cytotoxic lymphocytes express oxytocin
receptors, it has been inferred that oxytocin might play anactive role in T lymphocytes differentiation and focal
adhesion [20].
In the adipose tissue, oxytocin has an insulin-like
activity, increasing glucose oxidation and lypogenesys[113]. Both oxytocin and vasopressin increase insulin
release from the pancreas [114]. Patients with obesity havean altered glucose tolerance and hyperinsulinism, and they
seem to have four-fold more oxytocin than normal-weightindividuals [115]. The plasma level of oxytocin increases in
hypoglycemia [116].
At the level of the gastro-intestinal tract, oxytocin
Fiziologia - Physiology • 2016.26.2(90)increases gastric synchronization in stress conditions,
reducing the phenomenon of delayed gastric emptying
following restraint test in rodents [117]The role of oxytocin at the level of the adrenal gland is
not yet certain, even though there are numerous
oxytocinergic and vasopressine receptors at this level.
High doses of oxytocin have inhibited the aldosteronsecretion mediated by acetylcholine in experimental
conditions [118], whereas small doses have stimulated
aldosteron secretion [119]. It has also been stipulated thatoxytocin might decrease or modulate cortisole release fromthe adrenal gland [120]. Recently, it has been shown thatchronic oxytocin administration increases the adrenal
concentration of chatecholamines through increase in theamount of vesicular monoamine transporter 2 [121].

CONCLUSIONS
Overall, oxytocin has numerous functions, both in thecentral nervous system, as well as in the other organs of
the body. All of its functions seem to line up in modulatingsocial and mating behaviors, particularly throughinfluencing anxiety and empathy. The important role thatboth oxytocin and vasopressin play in the autonomic
response is another argument for their importance inhuman behavior and their potential usage as modulators ofspecific attitudes. Still, many effects are insufficiently
understood and long-term oxytocin supplementation
remains a central theme of psychiatric and neurobehavioral
research in terms of applicability and safety.
Another important issue to address is that of dose –relationship and adjustment for cronic treatment, as thephenomenon of receptors internalization makes it difficult
to maintain long-term effects, without getting side effects
due to the peripheral actions.
Even if oxytocin is the first hormone to be synthesized
artificially and one of the most constant substances in all
the animals, the research in the field is far from complete,particularly in the current context of constant increase indisorders that include social functioning deficits.

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SĂ VORBIM DESPRE OXITOCINĂ – CARACTERISTICILEFIZIOLOGICE ALE SISTEMULUI OXITOCINERGIC
Rezumat. Oxitocina a fost intens studiată în ultimele două decade, in mod special pentru implicarea sa în modularea
comportamentleor pro-sociale. De la rolurile identificate inițial în parturiție și alăptare, se știe astăzi că oxitocina
reprezintă un factor cheie în formarea perechilor, în atașament și Îîn funcționarea socială. În contextul unei creșteri
alarmante a incidenței patologiilor psihiatrice care asociază deficite de socializare, mecanismele fiziologice de acțiune
a oxitocinei au ieșit în prim-plan. Mai mult, a fost demonstrat că circuitele oxitocinergice sunt atipice în variate patologii
psihiatrice, cu precădere în depresie, tulburări de spectru autist și anxietate. Și alte patologii, precum tulburarea depersonalitate borderline, tulburarea obsesiv-compulsivă și tulburările schizofreniforme asociază alterări de secreție saude transducție a semnalului în cadrul circuitelor oxitocinergice. Scopul acestui articol este de a trece în revistă cele mai
recente descoperiri în domeniul studiului oxitocinei.
Cuvinte-cheie: oxitocină, vasopresină, efecte centrale și periferice

Fiziologia - Physiology • 2016.26.2(90)13NEW MARKER THAT CORRELATESEPIDEMIOLOGICAL INDICATORS WITHNONINVASIVE MARKERS IN CHRONIC HEPATITIS C
Claudiu Popescu1,2, Ioan Romoșan1,3, Vasile Mălăiuș4, Voichița Stoicoiu3
1. University of Medicine and Pharmacy „Victor Babeș” Timișoara, Department of Internal Medicine I, Discipline of Internal Medicine IV;
2. Military Unit 01109 Timișoara;
3. Clinic of Internal Medicine, Railway Clinical Hospital Timișoara;
4. Gastroenterology Section, Clinical Emergency County Hospital Târgu-Jiu

ABSTRACTAim and objectives: establishing a new marker, reproducible, quantifiable, that correlates various epidemiological indicators
to noninvasive indirect markers. Validating the new algorithm as noninvasive marker that predicts liver fibrosis stage.
Material and methods: the retrospective study, which includes a cohort of 133 cases with chronic hepatitis C. Inclusion
criteria: chronic hepatitis C diagnosis, gender, age, social environment, risk group, liver transaminases, total cholesterol,
triglycerides, antibodies to hepatitis C, liver biopsy, fibrosis stage (METAVIR).
Results: the greatest ratio was for the female gender (63.2%) and the age group 55-64 years (31.6%). The social
environment had the greatest ratio in urban areas (72.2%) and group risk indicator for risk-free group (67.7%). Diagnostic test
parameters had very good values for sensitivity test, early fibrosis (91.8%), advanced fibrosis, with a maximum for severe
fibrosis - F3 (97.8%). The accuracy of diagnostic test ranged between 93.07 to 99.25. Specificity of the test had encouraging
results, ranging from 93.90 to 100.00, which gives a strong predictive diagnostic test and a screening role.
Conclusion: the algorithm correlates epidemiological indicators with biochemical parameters, enabling evaluation of fibrosis
through an easy to apply methodology, with a strong predictive role, particularly for excluding severe fibrosis, advanced
fibrosis and cirrhosis.
Keywords: chronic hepatitis, Hepatitis C virus, epidemiology, fibrosis, noninvasive markers

INTRODUCTION
The patient with persistent infection with HCV has toface, in addition of suffering the disease the limitation incurrent activities, social discrimination in various kinds ofactivities such as the health care system predominantly, theworkplace or other kinds of social activities. In the sametime with discrimination, is limited the access to information(including medical informaion), prevention services, support,
testing, treatment and medical care [1].
According to the sero-prevalence study, conducted during
2006 - 2008, for persons with age between 18-69 years, theprevalence of hepatitis C resulted for Romania was 3.23% [6].
Also, the estimated prevalence in 2013 was 5.6%,
according to the sero-prevalence survey conducted by the
National Institute of Public Health (NIPH) [11].
It notes a discrepancy between the data collected by
the national collection systems, the results of prevalence
studies, but also between different prevalence studies [5, 9].
Liver biopsy has been considered for many years „gold
standard” because of the possibility of direct histologicalassessment of severity of liver disease.

In the same time, with the emergence of the possibilityof assessment by noninvasive markers of liver fibrosis,
between clinicians occurred conflicting views on liver biopsy
versus noninvasive markers, due both absolute and relative
contraindications(bleeding,pain,coagulopathies,
comorbidities) and other considerations [7].
Basically, at this time, after the emergence of new
non-invasive techniques, we cannot speak about a „gold
standard“ in diagnosing and assessing liver fibrosis, theexisting methods are all imperfect reference methods,
leaving room for new methodologies, noninvasive,
independent, which exceeds the current limits [10].

AIM AND OBJECTIVES
Establishing a new marker, reproducible, quantifiable,that correlates various epidemiological indicators tononinvasive indirect markers. Validating the new algorithm
as noninvasive marker that predicts liver fibrosis stage.

Adresă de corespondență: Doctorand Claudiu Popescu, Unitatea Militară 01109 Timișoara, Str. Chişodei, nr. 125, Timișoara,
România, e-mail: claudiu_popes_ro@yahoo.com

14

Fiziologia - Physiology • 2016.26.2(90)MATERIAL AND METHODSRetrospective study, developed between 2010 – 2015, in
the gastroenterology and hepatology clinic of Clinical
Emergency County Hospital „Pius Brânzeu Timișoara andgastroenterology section of County Emergency Hospital
Târgu-Jiu, includes a cohort of 133 cases with chronic
hepatitis C (CHC). All personal data resulting from medical
documents that were the subject of the study were kept
confidential in compliance with rules of professional conduct.
Selection of cases was performed using the following
inclusion criteria: main diagnosis CHC, framing each casethe following parameters: gender, age, social environment,risk group (risk behavior), liver transaminases, totalcholesterol, triglycerides , anti hepatitis C positive, stage offibrosis score interpreted by METAVIR. Determination of the
fibrosis stage was performed by liver biopsy (LB).
Statistical processing of results and comparative data
analysis was performed using the following softwares:
Microsoft Excel version 2007, Anova One Way, respectively
SPSS version 22.0. Sublots analyze was performed bystatistical indicators: weight, median, standard deviation.
Testing the consistency of the samples was performed
using the independent Student t-test, bilateral, and testing
the combination of parameters by using the Chi square testof association. Statistical differences of the test wererepresentative when the p value was <0.05 at a confidence
interval of 95%.

Study group structure based on epidemiologicalindicators (Table 1) included the most important factors forour analysis: gender, age group, social environment, risk
group. They were represented tabulated below including:number of cases, ratio, median, standard deviation (whereapplicable), the statistic probability index (p).

GenderAge group

Socialenvironment
Risk groupMarker type
Glutamic
oxaloacetictransaminase(GOT, IU/L)
Glutamic
pyruvictransaminase(GPT, IU/L)
Platelet count(U/µL) x 103)

RESULTS

Criteriontype

In our study, risk group (risk behavior) included allcases that were exposed to known risk factors: bloodtransfusions (before 1992), blood derivatives (thalassemia,
haemophilia, before 1987), surgery, dental care, chronic
hemodialysis, organ transplantation (before 1992) othermedical maneuvers with / without continuity solutions(intravenous, intramuscular, bleeding, wound care),intravenous drug use (sharing needles, syringes), tattoos,
piercing (rarely) [2].
To determine the statistical probability for each indicator,
I split the indicator age to age groups and calculated foreach of the other indicators related to these sub-indicators(Table 1). Thus for gender indicator, the value was 0.1299,
without statistical significance, for age 0.03493, for social
environment 0.0371, with statistical significance, and for risk
group indicator 0.0706, without statistical significance.

Number of cases
Male
Female
5-24
years
25-34
years
35-44
years
45-54
years
55-64
years
>65 years
Urban
Rural
Yes
No

49
84
0
13
18
39
42
21
96
37
43
90

Ratio
(%)

Median ±deviation(SD)

P value

36.8
63.2

-

0.129953 ±
12.13

0.03493-0.0371-0.0706

0
9.8
13.5
29.3
31.6
15.8
72.2
27.8
32.3
67.7

Table 1 Study group structure based on epidemiological indicators
Fiziologia - Physiology • 2016.26.2(90)Totalcholesterol(mg/dL)
Triglycerides(mg/dL)

Cases no.
NormallevelsHighlevelsNormallevelsHighlevelsNormallevelsLowlevelsHighlevelsNormallevelsHighlevelsNormallevels

Ratio
(%)

19

14.3

114

85.7

18

13.5

115

86.5

112

84.2

21

15.8

105

78.9

28

21.1

112

84.2

21

15.8

Median ±standard
deviation(SD)

P value

52 ±
35.93

0.011169 ±60.99

0.0086

213 ±
47.21

0.0162

162 ±
46.48

0.018386 ±
41.82

0.0097

Table 2 The value of indirect noninvasive markers
in the study group

Among the noninvasive indirect markers most relevant
to our study (Table 2), liver transaminases (GOT, GPT)
accounted comparative biochemical parameters, both
regression logic tests and as a component for othernoninvasive markers (APRI, FIB 4).
Other biochemical parameters from the analyzed ones
were represented by: platelet count, total cholesterol,
triglycerides, which we determined both the number ofcases normal/high levels, median and standard deviation
for the study group.
To determine the statistical probability for eachbiochemical parameter, we divided this parameter to
sub-parameters normal levels, high/low levels, we
correlated with age groups sub-indicator and I determined15

the p value, for each of these sub-indices based on theother parameters (Table 2).
Thus, for GOT parameter, the value was 0.0111, 0.0086
for GPT, 0.0183 for total cholesterol and 0.0097 fortriglycerides parameter, they all statistically significant.
In determining the hepatic fibrosis stage, we compared(Table 3) invasive and noninvasive techniques, determinedby several methods: LB, APRI, FIB 4 C-HEP (new marker)
and we split the group into two categories, depending on
the fibrosis stage: early fibrosis for fibrosis stage F0 to F2
and advanced fibrosis for fibrosis stage F2-F3 to F4.
Fibrosisstage(METAVIR)
F0 (without
fibrosis)F0 - F1F1 (mild
fibrosis)
F1 – F2
F2(significant
fibrosis)
F2 – F3F3 (severfibrosis)
F3 – F4F4 (hepatic
cirrhosis)

Method type/ number of cases
LB

APRI

FIB 4

C-HEP

0

0

0

5

4

24

50

5

11

0

0

6

0

0

0

13

41

0

0

22

0

62

48

0

44

0

0

43

0

0

0

1

33

47

35

38

Fibrosis
category

Early
fibrosis

AdvancedfibrosisTable 3 Comparative analysis of the fibrosis stage
in the study group

Following multiple analysis of logic regression, we
determined the most important parameters with predictive
value for hepatic fibrosis, thus, creating a new algorithm.
The new algorithm was calculated using the following
formula:C-HEP= total cholesterol (mg/dL) X (platelet
count)y(U/µL) + 1/triglycerides (mg/dL) + 1/age (years) –gender X z – risk group X w, where y, z and w represent
numerical coefficients resulting from calculation.
Parameter label
Sensibility (%)
Specificity (%)
Accuracy (%)
Positive
predictive value(%)
Negative
predictive value(%)
Minimum cutoff
value
Maximum cutoff
value

F0-F1
75
95.93
93.07

F0-F2
91.80
93.90
93.07

F3
97.78
100
99.25

F4
86.84
95.24
93.07

91.80

75

100

86.84

93.90

95.93

98.89

95.24

≥ 0.00≥ 0.00≥ 0.59≥ 0.75≤ 0.31≤ 0.58≤ 0.72≤ 1.00

Table 4 Diagnostic test parameters for different fibrosis stage
in the study group

16

The predictive value for the fibrosis stage F0-F2 (early
fibrosis) was determined (Table 4) dividing the study sample
into two groups: the group of fibrosis cases F0-F2 and thegroup of fibrosis cases F3-F4 (advanced fibrosis).
The predictive value for the fibrosis stage F3 (severe
fibrosis) was determined dividing the study sample into twogroups: the group of fibrosis cases F3 and the group of
fibrosis cases F0 to F2 and F4.
The predictive value for the fibrosis stage F4 (hepatic
cirrhosis) was determined dividing the study sample into
two groups: the group of fibrosis cases F4 and the group of
fibrosis cases F0 to F3-F4.
To determine the fibrosis stage with the new algorithm,
we have used numbering from 0 to 4, which iscorresponding to the degrees of fibrosis METAVIR from F0
to F4.

DISCUSSION
Analyzing the structure of the study group revealed thatthe greatest ratio was for the female gender (63.2%), datathat are consistent with other studies [3]. Age group 55-64
years (31.6%) was the most representative, the averageage in the study group was 54 ± 12.13 years, data partiallyconfirmed in other studies [3].
Other elements analyzed in the study group were thesocial environment, with the most important ratio in urban
areas (72.2%), data confirmed by other studies [4], with thepossible explanations and more complete information andmuch better addressability for urban residents.
Risk group indicator (risk behavior) had the greatest
ratio for the group without risk behavior (67.7%) and for risk
group was 32.3%, which is below the data reported by other(55%) [2].
Indirect non-invasive markers (biochemical parameters)
were analyzed from the point of view of ratio, and from thepoint of view of normal or high / low, resulting in hepatictransaminases a ratio of 85.7% for TGO respectively 86.5%
for TGP, normal levels, data are consistent with otherstudies [4,12].
Among other biochemical parameters, the most relevant
for our study were: total cholesterol, with a ratio of 78.9%
for the number of cases with normal levels and averagevalue 162 ± 46.48, serum triglycerides with a ratio of 84.2%
for the group with normal levels and average value 86 ±41.82, and for platelet count, for the group with normal
levels the ratio was 84.2% and average 213 ± 47.21, allvalues determined are comparable to values of otherspecialized studies [8].
From the comparative analysis of the fibrosis (METAVIR)
between PBH and other noninvasive markers, resulted: forearly fibrosis 56 cases (42.1%) PBH, 51 cases (38.3%) C-HEP,
24 cases APRI (18. 1%), 50 cases (37.6%) FIB 4 and foradvanced fibrosis 77 cases (57.9%) PBH, 82 cases (61.7%)
C-HEP, 109 cases (82%) APRI, 83 cases (62.4%) FIB 4.
Fiziologia - Physiology • 2016.26.2(90)Discordance for CHEP was 5 cases (8.9%) for early
fibrosis, the 5 cases representing virtually false negative
results, for advanced fibrosis 5 cases (6.5%), representing
false positive results, the ratio of false negative diagnosis
was 12.5%.
The analysis of the diagnostic test parameters, showedvery good sensitivity of the test values both for early fibrosis(91.8%), as well as advanced fibrosis, with a maximum
severe fibrosis - F3 (97.8%). The accuracy of the diagnostic
test ranged between 93.07 to 99.25, with a maximum forearly fibrosis (99.25).
Specificity of the test had encouraging results for all
types of fibrosis, with values ranging from 93.90 to 100.00,
which along with the negative predictive value (93.90 to
98.89), gives a strong predictive diagnostic test for hepatic
fibrosis and a screening part for undiagnosed cases.
Considering the promising results obtained so far andthe costs extremely low (all medical tests used are includedin the basic package or vouchers offered by medical
analysis laboratories), we think it would be appropriate tocontinue the research through a new prospective, casecontrol, validation diagnostic test and, based on the results,
to validate extensively the test as a screening test,
particularly in the family doctor's office.

CONCLUSION
The imagined algorithm correlates very well epidemiologicalindicators of biochemical parameters, enabling theassessment of fibrosis through an easy to apply
methodology. Allows the use of very simple diagnostic test
in clinical practice, with a strong predictive particularly for
excluding fibrosis (severe and advanced) and chirrosis.
ACKNOWLEDGEMENTS:
The author gratefully thanks the professor doctor Ioan Sporea,
chief of gastroenterology and hepatology clinic of Clinical
Emergency County Hospital „Pius Brânzeu" Timişoara, themanager of Public Health Department Gorj and Mr. Ion
Gheorghiţoiu, doctor, for their support in achieving thisscientific article.

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burden of a population: chronic hepatitis C. General population
screening - Medicine in Evolution, Volume XXII, No. 2, 2016
4. Claudiu Popescu, Ioan Romoșan, Matilda Săndesc, et al.
Multicenter comparative analysis of liver fibrosis in chronic
hepatitis C infection. The role of noninvasive markers - Medicine in
Evolution, Volume XXII, No. 3, 2016 (in process of publication)
5. E. F. Duffell, M. J. W. van de Laar and A. J. Amato-Gauci.
Enhanced surveillance of hepatitis C in the EU, 2006–2012 Journal of Viral Hepatitis, 2015, 22, 590-595.
6. Gheorghe L, Csiki IE, Iacob S, et al. The prevalence and risk
factors of hepatitis C virus infection in adult population in Romania:
a nationwide survey 2006 – 2008 - J Gastrointestin Liver Dis. 2010
Dec, 19(4):373-9.
7. Giada Sebastiani, Konstantinos Gkouvatsos, Kostas Pantopoulos.
Chronic hepatitis C and liver fibrosis. World J Gastroenterol 2014
August 28; 20(32): 11033-11053.
8. G. Sebastiani, P. Halfon, L. Castera, et al - Comparison of threealgorithms of non invasive markers of fibrosis in chronic hepatitis C Aliment
Pharmacol
Ther.
2012;35(1):92-104.
(http://www.medscape.com/ viewarticle/755974).
9. National Institute of Public Health. National Center for
Surveillance and Control of Communicable Diseases. Report for2013. Analyses of trends of communicable diseases insurveillance.
10. Mireen Friedrich–Rust, Mei–Fang Ong, Swantje Martens, et al.
Performance of Transient Elastography for the Staging of Liver
Fibrosis: A Meta-Analysis - Gastroenterology, Volume 134, Issue 4,
960 - 974.e 8.
11. Popovici O., Molnar G., Janta D., et al. Seroepidemiologic
prevalence study results for infection with hepatitis B and hepatitis
C - National Conference of Microbiology and Epidemiology,
Bucharest, 13 to 15 November 2014.
12. Vlad Ratziu, Julien Massard, Frederic Charlotte, et al.
Diagnostic value of biochemical markers (FibroTest-FibroSURE)
for the prediction of liver fibrosis in patients with non-alcoholic fatty
liver disease - BMC Gastroenterology 2006, 6:6
doi:10.1186/1471-230X-6-6

17

MARKER NOU DE CORELARE A INDICATORILOREPIDEMIOLOGICI CU MARKERII NEINVAZIVIÎN HEPATITA CRONICĂ CU VIRUSUL HEPATITIC CREZUMAT
Scop și obiective: stabilirea unui marker nou, reproductibil, cuantificabil, de corelare a indicatorilor epidemiologici cu
markerii neinvazivi indirecți. Validarea noului algoritm ca marker neinvaziv cu rol predictiv pentru stadiul fibrozei
hepatice.
Material şi metode: studiu retrospectiv, ce cuprinde o cohortă de 133 cazuri cu hepatită cronică cu virus hepatitic C.
Criterii de includere: diagnosticul de hepatită cronică cu virus hepatitic C, gen, vârstă, mediu social, grup de risc,
valoare transaminaze hepatice, colesterol seric total, trigliceride serice, anticorpi anti virus hepatitic C, puncție hepatică,gradul de fibroză (METAVIR).
Rezultate: ponderea cea mai mare a fost pentru genul feminin (63,2%) și grupa de vârstă 55-64 ani (31,6%). Mediul
social a avut ponderea cea mai importantă pentru mediul urban (72,2%), iar indicatorul grup de risc, pentru grupul fărărisc (67,7%) Parametrii testului diagnostic au avut valori foarte bune pentru sensibilitatea testului fibroza incipientă
(91,8%), fibroza avansată, cu un maxim pentru fibroza severă - F3 (97,8%). Acuratețea testului diagnostic a avut valori
cuprinse în intervalul 93,07 – 99,25. Specificitatea testului a avut rezultate încurajatoare, cuprinse între 93,90 – 100,00,
ceea ce conferă testului diagnostic un puternic rol predictiv și un rol de screening.
Concluzii: algoritmul corelează indicatorii epidemiologici cu parametrii biochimici, permițând evaluarea fibrozei printr-o
metodologie ușor de aplicat, cu un puternic rol predictiv, în special pentru excluderea fibrozei severe, avansate și acirozei hepatice.
Cuvinte cheie: hepatită cronică, virus hepatitic C, epidemiologie, fibroză, markeri neinvazivi

18

Fiziologia - Physiology • 2016.26.2(90)COMPLICATIONS OF CHEMOTHERAPYIN MULTIPLE MYELOMA
Cristina Sorica-Romanescu1, Ovidiu Potre1, Adina Bucur2, Ioana Ionita1,
Monica Pescaru1, Hortensia Ionita1
1.University of Medicine and Pharmacy ”Victor Babes” Timisoara, Department of Haematology
2.University of Medicine and Pharmacy ”Victor Babes” Timisoara, Department of Public Health and Management

ABSTRACT:Introduction: Multiple myeloma may cause a large range of problems, which are considered to be complications of thispathology.(1) Plasma cells proliferation interferes with normal production of blood cells leading to leukopenia, anemia andthrombocytopenia.
Aim: This paper aim is to assess complications in multiple myeloma patients, as well as to establish a correlation with thetreatment type they underwent.
Methodology: The study was performed on a group of 77 patients diagnosed with multiple myeloma between January 1st
2011 and December 31st 2015 in the Hematology Department of the City Clinical Emergency Hospital Timisoara.
Results: The group was studied in terms of anthropometric data, as well as of hematological, biochemical and immunologicalparameters. Complications present both at the time of diagnosis and after the start of polychemotherapy were shown, withstatistically significant correlations being established between complications and type of treatment, as well as with the
response to treatment.
Conclusions: This study shows that regardless of the type of treatment delivered, complications arise from the treatment
itself. The most common complications of patients diagnosed with MM are bone pain, followed by anemia, CKD andhypercalcemia. Despite the complications that may occur after polychemotherapy with Velcade and Dexamethasone, thistherapy significantly improves both survival and quality of life of patients.
Key words: myeloma multiple complications, polychemotherapy

INTRODUCTIONMETHODOLOGY

Multiple myeloma may cause a large range ofproblems, which are considered to be complications of thispathology.(1) Plasma cells proliferation interferes withnormal production of blood cells leading to leukopenia,
anemia and thrombocytopenia. Cells may form soft tissue
masses - plasmacytomas or lytic lesions in the skeleton (2).
The most important complications of multiple myeloma arebone pain, hypercalcemia, kidney failure and bone marrowcompression (3). Aberrant antibodies produced lead to theimpairment of humoral immunity; as a result, a great
prevalence of infections, particularly with encapsulatedorganisms such as Pneumococcus (4) is seen in thesepatients. Overproduction of these antibodies may lead to
hyperviscosity, amyloidosis and kidney failure. All thesecan be caused by both the disease itself and thechemotherapy patients underwent.(5)The study was performed on a group of 77 patientsdiagnosed with multiple myeloma between January 1st
2011 and December 31st 2015 in the Hematology
Department of the City Clinical Emergency Hospital
Timisoara. The database compiled in the Microsoft Excel
2010 programme comprised anthropometric data ofpatients, results of laboratory investigations, type oftreatment, response to treatment, as well as complications
present at the time of diagnosis and after the start oftreatment.
The recorded data were processed with the SPSS 17.0
programme. Data were shown as frequency andpercentage, while comparison between groups was
performed using Oneway Anova.

RESULTS
AIM
This paper aim is to assess complications in multiple
myeloma patients, as well as to establish a correlation withthe treatment type they underwent.

The study group of 77 patients was divided into two
categories according to age (under 60 and over 60
years of age), whilst gender distribution showed
prevalence of women. (Table I)

Correspondence author: Cristina Sorica-Romanescu, tel: 0745325848, email: cris24sorica@gmail.com

Fiziologia - Physiology • 2016.26.2(90)19Table I Anthropometric data of patientsParameter
Age
Sex

Values40-60 years>61 years
M
F

Frequency
40
37
35
42

Percentage
51.9
48.1
45.5
54.5

Several hematological, biochemical and immunologicalparameters were measured in order to establish thediagnosis and staging were determined (Table II)

Table II Hematological, biochemical and immunological parameters
Parameter
Hb (g/dl)Values.<6.5
6.5-7.9
8-10
10-13
13-17
<10.000
3
Leukocytes (mm )>10.000
Platelets (mm3)50.000-100.000
100.000-150.000
150.000-410.000>150.000FAS(u/l)35-105>105
Peripheral smear
FISIC
PLASMOCITEESR (mm/h)
3-10 mm10-60 mm
>60 mmLDH (u/l)
100-190>190<100
Fibrinogen (mg/dl)
200-400>400
Serum potassium (mmol/l)<3.5
3.5-5.1>5.1
Serum calcium (mg/dl)<8.8
8.8-10.2>10.2
Creatinine (mg/dl)
<0.8
0.8-2
>2
Urea (mg/dl)
15-39
>39
<3.5
Albumin (g/dl)
3.5-5>5Total proteins (g/l)
<64
64-83
>83
ELFO
LambdaKappa
Beta 2 macroglobulin (mg/l) <3.5
3.5-5.5>5.5
C-reactive protein (mg/l)+
IgG (g/l)AbsentPresent
Ig A (g/l)
AbsentPresent
IgM (g/l)AbsentPresent

20

No. of patients
1
7
22
32
15
43
34
7
11
56
3
44
33
55
22
13
11
53
58
18
1
27
50
14
51
12
23
14
40
29
25
23
42
35
39
30
8
10
38
29
25
52
29
23
25
15
62
23
54
56
21
75
2

Percentage
1.3%
9.1%
28.6%
41.6%
19.5%
55.8%
44.2%
9.1%
14.3%
72.7%
3.9%
57.1%
42.9%
71.4%
28.6%
16.9%
14.3%
68.8%
75.3%
23.4%
1.3%
35.1%
64.9%
18.2%
66.2%
15.6%
29.9%
18.2%
51.9%
37.7%
32.5%
29.9%
54.5%
45.5%
50.6%
39.0%
10.4%
13.0%
49.4%
37.7%
32.5%
67.5%
37.7%
29.9%
32.5%
19.5%
80.5%
29.9%
70.1%
72.7%
27.3%
97.4%
2.6%

Fiziologia - Physiology • 2016.26.2(90)Distribution of patients by type of treatment they
underwent is shown in Table III.
Table III Distribution of patients by type of treatment
Treatment

Frequency

Percentage

VAD
VELCADE-DEXA
VELCADE-DEXA-CAELYX

24
42
11

31.2
54.5
14.3

Total

77

100.0

The most common complications in these patients
were: bone pain -with bone fractures seen in more thanhalf of the patients (61%) -, followed by anemia (48.1%)
and CKD (32.5%); myelosuppression was the least
common complication (15.6%).

Although the number of complications increases after
the start of chemotherapy, no direct correlation could beestablished between the type of treatment and occurrenceof complications, as these are caused by many factors.
However, a statistically significant difference in theoccurrence of hypercalcemia in patients undergoing VAD
treatment only compared to those on Velcade-Dexa-Caelyx
treatment seems to exist, as hypercalcemia is morecommon in the latter group (p=0.024). The same statisticallysignificant change (p=0.046) is also seen between the
Velcade –Dexa and Velcade-Dexa-Caelyx.
When comparing VAD therapy with Velcade-Dexa andVelcade –Dexa- Caelyx, respectively, it seems that the rateof response to Velcade-Dexamethasone chemotherapy ishigher than that seen with the other therapies, as follows:
52.2% of the patients have a partial response, 18% haveprogressive disease, while only 10.8% showed a complete
response. The lowest survival rate was found in patientswith hypercalcemia (35 months) and bone pain (42months).

DISCUSSIONS

Fig. I Distribution of complications in multiple myeloma patients

Some of the complications of multiple myeloma werealready present at the time of diagnosis, namely: anemia,
thrombocytopenia, hypercalcemia, CKD and infections,while others started after the initiation of chemotherapy:
myelosuppression, hyperviscosity syndrome and bone pain
– fractures.
Table IV Frequency of complications according to the time ofoccurrence
Parameter

AnemiaThrombocytopenia
Hypercalcemia
Infections
Myelosuppression
CKD
Hyperviscosity
syndrome
Bone pain - fractures

Complication
present at thetime of diagnosis
8 (10.4)
7 (9.1)14 (18.2)
34 (44.2%)
023 (29.9)0Complication
arising after thestart of treatment
37 (48.1)
18 (23.4)
23 (29.9)21 (27.3)
12 (15.6)
25 (32.5)
18 (23.4)

047 (61)Fiziologia - Physiology • 2016.26.2(90)Patients with multiple myeloma develop two types ofcomplications: some are disease-related and others arecaused by the polychemotherapy they received. The mostcommon complications in both instances are:hypercalcemia, kidney failure, infections and skeletal
lesions. [6,7] The importance of these complications was
discussed in several clinical trials. One of these trials,
performed in UK, studied the rate of deaths at 60 days after
the diagnosis and showed premature death occurred in 10%of the patients due to infections (50%) and chronic kidneydisease (28%). [8].
In our study, the most common complications arising
after the start of polychemotherapy are bone pain, anemia,
hypercalcemia and CKD, premature death being recordedin patients developing hypercalcemia (at approximately 35
months from diagnosis) and bone pain (42 months fromdiagnosis). However, we were not able to establish a direct
correlation between the type of treatment and theoccurrence of complications.

CONCLUSIONSThis study shows that regardless of the type oftreatment delivered, complications arise from the treatment
itself. The most common complications of patientsdiagnosed with MM are bone pain, followed by anemia,
CKD and hypercalcemia. Despite the complications that
may occur after polychemotherapy with Velcade andDexamethasone, this therapy significantly improves bothsurvival and quality of life of patients.

21

REFERENCES
1. Ioniţă Hortensia, 2015 – Hematologieclinică, EdituraLitoUmfTimişoara.
2. Bergsagel Pl., 2003 - Prognostic factors in multiple
myeloma:it’sin the genes. Clinical cancer research.
3. BarlogieB., ZangariM., 2005 - Velcade-Dexamethasonefor
advanced and refractory multiple myeloma.
4. Tkachuk, Douglas C.;Hirschmann, Jan V. 2007. Wintrobe'sAtlasof Clinical Hematology , 1stedition.
5. A.V. Hoffbrand, P.H.A. Moss.2011. Essential Haematology,
6thedition.

6. Kyle RA, Rajkumar SV. Multiple myeloma. N Engl J Med 2004;
351:1860.
7. Smith A, Wisloff F, Samson D, et al. Guidelines on the diagnosis
and management of multiple myeloma 2005. Br J Haematol 2006;
132:410.
8. Augustson BM, Begum G, Dunn JA, et al. Early mortality after
diagnosis of multiple myeloma: analysis of patients entered onto
the United kingdom Medical Research Council trials between 1980
and 2002--Medical Research Council Adult Leukaemia Working
Party. J Clin Oncol 2005; 23:9219.

COMPLICATII ALE CHIMIOTERAPIEIIN MIELOMUL MULTIPLU
ABSTRACT:Introducere: Mielomul multiplu poate determina o larga varietate de probleme, considerate complicatii ale acestei
patologii. (1) Proliferarea plasmocitelor interfera cu productia normala de celule sanguine determinind leucopenie,
anemie, trombocitopenie.
Scop: Scopul acestei lucrari este evaluarea complicatiilor pacientilor cu mielom multiplu precum si stabilirea unei
corelatii cu tipurile de tratament efectuate.
Metodologie: Am luat in studiu un lot de 77 de pacienti diagnosticati cu mielom multiplu in perioada 1 ianuarie 2011-31
decembrie 2015 in cadrul Sectiei de Hematologie a Spitalului Clinic Municipal de Urgenta Timisoara.
Rezultate: Lotul a fost studiat din punct de vederea al datelor antropometrice precum si a parametrilor hematologici,
biochimici si imunologici. S-au evidentiat complicatiile prezente in momentul diagnosticului precum si dupa inceperea
polichimioterapiei evidentiindu-se corelatii semnificative statistic intre complicatii si tipul tratamentului precum si
raspunsul la tratament.
Concluzii: Acest studiu releva faptul ca, indiferent de tipul tratamentului efectuat complicatiile apar ca urmare aacestuia. Cele mai frecvente complicatii intalnite la pacientii diagnosticati cu MM sunt durerile osoase, urmate deanemie, IRC si hipercalcemie. In ciuda complicatiilor care pot surveni secundar polichimioterapiei cu Velcade si
Dexametazona, aceasta terapie imbunatateste semnificativ atat supravietuirea pacientilor cat si calitatea vietii
acestora.
Key words: complicatiile mielomului multiplu, polichimioterapie

22

Fiziologia - Physiology • 2016.26.2(90)IN VITRO TESTING THE CYTOTOXIC EFFECTOF TITANIUM-HYDROXYAPATITE BIOMATERIAL
Roxana Voisan1, Mirabela Cristea2, Gabriela Tanasie2, 3, Florina Bojin2, 3,
Daciana Nistor2, 3, Simona Anghel2, 3, Mihai Ionac1
1 Surgery II Department, University of Medicine and Pharmacy Victor Babes Timisoara
2 Functional Sciences Department, University of Medicine and Pharmacy Victor Babes Timisoara
3 OncoGen Center, Emergency Clinical County Hospital Pius Brinzeu, Timisoara

ABSTRACTThe use of cellular compounds, sustained by a proper scaffold, could lead to positive results in the repair of joint and bone
tissue, which will result in an improvement of surgical technique. Such 3D structures must have the biocompatibility feature.
As much as important are the biocompatibility properties of this matrices as well as their ability to stimulate the survival anddifferentiation of cells and the normal restoration of the injured tissue. The cytotoxicity test is one of the screening tests use for
evaluation of a new sensitized biomaterial for tissue engineering and/or production of implantable devices. The present study
aimed to investigate the in vitro cytotoxic effect of a titanium-hydroxyapatite biomaterial on a primary culture of mesenchymal
stem cells (MSCs). Our experiments tested a novel synthesized biomaterial, a nanostructured matrix of hydroxyapatite
reinforced with particles of titanium (HAP-Ti). The test were performed in comparison with another biomaterial made fromtitanium which undergone a process of anodization (Ti-a). Some samples were used for preparation of an extract frommaterial. The direct contact cytotoxicity test was performed using pads of biomaterials (with and without conditioning by
incubation with culture media) as well as extracts. The plates were examined in fluorescent light after using a live/death dye(acridine orange/ethidium bromide). Our experimental data indicate a minimum impact of direct contact with both biomaterials
pads on the MSCs growth and proliferation. Even more, when the pads were previously conditioned by incubation with culture
media, the MSCs proliferation appear to be stimulated. When the extracts were used, the changes are more extensive, this ispossible due to the ability of extraction media to release more compounds able to a harmful effect on living cells. The cells
injury appear to be more intense in contact with extracts than the plates of materials and for evaluating the amplitude of thiseffect we performed further the MTT test. Analysis of results obtained using the MTT test performed comparatively indicate
higher rate of cell survival when the extract of titanium combined hydroxyapatite was used. This suggests that the tested
material may be a better proposal regarding use in clinical applications for patients with bone injury which lost high amount of
normal tissue. Nevertheless the material should be optimized.
Key-words: in vitro cytotoxicity tests, direct contact, extract, MTT, titanium-hydroxyapatite biomaterial

INTRODUCTION
Tissue engineering represent a multidisciplinary field ofstudy, comprising complex knowledge from biology,
engineering, medicine having as purpose to improve thehealth and quality of life by restoring, maintaining or
improving the function of a tissue or an organ. One of themain areas of tissue engineering is the synthesis of
structures able to replace normal human tissue, evenentire organs. Such structures have two main components,
the scaffold or the matrix and the cells [1]. A large variety ofhuman cells could be used, starting from specific adult
differentiated cells coming from the tissue which should
be replaced, including also different types of adult stem
cells, more or less committed into the desired lineage up
to embryonic stem cells and induced pluripotent stem
cells [2].

The use of cellular compounds, sustained by a proper
scaffold, could lead to positive results in the repair of joint
and bone tissue, which will result in an improvement ofsurgical technique. Such 3D structures must have thefeatures of biocompatibility (not to generate an
inflammatory response, not to be immunogenic and not totrigger cytotoxic reactions), to be biodegradable, possible
to be sterilized, must permit the cells multiplication and the
production of extracellular matrix components, thus
providing the necessary nutrition [3]. The mechanicalproperties of these sustaining structures have to insure anormal development for the further physical activities of thepatients [4]. As much as important are the biocompatibility
properties of this matrices as well as their ability tostimulate the survival and differentiation of cells and thenormal restoration of the injured tissue [5]. The cytotoxicity
test is one of the screening tests use for evaluation of a

Received April 12th 2016. Accepted May 2nd 2016. Address for correspondence: Assoc. Prof. Gabriela Tanasie, Discipline of
Physiology, University of Medicine and Pharmacy “Victor Babes”, 2 E. Murgu Square, 300041 Timisoara, Romania, phone:+40256490507, e-mail: gtanasie@umft.ro.

Fiziologia - Physiology • 2016.26.2(90)23new sensitized biomaterial for tissue engineering and/or
production of implantable devices. The tests comprising a
set of assays in which is evaluated if the in vitro contact ofliving mammalian cells with the proposed biomaterial could
interfere with normal cell functions [6]. Three types ofcytotoxicity test are stated in the International Organizationfor Standardization 109993-5: extract or elution, directcontact and indirect contact tests [7]. These standards
could be supplemented with different other complementary
methods or could be amended in regard of cell type used
for test as well as the use of more advanced techniques or
equipment [8, 9].
The present study aimed to investigate the in vitro
cytotoxic effect of a titanium-hydroxyapatite biomaterial ona primary culture of mesenchymal stem cells (MSCs).

MATERIAL AND METHODS
Preparation of biomaterial samples and extracts
Our experiments tested a novel synthesizedbiomaterial, a biocomposite nanostructured matrix ofhydroxyapatite reinforced with particles of titanium(HAP-Ti). The test were performed in comparison withanother biomaterial made from titanium which undergone aprocess of anodization (Ti-a). The materials specimens
were kindly provided by the colleagues from Faculty of
Mechanics, Craiova University, Romania.
From each type of material were prepared 0.6 mm squared
samples. As negative control was used an inert material,
glass slides, prepared at the same dimensions. Priorutilization the samples were sterilized by autoclaving.
Some samples were used for preparation of an extractfrom material: the samples were placed in 5 ml culture
media and incubated (37ºC, 5% CO2) for 3 days. Daily, therecipients were placed in an orbital shaker for 10 minutes.
After this period of time the culture media was harvested
followed by centrifugation (10 minutes, 300g) and theextracts were used in subsequent experiments.
Cell culture
For cytotoxicity testing a primary culture ofmesenchymal stem cells (MSCs) derived from Hoffa’s fat
pads were used. The tissue was harvested from a malepatient, age 33, undergoing arthroplasty after traumaticknee injury. The written informed consent was priorobtained from patient. The tissue was cut into very small
parts and processed via enzymatic degradation bycollagenase. The MSCs were isolated based on theirplastic adherence property, using alpha-minimum essential
medium (MEM; Gibco BRL, Invitrogen), supplemented with
10% fetal calf serum (FCS; PromoCell, Heidelberg,
Germany) and 1% penicillin-streptomycin (Pen/Strep,
10,000 IU/mL; PromoCell, Heidelberg, Germany). Cells
were cultured in 5 % CO2 atmosphere at 37ºC, medium
being replaced every three days. At the 80% confluence

24

MSCs were plated in T75 culture flasks at a density of
10,000 cells/cm2, after the previous trypsinization using
0.25% Trypsin-EDTA solution (Sigma Aldrich Germany)
followed by centrifugation (10 minutes, 300g). The MSCs atpassage 3 and 4 were used in the experiments.
Direct contact cytotoxicity test
The MSCs were placed in 24 wells plates at 5,000
cells/cm2; after confluence (3-4 days), the 0.6 mm squared
samples of tested materials (HAP-Ti, Ti-a, glass slides)
were placed in the middle of each well, above the cell layer.
Previously, a lot of samples from each material wasconditioned by incubation for 48 hours in culture media. In aparallel set of experiments instead of material samples theextraction was used by adding in each well 0.5 ml ofcorresponding extract. The plates were incubated at 37ºC
for 5 days being daily evaluated at an inverted microscope(Nikon Eclipse E800). Eventually the culture plates were
examined in fluorescent light after using a live/death dye(acridine orange/ethidium bromide). Briefly, the culture
media was discarded and after a washing step withphosphate buffer the acridine orange/ethidium bromide
solution was added in the dark room for 3 minutes. After thisperiod the China ink was added for contrast and theexamination procedure was done. Live cells appear ingreen fluorescence (with acridine orange), and dead cells
appear fluorescent orange (ethidium bromide).
MTT test
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay is based on the assessment of
mitochondrial activity due to the conversion of tetrazolium
salt MTT (yellow) to formazan (purple). The MSCs wereseeded on 96-well culture plate at a density of 7,000 /wellin triplicate. The cells were incubated for 24 hours and thenthe medium was changed with fresh medium completedwith four different concentrations of each type ofbiomaterials extracts: 25%, 50%, 75% and 100%; dimethyl
sulfoxide (DMSO) was used as control. After 24 hours the
MTT reagent solution was added and the next steps weredone according to manufacturer protocol (Sigma-Aldrich,Germany).The absorbance was read at 570 nm using aspectrophotometric plate reader (Bio-Rad, Tokyo, Japan)
with a reference wavelength of 655 nm to subtract
background. The results were considered as the averagevalues from triplicate readings. The percent of cell viability(% viable cells) was computed as mean absorbance ofsample x 100/mean absorbance of control.

RESULTS
Direct contact cytotoxicity test
To highlight and quantify the direct cytotoxic effect, theMSCs cultured to confluence were put in contact withmaterial pads or material extracts, from both HAP-Ti and
Fiziologia - Physiology • 2016.26.2(90)Ti-a. The images, analyzed by fluorescence microscopy
after staining with acridine orange/ethidium bromide
showing green proliferating viable cells viable and dead or
suffering cells are colored orange.
The addition of biomaterials extracts seems to suppress
the normal cell proliferation. Comparison of the figure 1A andfigure 1B indicate that cell viability was stronger altered dueto the interaction with Ti-a extract, while the HAP-Ti extract
preserve a higher number of viable cells.
MSCs cultured in contact with Ti-a pads had shown abetter rate of viability in comparison with extract, the effectbeing more pronounced when conditioned pads were used
(Figure 1C and Figure 1E). Was observed also a tendencyof cells to orientate corresponding to architecture ofbiomaterial surface, with higher proliferation at the pads
margin and angles.
The direct contact of HAP-Ti pads with cultured cells
seems to not interfere with normal cell proliferation andgrowth. When unconditioned pads were used (Figure1D) asmall percent of dead/apoptotic cells diffusely scattered on
the plate was observed, while the contact with previously
conditioned pads by 48 hours interaction with culture
media seems to favor the MSCs proliferation. The cells
appear to have a multilayers disposal with very small
percent of apoptotic cells localized probably in the areas ofcells crowding (Figure 1F).

A. MSCs cultured with Ti-a extract,ob. 20x

B. MSCs cultured with HAP-Ti
extract, ob. 10x

C. MSCs in direct contact with
Ti-a unconditioned pads, ob. 20x

D. MSCs in direct contact with
Ti-a unconditioned pads, ob. 20x

MTT test
The MTT test was performed in order to have aquantification of the cytotoxic effect induced by the extractsof both types of biomaterials. The progressively increasing
concentration of extracts was used. The culture media
without any extract addition was used as a positive control,
and the negative control was represented by DMSO. The
Table I presents the results of MTT teste expressed aspercent of viable cells.
Table I. The MTT test for MSCs cultured in the presenceof HAP-Ti and Ti-a extracts (values are computed as the meanof 3 identic experiments)

Cells
viability(%)

HAP-Tiextract
Ti-a
extract

100%
extract75%
extract

50%
extract

25%
extract

DMSO(negative
control)

Positive
control

84.65390.15693.58798.2139.237

100

75.35680.65285.36292.663

9.237

100

Analyzing the obtained results we observed a higher
cell survival rate when the HAP-Ti extract was used incomparison with Ti-a extract, indicating that titaniumhydroxyapatite biomaterial appears to have less effect on
the function and survival of MSCs in culture. In both casesa good correlation dose-effect can be observed, as the
proportion of extract decreases the cell survival rateincreases (Figure 2).

Fig. 2. Comparative cytotoxic effect of HAP-Ti and Ti-a extracts

DISCUSIONS AND CONCLUSION

E. MSCs in direct contact with
Ti-a conditioned pads, ob. 10x

F. MSCs in direct contact with
HAP-Ti conditioned pads, ob. 20x

Fig.1. Direct contact test. Fluorescence micrographs.
Acridine orange/ethidium bromide staining.
Fiziologia - Physiology • 2016.26.2(90)Tissue engineering using a combination of scaffolds
and cells, especially stem cells, allows exploiting two
important qualities of these cells: high proliferation capacityand plasticity of these immature cells. The use ofembryonic stem cells is limited by ethic reasons, and also
by the immune response, that could lead to transplant
rejection [10]. An alternative approach seems to be the useof stem cells. There are several attempts to create a mixed25

implant for tissue replacement using a three dimensional
porous structure seeded with different type of stem cells.
This suggest that it could be a good approach to use stem
cells for in vitro testing of biocompatibility and the active
properties of some new proposed biomaterials. This was
also the reason for choosing a primary culture ofmesenchymal stem cells for the in vitro cytotoxicity test
applied in the present experimental study.
Despite the evolution of surgical techniques, thereconstruction of large bone defects remains a major
challenge for which a tissue engineering approach can bea solution. Different biomaterials have been proposed asbone substitutes with conflicting results. Among these,hydroxyapatite and other ceramics have shown the best
results due to their osteoinductive properties, absence of
immune response, risk of infection and unlimited availability.
On the other hand titanium alloys were very wide used in
the same purpose and the titanium based biomaterials
became more and more adapted to the requirements inthis field. For instance, Dabrowski et. al. proposed a highly
porous titanium structures received from powder
metallurgy process. In that study, a relatively highpermeability was observed for highest values of porosity.
Comparing to cast titanium, the porous titanium was low
resistant to corrosion. The mechanical parameters of theinvestigated samples were similar to those for cancellous
bone. The development of high-porous titanium materialshows high potential to be modern material for creating a
3D structure for bone regeneration and implant fixation [11].
In the present study we compared the cytotoxic effect of atitanium based material with a material with more complex
structure, containing both hydroxyapatite and titanium
particles. Although some preliminary data indicate asuperiority of the second material regarding mechanicalstrength and other physical and chemical properties, theabsence of cytotoxic effect is crucial for further
development and improvement of this material.
Cytotoxicity testing allows determining whether acompound or extract contains significant quantities of
biologically harmful chemicals. Cytotoxicity test methods
are useful for screening because they serve to separate
toxic from nontoxic materials, providing predictive evidenceof compound safety [12]. Testing the effects of compounds
on the viability of cells grown in culture is widely used as apredictor of potential toxic effects in whole animals [13] so
the in vitro tests should be used as first screening when a
new type of biomaterial is tested.
The direct contact test is a high sensitivity method
which evaluate the morphological changes and detect thechanges in the number of cells; it can directly reflect the
impact of testing the medical devices on the cells [6] and isvery suitable for biomaterials intended to be used in boneor dental tissue reparation. The test cannot be used forless dense medical devices, which float in the medium anddo not stay in direct contact with the cell layer [14]. Our
experimental data indicate a minimum impact of direct26

contact with both biomaterials pads on the MSCs growthand proliferation. Even more, when the pads werepreviously conditioned by incubation with culture media,
the MSCs proliferation appear to be stimulated. Thissuggest that our tested biomaterials interact with somecompounds from culture media and we could speculatethat some peptides remains on the material surface andimprove the cells adherence and proliferation [15]. Whenthe extracts were used, the changes are more extensive,
this is possible due to the ability of extraction media torelease more compounds able to a harmful effect on living
cells. For this reason it is necessary to continuously
improve the process of developing these materials, in orderto prevent the release of various compounds with potential
cytotoxic or pro inflammatory effect [16]. Our finding after
performing the direct cytotoxicity test allow us to sustain asuperiority of HAP-Ti versus Ti-a material. In contrast,
comparing a titanium surface having a porous titanium
plasma-sprayed coating, a titanium surface with a deep
profile structure, an uncoated titanium substrate and atitanium substrate with a porous hydroxyapatite
plasma-sprayed coating other researchers found thatstromal cells cultured on Ti-HA showed a delayed growth
pattern, may be related to its high phosphate ion release[17]. The cells injury appear to be more intense incontact with extracts than the plates of materials and forevaluating the amplitude of this effect we performed further
the MTT test.
MTT test and other types of assays used in the testing
the material effect on cell populations in vitro, are indicatedto be performed in the first 12-24 hours after exposure. The
MTT assay is based on the analysis of metabolic activity of
the cell populations, including the process of apoptosis, byanalyzing the light absorption ability at the surface of thematerial. It is not a test to follow to the long term cell
culture. The MTT assay was confirmed to be feasible, rapid
and reproducible. Moreover, it showed a good correlation
with other in vitro proliferation assays, such as the
3H-thymidine uptake assay [18].
Regarding the cytotoxic effect of extract obtained byincubating hydroxyapatite-titanium plates in the culture
medium, our experiments have demonstrated that it isrelatively small in amplitude, with a good correlation
between dose and effect, which demonstrates that
although low cytotoxicity it is present and isdose-dependent. Probably stability of tested material could
be improved in the future so this will release virtually zerocompounds in the environment fluid in which it is placed,
which will result in increased biocompatibility of thematerial.
Analysis of results obtained using the MTT test
performed compared the two types of materials, test
material and anodized titanium indicate higher rate of cell
survival when the extract of titanium combinedhydroxyapatite was used. This suggests that the tested
material may be a better proposal regarding use in clinicalFiziologia - Physiology • 2016.26.2(90)applications for patients with bone tissue destructive
processes. Nevertheless the material should be optimizedin order to ensure a good tissue integration of devices
made from this material that does not expose to the risk ofa possible immune reactions and rejection.

10.

11.

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Biomaterials. 1993;14(5):359-64.

INVESTIGAREA EFECTULUI CITOTOXIC IN VITROA UNUI BIOMATERIAL DE TITAN COMBINAT CU
HIDROXIAPATITĂREZUMAT
Utilizarea diverselor tipuri celulare, susținută de un sistem matricial adecvat, ar putea conduce la rezultate pozitive înrepararea țesutului osos și articular, ceea ce va duce la o îmbunătățire a tehnicii chirurgicale. Astfel de structuri 3D
trebuie să aibă caracteristica de biocompatibilitate. La fel de importante ca proprietățile biocompatibilitate ale acestei
matrici este și capacitatea lor de a stimula supraviețuirea și diferențierea celulelor și restabilirea normală a țesutuluirănit. Testul de citotoxicitate este unul dintre testele de screening folosesc pentru evaluarea unui biomaterial nou
sintetizat pentru ingineria tisulară și/sau producerea dispozitivelor implantabile. Prezentul studiu a urmărit investigareaefectului citotoxic in vitro a unui biomaterial de titan combinat cu hidroxiapatită pe culturi primare de celule stem
mezenchimale (CSM). Experimentele noastre au testat o matrice nanostructurată de hidroxiapatită armată cu particulede titan (HAP-Ti). Testul a fost efectuat în comparație cu un alt biomaterial realizat din titan supus unui proces deanodizare (Ti-a). Anumite probe au fost folosite pentru prepararea unui extract din material. Testul de contact direct afost realizat folosind fragmente de biomateriale (cu și fără condiționare prin incubare cu medii de cultură), precum și
extracte. Plăcile au fost examinate în lumină fluorescentă după utilizarea unui colorant vital (acridin orange/bromură deetidiu). Datele noastre experimentale indică un impact minim al contactului direct cu ambele tipuri de biomateriale
Fiziologia - Physiology • 2016.26.2(90)27asupra creșterii și proliferării CSM. Mai mult, atunci când plăcuțele au fost condiționate anterior de incubare cu medii
de cultură, proliferarea CSM par să fie stimulată. Atunci când s-au utilizat extractele, modificările sunt mai extinse,
acest lucru este posibil datorită capacității de extracție a mediului de cultură pentru a elibera mai mulți compuși capabili
să aibă un efect nociv asupra celulelor vii. Leziunile celulare par a fi mai intense în contact cu extracte decât cu plăcile
de materiale și pentru evaluarea amplitudinii acestui efect am efectuat in continuare testul MTT. Analiza rezultatelorobținute cu ajutorul testului MTT indică o rata mai mare de supraviețuire a celulelor atunci când a fost utilizat extractul
de titan combinat cu hidroxiapatită. Acest lucru sugerează că materialul testat poate fi o mai bună propunere în ceea
ce privește utilizarea în aplicații clinice, la pacientii cu leziuni osoase, care au pierdut o mare cantitate de tesut normal.
Cu toate acestea, materialul trebuie să fie supus unor procese de optimizare.
Cuvinte-cheie: teste in vitro de citotoxicitate, contact direct, extract, MTT, biomaterial titan-hidroxiapatită

28

Fiziologia - Physiology • 2016.26.2(90)NEUROPHYSIOLOGICAL AND NEUROPSYCHOLOGICAL ANALYSIS OF COGNITION IN NEW
HYPOTHYROID FEMALES
Pravriti Mishra1, Preethi Bl1, Pramila Kalra2
Department of Physiology, M.S. Ramaiah Medical College, Bengaluru, India
Department of Endocrinology, M.S. Ramaiah Medical College, Bengaluru, India

ABSTRACT
Hypothyroidism is a common thyroid disorder with a prevalence of about 4% in the Indian population. Hypothyroidism
has been associated with neuropsychiatric symptoms affecting mood & cognitive functions. A relationship has beensuggested between thyroid state and cognition, particularly slowed information processing speed, reduced efficiency inexecutive functions, specific retrieval defects, and poor learning.
Aim: To evaluate cognition in newly diagnosed hypothyroid patients & compare with normal euthyroid individuals.
Methods: P300 was recorded on 29 newly diagnosed hypothyroid female patients by Galileo NT evoked potential
recorder using standard odd ball paradigm of acoustic stimuli. Electrodes were put according to the 10-20 International
System.
Cognitive functions were further assessed using colour trails test, triads test & digit vigilance test.
Similar procedures were repeated on 29 age & education matched normal euthyroid females.
Statistical Analysis was done using SPSS 17 & independent sample “t” test.
Results: All the tests showed statistically significant difference (p <0.05) in hypothyroid patients as compared tocontrols.
Conclusions: Cognitive decline was observed in hypothyroid patients at diagnosis. This can be detected
electro-physiologically as well as by institution of appropriate cognitive function tests. Early detection & intervention
may prevent further cognitive decline.
Keywords: cognition; P300; hypothyroidism; evoked potentials, Cognition in hypothyroid patientsINTRODUCTION
Hypothyroidism is a common thyroid disorder with aprevalence of about 4% in the Indian population [1].
Hypothyroidism has been associated with neuropsychiatric
symptoms affecting mood & cognitive functions. A
relationship has been suggested between thyroid state andcognition, particularly slowed information processing speed,
reduced efficiency in executive functions, specific retrieval
defects, and poor learning.
Hypothyroidism has also been associated with anincreased susceptibility to depression and reductions inhealth-related quality of life [2]. These effects of low thyroid
function have been attributed to varied roles of thyroid
hormone in brain function & metabolism.
Few studies also suggest that the cognitive decline
associated with hypothyroidism is subtle and any
significant mood or cognitive distress should be evaluated& treated independently [3].

Limited literature is available on cognitive status ofhypothyroid patients at the time of diagnosis before
initiation of thyroid replacement therapy. Evoked potential
studies in late-onset hypothyroidism have shown variable
results. Some studies claim no difference [4], while others
have found prolonged latencies or reduced amplitudes ofdifferent evoked potentials in hypothyroids as compared toeuthyroid controls [5]. In the present study we have tried toevaluate the cognitive status of hypothyroid patients
electrophysiologically as well as by cognitive function tests.
Aims & Objective
To evaluate cognition in hypothyroid patients &compare with normal euthyroid individuals. To assess the
cognition of newly diagnosed hypothyroid patients bymeasuring P300 latencies & institution of cognitive function
tests. To compare the cognition of newly diagnosed
hypothyroid patients with normal subjects.

Received April 12th 2016. Accepted May 2nd 2016. Address for correspondence: Associate Professor Pravriti Mishra,
Department of Physiology, M.S. Ramaiah Medical College, Bengaluru, India; e-mail: mishra.pravriti@gmail.com and Associate
Professor Preethi BL, Department of Endocrinology, M.S. Ramaiah Medical College, Bengaluru; e-mail: blpreethi97@gmail.com

Fiziologia - Physiology • 2016.26.2(90)29MATERIALS AND METHODS
Sample size
Based on previous study conducted by Shama et al [6],and considering effect size of 0.7, α error of 5% and power of
80%, sample size was calculated to be 29 in each group.
The study subjects were divided into 2 groups.
Group I: euthyroid healthy females without co-morbidities
Group II: newly diagnosed hypothyroid patients who
have not started thyroid replacement
Inclusion criteria
29 newly diagnosed hypothyroid female patients
between the ages 18-55 years were taken as cases.
Equal number of age & education matched euthyroid
females were taken as controls.
Exclusion criteria
Hypothyroid patients undergoing thyroid replacement
treatment, patients with known neurodegenerative
diseases, demyelinating neuromuscular disorders,chronically ill, those taking drugs acting on central nervous
system or having habitual history of smoking and alcohol
drinking were excluded.
Testing procedures and protocol were explained andconsent obtained from the cases and controls. All thesubjects were generally and systemically examined to ruleout the presence of other comorbid conditions.
The study subjects were evaluated by general history,
clinical examination and serum levels of fT3, fT4 and TSH.
Cognitive functions of the subjects were
electro-physiologically evaluated by P300.
Digit Vigilance test, Colour trails test & Triads test of
the NIMHANS neuropsychological battery [7] were used toassess different domains of attention.
P300
The P300 wave is a measurable direct reaction of thebrain to a certain sensory, cognitive or mechanical stimulus.
It belongs to ERP (event related potentials), which areelectrophysiological responses to stimuli. P300 latencyreflects the time required for processing of external stimuli
and increases with the reduction of cognitive capacity in the
allocation of attention and memory resources.
The 10-20 International system of standard electrodeplacement was followed and the latencies of the P300
waves were recorded and analyzed using Galileo NT
Evoked potential recorder having P300 protocol. Auditory
stimuli were presented to the subjects according to theoddball paradigm. The test result was obtained as a functionof focusing attention on the rare stimulus.
Digit vigilance test
It is a test of sustained attention.
In this test the subject has to focus on the target digits(6&9) among other distracter digits.

30

The subjects had to strike off the digits 6 & 9 on the
sheet as quickly as they could and the time taken to finish
the task was taken as a score.
Colour trails test
It is a test of focussed attention and has two parts.
Part I: Colour trails test 1(CT 1) consists of a sheet inwhich numbers 1-25 are randomly spread, with odd
numbers in pink circles & even numbers in yellow circles.
The subject is asked to point to successive numbers inascending order from 1-25.
Part II: Colour trails test 2(CT 2) consists of numbers
from 2-25 printed twice, once on yellow circles & once onpink circles arranged randomly on a sheet. The subject is
asked to point to numbers in alternating colours with thesuccessive numbers being in an ascending order.
Appropriate practice sheets were first instituted to thesubjects before instituting the test sheets. The time taken by
the subjects to finish the task was considered as a score.
Triads test
It is a test of divided attention.
It combines a verbal triad task with a tactual number
identification task. The subject is blindfolded in the test.
The verbal triad task consists of 48 concrete nouns
grouped into 16 word triads. In each triad two words belong
to one category while the third one does not. The subject
names the odd word.
In the tactual number identification task, an Arabic
numeral single or double digit is written on the subject’s
non-dominant hand. The subject identifies the number bycalling it out.
The number of mistakes in calling out either the wordor the number was taken as a score.
Statistical analysis
Data were analyzed using SPSS software version17.
All the results were expressed in terms of mean & standard
deviation. The mean difference in the latency of P300 &scores of cognitive function tests were tested using
independent sample‘t’ test.
RESULTS
Both the groups were similar on the basis of age, height,
weight & educational level. On analysis, the latency of P300 ofcontrol group was found to be 317.98 ± 16.67 while that ofhypothyroid females was 378.01 ± 28.02 & the difference wasfound to be statistically significant (p=0.000).
The scores of cognitive function tests i.e. triads test,
digit vigilance test and colour trails test were also found tobe significantly higher in hypothyroid subjects as comparedto euthyroid controls.

Fiziologia - Physiology • 2016.26.2(90)Table I. Baseline parameters of hypothyroid patients & controls
Parameter

Cases

Controls

Mean±S.D.

Mean±S.D.

Age (years)

29.9 ± 6.74

26.89 ± 6.57

0.311

Height (cm)

156.7±5.18159.9±5.36

0.191

Weight (kg)

57.5 ± 8.33

55.78 ± 7.08

0.616

Serum T3

0.51 ± 0.48

1.35 ± 0.34

*0.000

Serum TSH

33.23 ± 35.01

2.51 ± 1.16

*0.012

P value*P<0.05 = significant difference

Table II. Cognitive parameters of hypothyroid patients & controls
Cases

Controls

Mean±S.D.

Mean±S.D.

P300 (ms)378.01± 28.02

317.99±16.67*0.000Digit vigilance
test (s)282.36±100.95

234.53±76.620*0.019Colour trails
test 1 (s)75.38±28.19

48.39±16.33*0.000Colour trails
test 2 (s)182.62±51.35

116.42±29.66*0.000

Triads test
(errors)5.6±2.66

3.36±1.73*0.000

Test

Fig. 3. Comparison of Colour trails test 2 scores(s) between
controls (1) & newly diagnosed hypothyroid cases (2)P value*P<0.05 = significant difference

Fig. 1. Comparison of P300 latency (ms) between controls (1)& newly diagnosed hypothyroid cases (2)Fig. 4. Comparison of Triads test scores (errors) between controls(1) & newly diagnosed hypothyroid cases (2)Fig. 5. Comparison of Digit Vigilance Test scores (s) between
controls (1) & newly diagnosed hypothyroid cases (2)DISCUSSION

Fig. 2. Comparison of Colour trails test 1 scores(s) between
controls (1) & newly diagnosed hypothyroid cases (2)Fiziologia - Physiology • 2016.26.2(90)The latency of P300 is indicative of stimulus processing& evaluation. The presence & timing of P300 has often
been considered as a marker of cognitive function indecision making processes.
In our study we found that the latency of P300 was
significantly prolonged in newly diagnosed hypothyroid
patients as compared to euthyroid individuals. Also the
hypothyroid patients had greater scores in all the cognitive
function tests instituted in the study (greater the score,greater being the time taken to finish the task, so slower
the person’s cognition).

31

Our study impresses the fact that cognitive decline ispresent in newly diagnosed cases of hypothyroidism andthis may be detected electrophysiologically as well as byinstitution of appropriate cognitive function tests.
Our finding is similar to studies conducted by Sharma
et al [8] & Tutuncu et al [9], who found prolonged P300
latencies in hypothyroid patients.
Krausz Yodphat et al [10] in their study on hypothyroid
individuals had observed deficits in cerebral blood flow toregions mediating attention, motor speed, memory, andvisuo-spatial processing. This may be a cause behindprolonged P300 latencies & delayed completion of tasks
involving cognition.
Hypothyroidism has been associated with reduced
synthesis of neurotransmitters & receptors. This can be acause of the reduced cerebral responsiveness in hypothyroid
subjects as compared to the euthyroid state [11].
Cognitive decline in hypothyroidism can be attributed tochanges in relative regional cerebral glucose metabolism in
hypothyroid patients [11,12], adverse effects of high levels
of TSH [14], thyroid hormone interaction with intrinsic
regulatory circuits and the systemic effects of thyroid
hormone on metabolic pathways [15]. Slowness ofcognition in hypothyroidism may be caused by theimbalance between oxidative stress & antioxidant
mechanisms in the brain [16] or reduced conduction
velocity of neural impulses due to reduced density ofvoltage-gated sodium current [17].

CONCLUSION
Hypothyroidism leads to early impairment of cognitive
functions which may be due to multiple factors. These canbe diagnosed electrophysiologically as well as by cognitive
function tests. Early detection will help in early institution ofinterventions to prevent progression of cognitive decline.
Acknowledgements
The authors would like to thank to Arun Kumar M,
Department of Physiology, M.S.Ramaiah Medical College,
Bengaluru and Jaisri G, Department of Physiology,
International Medical School, Bengaluru

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Fiziologia - Physiology • 2016.26.2(90)EVALUATION OF THE IN VITRO SECRETORYPROFILE OF MESENCHYMAL STEM CELLSIN CONTACT WITH A TITANIUM-HYDROXYAPATITEBIOMATERIAL
Roxana Voișan1, Gabriela Tănasie2,3, Mirabela Cristea3, Laura Marusciac2,
Marius Georgescu2, Carmen Tatu2, 3, Mihai Ionac1
1 Surgery II Department, University of Medicine and Pharmacy Victor Babes Timisoara
2 Functional Sciences Department, University of Medicine and Pharmacy Victor Babes Timisoara
3 OncoGen Center, Emergency Clinical County Hospital Pius Brinzeu, Timisoara

ABSTRACTThe roles of mesenchymal stem cells (MSCs) in the processes of tissue regeneration and reparation were debatedvery much in the last period. The MSCs seems to sustain the tissue restoration by maintaining such environmental
conditions which favor this process due to their ability to release cytokines and even growth factors. Starting from thisdata we developed an experimental protocol aiming to evaluate the changes of the MSCs secreted substances in to
the culture media after in vitro interaction with a titanium-hydroxyapatite (HAP-Ti) biomaterial as well as a control
biomaterial, anodized titanium (Ti-a). From each biomaterial were prepared squared 0.6 mm plates as well as an
elution. The secretion of MSCs was evaluated either after direct contact either after elution addition in culture media. An
immunoenzymatic assay (ELISA) was used for following cytokines and growth factors: tumor necrosis factor alpha(TNF-alpha), interferon gamma (IFN-gamma), interleukin 12 (IL12), interleukin 4 (IL4), interleukin 10 (IL10), tumor
growth factor beta (TGF-beta) and vascular endothelial growth factor (VEGF). According to the data obtained in our
experiments, evaluation of secretory profile in MSCs in vitro into contact with the tested biomaterials indicated that cells
releases into the culture medium higher concentrations of TNF alpha and IFN gamma, changes with no statisticalsignificance except elution prepared from anodized titanium (Ti-a). The effect, even present is less extensive in thecase of HAP-Ti, which demonstrates its better quality compared to the material used as a positive control. In addition,
an increased release of interleukins with suppressor and anti-inflammatory effects (IL10, IL4) appears suggesting a
possible better tissue integration of this material. The in vitro MSCs contact with tested biomaterials appear to stimulate
the release into the environment of VEGF, the highest concentration being found in tests with elution of HAP-Ti.
Overall, our results suggested that the proposed materials are not very well structured and need to be optimized inorder to be more stable. However it appears that HAP-Ti is less reactive than Ti-a suggesting that HAP-Ti should be
the chosen material for future improvements.
Key words: mesenchymal stem cells, in vitro secretory function, titanium-hydroxyapatite, in vitro cells-biomaterialinteraction

INTRODUCTION
The roles of mesenchymal stem cells (MSCs) in theprocesses of tissue regeneration and reparation weredebated very much in the last period. Due to their large
plastic ability, being able to give rise to all mesodermal
lineages, the MSCs were considered to have a structuralfunction in this process by replacing the normal tissue
functional cells [1]. Some studies found that thisreplacement could be developed only in to a small extend
and this not explain their positive contribution to the healing

process [2]. The MSCs seems to sustain the tissue
restoration by maintaining such environmental conditions
which favor this process due to their ability to releasecytokines and even growth factors [3]. On the other hand,some implantable devices are used for supporting the lackof normal tissue, especially for bone replacing after severe
traumatic injuries. After the placement of implant at the
injury sites, ones of the first interaction involves themesenchymal stem cells, which, depending on thebiomaterial properties, could support the tissue tolerance
and the tissue integration of such devices [4,5]. It is

Received May 10th 2016. Accepted June 2nd 2016. Address for correspondence: Assoc. Prof. Gabriela Tanasie, Discipline of
Physiology, University of Medicine and Pharmacy “Victor Babes”, 2 E. Murgu Square, 300041 Timisoara, Romania, phone:+40256490507, e-mail: gtanasie@umft.ro.

Fiziologia - Physiology • 2016.26.2(90)33supposed that an important role in this interaction is playedby the biological active molecules released from MSCs at
the implantation site [6, 7]. Starting from this data wedeveloped an experimental protocol aiming to evaluate thechanges of the MSCs secreted substances in to the culture
media after in vitro interaction with a titanium-hydroxyapatite biomaterial.

MATERIAL AND METHODS
1. MSCs isolation and culture
The MSCs were previously isolated from human
Hoffa’s fat pad. The samples were harvested during thejoint arthroplasty intervention after obtaining the written
informed consent of the patients. The donors were 5 malepatients, age 38±7, suffering traumatic knee injuries andno having other associated chronic diseases. The tissue
samples were mechanically and chemically (collagenase
digestion) processed and MSCs were isolated based on
their plastic adherence property. Cells were maintained inculture for 3 passages and after this period were stored inliquid nitrogen. For our study the frozen MSCs were
thawed rapidly (less than 1 minute) in a 37°C water bath.
The cells were placed further in 24 wells culture plates at adensity of 5,000 cells/cm2 in alpha-minimum essential
medium (MEM; Gibco BRL, Invitrogen), supplemented with
10% fetal calf serum (FCS; PromoCell) and 2%
Penicillin/Streptomycin mixture (Pen/Strep, 10,000 IU/ml;
PromoCell), by incubation at 37ºC in 5% CO2 atmosphere,
medium being replaced every three days. When thesemi-confluence was obtained the biomaterials samples(0.6 mm squares) were placed in each well; after 4 days,
the supernatant was harvested and stored at -20°C forcytokine assay. In parallel a similar set of experiments wasdone using biomaterials elution, which was added at thesemi-confluence (0.5 ml elution/well).
2. The biomaterials samples and elution preparation
The biomaterial studied in this experimental setting washydroxyapatite reinforced with titanium particles (HAP-Ti);also we used a positive control biomaterial, the anodized
titanium (Ti-a). The samples were kindly given by Oana
Gingu from the Faculty of Mechanics, Craiova University andwere manufactured as prototypes. The samples weresterilized by autoclaving. From each biomaterial wereprepared squared 0.6 mm plates as well as an elution. For
elution preparation, the biomaterials specimens were placedin MSCs culture media and incubated at 37ºC in 5% CO2
atmosphere for 7 days. After this period of time the culture
media was harvested followed by centrifugation (10 minutes,300g) thus becoming the elution.
3. MSCs secretion evaluation
The secretion of MSCs was evaluated by using an
immunoenzymatic assay (ELISA) and specific ELISA

34

Immunoassay kits (R@D systems) for following cytokinesand growth factors: tumor necrosis factor alpha(TNF-alpha), interferon gamma (IFN-gamma), interleukin
12 (IL12), interleukin 4 (IL4), interleukin 10 (IL10), tumor
growth factor beta (TGF-beta) and vascular endothelial
growth factor (VEGF).
4. Statistical analysis
All the experiments were done in triplicate. The obtained
data were analyzed for statistical relevance using Excel
Microsoft Office 2007 software (Microsoft Corporation,
Redmond, WA, USA). The central tendencies of thevariables were expressed as a mean (M), and thedispersion ones as a standard deviation (SD). In order toperform the statistical comparisons, Student's t test was
conducted, considering significant differences for p < 0.05.

RESULTS
The pro-inflammatory cytokines evaluated in thepresent study were TNF-alpha, IFN-gamma and IL-12. The
results indicated that the in vitro contact of MSCs withbiomaterials determines an increase of TNF-alpha and
IFN-gamma in comparison with MSCs secretion when they
are cultured without any supplementary interactions. For
IFN-gamma, the increasing was statistically significant(P<0.05) when elution of Ti-a was used in comparison with
MSCs alone and in comparison with MSCs with HAP-Ti
elution (Table I and Figure 1).
Table I. The pro-inflammatory cytokines assay after in vitro
contact with biomaterials

MSCs
culturemedia
MSCs
Control
MSCs +
Ti-aelution
MSCs +
HAP-Tielution
MSCs +
Ti-a
MSCs +
HAP-Ti

TNF α (pg/ml)

IFN γ(pg/ml)IL-12(pg/ml)

M

0.852

0.568

0.025

SD

0.002

0.008

0.001

M

6.864

4.576

0.331

SD

0.216

0.088

0.078

M

8.016

5.183

0.289

SD

0.147

0.028

0.007

M

7.869

4.790

0.298

SD

0.428

0.142

0.062

M

8.918

4.868

0.272

SD

1.019

0.322

0.011

M

8.123333333

4.7516667

0.3316667

SD

0.518716364

0.2020314

0.0355903

Fiziologia - Physiology • 2016.26.2(90)Fig. 1. Comparison of pro-inflammatory cytokines
MSCs secretion in culture media

It seems that the tested biomaterials materials are notsufficiently stable in prolonged contact (7 days) with culture
media, they release some molecules which encourage
secretion of pro-inflammatory factors by cells cultured invitro. The effect is less extensive in the case of HAP-Ti
biomaterial, which demonstrates its better quality
compared to the Ti-a material used as a positive control.
In the experimental set-up, the IL-4 and IL-10
anti-inflammatory cytokines were also evaluated. The results,
showed in table II and Figure 2 indicated the property of MSCs
to release into the environment immunosuppressive andanti-inflammatory factors is somewhat suppressed by contact
with the Ti-a material, the effect having a statistical significancefor releasing of IL10 and being more evident when using thematerial as such, not elution. Though without having statisticalsignificance, it appears that the interaction between cells in
culture and HAP-Ti material tested in this study, induce anincrease in the release into the environment of IL4 4, and IL10
10, which advocates for a good tissue integration of thismaterial.
Table II. The anti-inflammatory cytokines assay after in vitro
contact with biomaterials

MSCs culture
media
MSCs Control

IL-4 (pg/ml)

IL-10 (pg/ml)

M

0.02

0.05

SD

0.004

0.003

M

0.233

1.142

SD

0.005

0.070

MSCs + Ti-aelution

M

0.21

1.038

SD

0.010

0.131

MSCs + HAP-Ti
elution

M

0.205

1.213

SD

0.013

0.057

M

0.191

0.778

SD

0.133

0.081

M

0.245

1.308

SD

0.039

0.249

MSCs + Ti-a

MSCs + HAP-Ti

Fiziologia - Physiology • 2016.26.2(90)Fig. 2. Comparison of anti-inflammatory cytokines
MSCs secretion in culture media

The assay for some growth factors secreted in culture
media by MSCs were shown in Table III.
Table III. The growth factors assay after in vitro contact
with biomaterials

MSCs culture
media
MSCs Control
MSCs + Ti-aelution
MSCs + HAP-Ti
elution
MSCs + Ti-a

MSCs + HAP-Ti

VEGF (pg/ml)

TGF β (pg/ml)

M

21.32

854.25SD

3.434

52.210M

128.406

2399.717

SD

1.887

13.130M

230.133

2805.966

SD

9.047

37.042M

230.8132672.663

SD

7.566

71.448M

177.153

2550.118

SD

16.51799.700M

237.7312419.843

SD

26.275147.360

An increased releasing of VEGF in culture media from
MSCs in the presence of biomaterials was noticed, both forelution and for material as it (Figure 3); the statisticalsignificance is grater when elution was used, being
stronger for the test material, HAP-Ti, compared to thematerial used as a positive control Ti-a. Regarding the
TGF beta levels in culture media, this growth factor had
elevated amounts in comparison with the control (Figure 4),
but the differences are statistically significant only inexperiments that use elution. It is known that TGF-β is amolecule with multiple actions on cell functions, favoring
cell migration and differentiation to osteogenic andchondrocytes lineage. The tested material, HAP-Ti, thatsignificantly improves its release in the culture medium,show that at least for in vitro conditions, the biomaterial
does not contribute to the differentiation of MSCs recruited
at the injury site in specific tissue cells.

35

Fig. 3. VEGF secretion

Fig. 4. TGF beta secretion

DISCUSSIONS AND CONCLUSION
The role of stem cells is not completely known but
based on experimental data some hypothesis regarding
stem cells functions was formulated. The potential of stem
cells to differentiate in vivo is a function of maintaining andregeneration of tissues, easily to observe in the tissues
with a great turn-over rate like blood; in this case thehematopoietic stem cell (HSCs) is a forming unit for all the
blood cell lines. In the mesenchymal stem cells (MSCs)
case there are not completely clarified the mechanisms of
migration and differentiation in target tissues.
The MSCs role in the adult human body is thegeneration of mesenchymal cell lines and afterwards,
through mesenchymal progenitors is involved indevelopment, maintenance and restoration of connective
and muscular tissue. Having these outstanding properties
MSC represents the best alternative for cell therapy
applications; they have self-renewal, great plasticity withdifferentiation potential in functional cell lines. Their
efficiency was proven in numerous therapeutic protocols:hematopoietic recovery, osteogenesis imperfecta, boneregeneration. Another advantage seems to be the fact that
MSCs could be obtained from autologous sources (bonemarrow) eliminating thus the complication of allogeneictransplantation. In past years were developed many
researches in the field of MSC and now we have lot of

36

information regarding plasticity, homing and differentiation
potential in vivo [8, 9].
Massive bone defects are an important problem fororthopedic surgeons, the best solution being the autograftbone harvesting but the sources used are limited andinvolve many risks such as infection, damage of peripheral
nerves and loss of function. Bone regeneration studies
using MSCs involves cultivation or cell loading on bonesimilar scaffold structures.
Scaffold structures are generally hard, porous allowing
MSCs to join, offering mechanical support, based onsynthetic or natural materials [10]. The results obtained so
far indicate the effectiveness of MSCs loaded scaffolds inbone reconstruction. Not every biomaterial can be used as
scaffold. It must determine the formation of cell matrix,
meaning that cells must interact with the scaffold, adhere,
spread and finally proliferate. Another important aspect isthat the biomaterial is not inert chemically or biologically. A
very important feature is the porosity of the biomaterial [11],it is needed for fluid flow through the material.
Many materials, such as metals, polymers, ceramics, areused as artificial implants according to the anatomical location.
Ceramic materials are most commonly used in dental andorthopedic surgery due to the ability to not cause severe
damage to the tissue [12]. Titanium and titanium alloys are thematerials widely used as components of an implant. Theirapplicability ranging from medical implant used in a patientwith severe orthopedic trauma or internal fixation of fractures
to those used for cardiovascular diseases like heart valve
prostheses and pacemakers [13,14]. From the physical pointof view, titanium has certain properties responsible forbiocompatibility like low electrical conductivity, increased
corrosion resistance and good thermodynamic properties atnormal pH [15].
For every material intended to be used for implantation
is important to be tested in order to avoid a biologicalreaction between the host tissue and the implant itself. The
in vitro tests are very much used, involving low costs, shortduration of time and reliability, all extremely important andabsolutely necessary issues [16].
The evaluation of secretory profile of mammalian cells
in contact with biomaterials is not included into thestandard biocompatibility tests required by ISO 10993 [17],but it could be considered as an additional optional test
which could give information about the bioactivity of thematerials tested. Depending on the cells type used for thisassay [18] some predictions regarding the possible in vivo
irritations and inflammatory reactions could be obtained[19]. The MSCs are known as a cell type endowed withimmunomodulatory properties [20] which present a large
variation depending on a source of MSCs and on
experimental settings used for evaluation. It has beenshown that the mesenchymal cells derived from adipose
cells, have different secretory patterns of TGFβ1 compared
to mesenchymal cells from the bone marrow, the latterbeing most commonly used in in vitro studies. In addition,
Fiziologia - Physiology • 2016.26.2(90)TGFβ1 is not responsible for the immunomodulatory abilityof mesenchymal cells in the presence of IFN-γ, however,other factors such as COX-2 and PGE2, are involved inthis function. The data suggest that environmental factors
can play a crucial role in determining which
immunomodulatory pathways are operational in MSCs [21].
Although currently there is lack of consistency in certain
areas of MSCs therapeutics, the potential of
immunomodulatory properties of MSCs is remarkable [22].
According to the data obtained in our experiments,
evaluation of secretory profile in MSCs in vitro into contact
with the tested biomaterials indicated that cells releases into
the culture medium higher concentrations of TNF alpha and
IFN gamma, changes with no statistical significance except
elution prepared from anodized titanium (Ti-a). The effect,
even present is less extensive in the case of HAP-Ti, which
demonstrates its better quality compared to the material
used as a positive control. In addition, an increased releaseof interleukins with suppressor and anti-inflammatory effects(IL10, IL4) appears suggesting a possible better tissue
integration of this material.
The in vitro MSCs contact with tested biomaterials
appear to stimulate the release into the environment of
VEGF, the highest concentration being found in tests withelution of HAP-Ti. It is known that VEGF supports theprocess of new vessels formation that is probably useful inrepairing tissue. Restoring and enhancing vascular structure
allows nutrients and oxygen to reach the area underreconstruction, thereby contributing to its correct recovery. It
seems that the formulation of HAP-Ti ensure optimization ofthis effect in comparison to the Ti-a material.
The present study is a preliminary in vitro assessmentof the interactions between two different types of
new-synthesized biomaterials for bone replacement and a
primary culture of human MSCs. Overall, our results
suggested that the proposed materials are not very wellstructured and need to be optimized in order to be morestable in aqueous environments thus avoiding potential
harmful reactions. However it appears that HAP-Ti is lessreactive than Ti-a suggesting that HAP-Ti should be thechosen material for future improvements.

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introductory overview. Ed. Eurobit Timisoara, 2011.
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Impact of Porosity and Electrolyte Composition on the
Surface Charge of Hydroxyapatite Biomaterials. ACS Appl.
Mater. Interfaces, 2016, 8 (1): 908-917.
12. Navarro M, Michiardi A, castan O, Planell JA. Bioomaterials
in orthopaedics. J. R. Soc. Interface, 2008; 5: 1137-1158.
13. Rajesh P, Muraleedharan CV, Komath M, Varma H. Pulsed
laser deposition of hydroxyapatite on titanium substrate withtitania interlayer. J. Mater. Sci. Mater Med. 2011;22(3):497-505.
14. Niespodziana K, et al. Synthesis of titanium alloys forbiomedical applications. Rev. Adv. Mater. Sci. 2008; 18:236-240.
15. Al-Mobarak NA, Al-Swayih AA, Al-Rashoud FA. Corrosion
Behavior of Ti-6Al-7Nb Alloy in Biological Solution for
Dentistry Applications. Int. J. Electrochem. Sci. 2011;
6(011):2031-2042.
16. Albulescu R, Codorean E, Albulescu L, Caraene G,
Vulturescu V, Tanase C. In vitro biocompatibility testing of
implantable biomaterials. Roum. Biotechnol. Lett, 2008; 13(4):3863-872.
17. International Standard ISO 10993-1, Biological evaluation of
medical devices - Part 1: Evaluation and testing within a risk
management process, June 2016.
18. Hsiao ST, Asgari A, Lokmic Z, Sinclair R, Dusting GJ, Lim SY,
Dilley RJ. Comparative analysis of paracrine factor
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21. Guiqian C, Chuxia D, Li YP. TGF-β and BMP Signaling in
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37

EVALUAREA IN VITRO A MODIFICĂRILOR INDUSEASUPRA PROFILULUI SECRETOR AL CELULELORSTEM MEZENCHIMALE ÎN CONTACT CU UN
BIOMATERIAL DE TITAN-HIDROXIAPATITĂREZUMAT
Rolurile celulelor stem mezenchimale (CSM) în procesele de regenerare și reparare tisulară au fost dezbătute
foarte mult în ultima perioadă. CSM par să susțină refacerea țesutului prin menținerea unor condiții de mediu
care favorizează acest proces, datorită capacității lor de a elibera citokine și chiar factori de creștere. Pornind dela aceste date am dezvoltat un protocol experimental cu scopul de a evalua modificările substanțelor secretate
de CSM în mediul de cultură după interacțiunea in vitro cu un biomaterial din hidroxiapatită ranforsată cu titan(HAP-Ti) precum și cu un biomaterial de control, titan anodizat (Ti-a). Din fiecare biomaterial am preparat
specimene pătrate cu latura de 0,6 mm, precum și un eluat. Secreția a fost evaluată fie după contactul direct cu
biomaterialul, fie după adăugarea de eluat în mediul de cultură. Am utilizat un test imunoenzimatic (ELISA)
pentru măsurarea următoarelor citokine și factori de creștere: factorul de necroză tumorală alfa (TNF-alfa),
interferon gamma (IFN-gamma), interleukina 12 (IL12), interleukina 4 (IL-4), interleukina 10 (IL10) , factor decreștere tumorală beta (TGF-beta) și factorul de creștere vasculo-endotelial (VEGF). Conform datelor obținute înexperimentele noastre, evaluarea profilului secretor al CSM in vitro în contact cu biomaterialele testate a indicat
faptul că celulele eliberează în mediul de cultură concentrații mai mari de TNF alfa și IFN gamma, modificări fărăsemnificație statistică cu excepția eluatului preparat din titan anodizat (Ti-a). Efectul, chiar prezent este mai puțin
extins în cazul HAP-Ti, ceea ce demonstrează calitatea mai bună în comparație cu materialul utilizat ca martor
pozitiv. In plus, apare o eliberare crescută de interleukine cu efecte supresoare si anti-inflamatorii (IL10, IL4),
care sugerează o posibilă integrare tisulară mai bună a acestui material. La contactul in vitro cu biomateriale
testate celulele par a stimula eliberarea în mediu a VEGF, cea mai mare concentrație fiind găsită în testele cu
eluat HAP-Ti.
În general, rezultatele noastre au sugerat că materialele propuse nu sunt foarte bine structurate și trebuie să fie
optimizate pentru a fi mai stabile. Cu toate acestea, se pare că HAP-Ti este mai puțin reactivă decât Ti-asugerând că HAP-Ti ar trebui să fie materialul ales pentru îmbunătățiri viitoare.
Cuvinte cheie: celule stem mezenchimale, funcția secretorie in vitro, titan-hidroxiapatită, interacțiunea in vitro
celule-biomateriale

38

Fiziologia - Physiology • 2016.26.2(90)PREDICTORS OF EXPERIENCING TROUBLE WITH
THE POLICE IN YOUNG DRIVERS AGED 18-20 YEARS,ALCOHOL AND DRUGS CONSUMERS RESIDINGIN TIMIS COUNTY, ROMANIA
Cristina Pantea1,2, Preda M.A, Ioana Tuță Sas2, Codruța Băcean Miloicov2,
Radu Bagiu2, Mihaela Popa2, Corneluța Fira Mladinescu2,
Cristina Petrescu2, Salomeia Putnoky2, Oana Suciu2, Brigitha Vlaicu2
1Clinical Emergency County Hospital „Pius Brinzeu” Timisoara
2”Victor Babeș” University of Medicine and Pharmacy Timișoara, Departament 14 - Microbiology

ABSTRACTThe research aimed to assess certain predictors for experiencing trouble with the police in cases of young people who
possess driver’s licenses and who are also alcohol binge drinkers, drugs or mixed alcohol and drugs consumers, inTimis County, Romania. The sample of 1605 young people aged between 18-20 years, 51.4% pupils and 48.6%
students, in which girls were significantly better represented, was applied a transversal population study. Of young
people who drive, 29.5%, reported trouble with the police, 15.8% of boys and 4.6% of girls. According to the alcohol
and/or drugs consumption pattern during the last 30 days, incident reporting is significantly higher in boys for any of theconsumption patterns and in girls in the case of drugs or mixed consumption, as compared to non-consumers. The
identified police trouble predictors were all consumption patterns, masculine gender, clash or fight experiences, money
loss, low school and work performance, previous experience as a victim of robbery or theft.
Keywords: young people, trouble with police, alcohol and drugs consumption

INTRODUCTION
The promotion of healthy practice during adolescenceand early adulthood, as well as the implementation of
measures to better protect young people from health risks
are critical for the prevention of health problems in adult lifeand for a good population health status and a better social
infrastructure for the future [1,2].
Worldwide, traffic accidents are the main cause of deathamong young people aged between 10-24 years and thesecond cause of disease and invalidity. Each year, over
400,000 persons under 25 years, which is more that 1000 aday, die on the roads. Boys are significantly more affected,
with a death rate three times higher than girls [3,4].
Examining historic trends of car accidents involving
young people, the authors [5] observed significantly higher
prevalences as compared to adult age groups anddepending on the number of kilometers driven. They also
observed there are 7 categories influencing driving
behaviour in young people: the driving ability, development
factors, behaviour factors, personality factors, demographic
factors, the perceived environment and the driving

environment. Due to this complex group of factors which
influence the driving behaviour of young people,
comprehensive and multilevel interventions to decrease
their exposure to hazardous driving conditions are needed.
Our study aimed to assess some predictors ofexperiencing trouble with the police for young drivers,
alcohol and/or drugs consumers in Timis County, Romania.

MATERIAL AND METHOD
We studied a group of 1605 young people aged
between 18 and 20 years, of whom 51.4% (825) are pupils,
and 780 are students. Boys represent 46.2% (381) of thepupils and 38.4% (299) of the students.
Possessing a driver’s license was declared by 29.5%(463) of participants, with significantly different
proportions depending on the study group, χ2(1)=94.45,
p<0.001, OR=3.04, CI95%=(2.419; 3.822): in pupils,
possession of a driver’s license was declared by 18.5%(149), and in students the percent is 40.9% (314). Boyspossess a driver’s license significantly more frequently

Received April 10th 2016. Accepted May 2nd 2016. Address for correspondence: Prof. Dr. Vlaicu Brigitha, Microbiology
Department, UMF ’’Victor Babes” Timisoara, B-dul Victor Babes, Nr. 16, Timişoara, phone: 0256/490412, 0256/492101; e-mail:vlaicu@umft.roFiziologia - Physiology • 2016.26.2(90)39than girls, in the group of pupils, χ2(1)=16.27, p<0.001,
OR=2.09, CI95%=(1.456; 3.016), as well as in the groupof students, χ2(1)=52.66, p<0.001, OR=3.01,
CI95%=(2.229; 4.082).
The work method was the transversal population study
based upon the CORT 2004 Questionnaire regarding
health risk behaviours in adolescents and young adults [6].
The questionnaire was validated by the Ethics Committeeof the „Victor Babeş” University of Medicine and PharmacyTimişoara.
The study was conducted after written approval was
obtained from the university teaching institutions in Timis
County.
Young subjects were only included in the study after
freely expressed consent was obtained from eachparticipant, according to individual rights.
Data were processed with the PASW 18 (SPSS18)
2010 programme. The threshold for statistical significancewas set at p<0.05, except for situations when the
Bonferroni correction was applied. For comparing ordinaldata, we used the Mann-Whitney and Kruskal-Wallis tests.
The Chi-square test was used in tables withordinal/nominal data. We also used logarithmic regression
for predicting various association patterns between risk
behaviours.

RESULTSIn our study, of the young driver’s license possessors,
15.8% (105) of boys and 4.6% (42) of girls reported
experiencing trouble with the police, with boys reporting
this type of incident significantly more frequently than
girls χ2(1)=56.80, p<0.001, OR=3.88, CI95%=(2.67-5.63).
When comparing subgroups of subjects, we found no
significant differences in reporting trouble with the police
between boys, either pupils or students, nor did we findsuch differences in girls. Comparing study subgroups, wefound no significant differences in reporting trouble withthe police between pupils and students, neither in girls,
p=0.717 nor in boys p=0.735 (Figure 1).
In boys, trouble with the police was declared by
7.7% (32) of non-consumers, 24.9% (43) of binge
drinkers, 29.2% (7) of drugs consumers and 44.7% (21)of those who practice mixed consumption. In girls, trouble
with the police is declared by 3.3% (26) of
non-consumers, 8.5% (6) of those who practice binge
drinking, 13.6% (3) of drug consumers and 30.0% (6) of
those practicing mixed consumption.
The association between experiencing trouble withthe police and the consumption pattern was tested with
the Kruskal-Wallis test, and results showed a statisticallysignificant difference in the reporting frequency of such
experiences depending on the manner of consumption inboys, H(3)=64.00, p<0.001, as well as in girls,
H(3)=39,61, p<0,001. We additionally investigated this40

result by applying the Bonferroni correction, and thethreshold for statistical significance was set at 0.0166.

Fig. 1. Percent gender and subgroup distribution of participants
depending on admitting the experience of trouble with the police

In boys, when compared to non-consumers, those whoreport practicing binge-drinking (U=29678, z=-5.66,
p<0.001, r=0.23), drugs consumption (U=3903, z=-3.58,
p<0.001, r=0.17) and those who admit mixed consumption(U=6134, z=-7.52, p<0.001, r=0.35) declared significantly
more frequently they had trouble with the police. The sizeof the association was small for mono-consumption andmedium for mixed consumption.
In girls, when comparing to non-consumers, those who
consume drugs (U=7819.5, z=-2.58, p=0.010, r=0.09) andgirls who admit practicing mixed consumption (U=5811,
z=-6.06, p<0.001, r=0.21) declared significantly more
frequently that they experienced trouble with the police.
The size of the difference is very small in girls who
consume drugs and small in those practicing mixed
consumption, as compared to non-consumers, p=0.027(Figure 2).

Fiziologia - Physiology • 2016.26.2(90)trouble with the police and correctly classifies 91.7% ofcases.
Only six independent variables significantly contributeto the model, i.e. gender (p=0.002), substanceconsumption pattern (p<0.001), clash or fight experience(p<0.001), money loss (p=0.003), low school and work
performance (p<0.001), the experience of being a victim ofrobbery or theft (p<0.001). The highest odds ratio is foundin those who consumed drugs and practiced binge drinking
versus non-consumers, with an OR=6.4, those who
consumed drugs vs those who never did (OR=3.9), those
who practiced binge drinking, vs those who neverconsumed alcohol (OR=2.9). Other predictors ofexperiencing trouble with the police are: clash or fight(OR=2.3), being a victim of a theft (OR=2.2), money loss
(OR=2.1), low performance at work or in school (OR=1.9).
The chances of boys reporting trouble with the police were
2.1 times higher than for girls, OR=2.1, when the otherfactors in the model remain constant. The originating group
did not significantly influence the prediction model (Table I).
Fig. 2. Percent and gender distribution of participants dependingon admitting trouble with the police, according to the consumption
pattern

After excluding, by repeated trials, variables linked tofamily and school environment which did not contribute to
the model, we applied logistic regression to demonstrate
the influence of gender, subgroup, pattern of high-risk
substance consumption and some manifestations of violent
behaviour on reporting trouble with the police and we foundthat the model containing these predictors is statisticallysignificant χ2(9)=206.2, p<0.001, indicating that the model
may discriminate between responders who did have theseexperiences and those who did not. The model explains
between 12.6% and 27.8% of the variance in reporting

DISCUSSION
In the study conducted in Timis County, Romania,
18-20 years old boys with driver’s license reported
incidents with the police almost 4 times more frequently
than girls (15.8% vs 4.6%). The reporting frequency ofsuch incidents is significantly higher for boys in any of theconsumption models, and for girls who consume drugs, aswell as for girls with mixed consumption, as compared to
non-consumers. In the multivariate prediction model forreporting incidents with the police 6 predictors wereidentified: substance consumption, masculine gender(OR=2.1), clashing or fighting experiences (OR=2.3),
money loss (OR=2.1), low performance in school and at

Table I. Coefficients of logarithmic regression for prediction of experiencing trouble with the police in young drivers
Exp(B)

95% CI for
Exp(B)
Lower

Upper

.000
.000

2.863

1.804

4.545

1

.002

3.892

1.639

9.243

32.308

1

.000

6.396

3.373

.203
.231
.234
.243

.047
9.888
12.339
8.880

1
1
1
1

.829
.002
.000
.003

1.045
2.070
2.278
2.061

.702
1.315
1.439
1.281

12.12
9
1.556
3.257
3.605
3.317

.634

.210

9.148

1

.002

1.885

1.250

2.841

.796
-4.609.224.282

12.640266.736

1
1

.000
.000

2.217
.010

1.429

3.438

Variables
Consumption model Binge drinking vs
non-consumer
 Drugs vsnon-consumer
 Binge drinking and drugs vsnon-consumer
Group (1)Gender (1)Clash or fightMoney loss or other typesof accidents
Low performance in schoolor at work
Victim of robbery or theft
Constant

B

SE

Wald

.236

43.97419.905

d
f
3
1

1.052
1.359

.441

9.482

1.856

.326

.044
.727
.823
.723

Sig.

Legend: B= β coefficient; SE=standard error of β coefficient; Wald= Wald coefficient; df=degrees of freedom; Sig.= p value; OR=odds ratio

Fiziologia - Physiology • 2016.26.2(90)41work (OR=1.9), experience as a victim of robbery or theft(OR=2.2). Regarding the consumption model, whencompared to non-consumers, the chance to report such
incidents was 6 times higher in those practicing mixed
consumption, 4 times higher in drugs consumers andalmost 3 times higher in binge drinkers.
The transition from adolescence to early adulthood ismarked by rapid development on multiple fronts. As aconsequence of the evolution of social and family
relationships, young people acquire autonomy, individuality
and responsibility during they journey towards adult life.
Even though most adolescents advance throughpredictable development stages, there is heterogeneity
within this population, with increased inter-individual
variability regarding the degree of maturity, even in thecase of similar ages. During this period when
responsibilities and pressure increase, young people arelacking the development capacities and abilities, but also
the financial and social resources of adults. This paradox
makes young people vulnerable to external pressures
which, for some of them, result in making wrong decisions
such as substance consumption and involvement inhazardous activities [7,8].
The health of adolescents is strongly impacted bysocial factors at a personal level, by family, community andat a national level. As health and health risk behaviours arecorrespondent between adolescence and adulthood, themanner social factors impact the health of adolescents isessential for the health of the entire population and for the
economic development of nations [9].
Adolescents exposed to multiple forms of psychologicaltrauma present an increased risk for psychiatric andbehaviour disturbances [10]. Testing this hypothesis, Fordet al [11] showed that participants who witnessed abuse inmultiple forms have more chances to present psychiatricdiagnoses and be involved in various forms of delinquency
than those who were traumatized by exposure to violenceor accidents. They demonstrated that the delinquency riskfor polytraumatized victims cannot entirely account for
post-traumatic behaviour disturbances, depression or
substance abuse, suggesting that repeatedly victimised
young people should be considered at risk for behaviour
problems and trouble with the police.
The results of a study testing potential predictors for
repeated victimizing, analysing two measurements in thesame population of certain information regarding drugs
consumption, alcohol consumption, depression, andinfliction of injuries found that the victim status upon firstassessment predicted violent injuries, delinquency, anddrugs consumption upon the second assessment, evenafter confounders control. For the masculine gender, theviolent injury was found to be a robust and consistent
mediator. For women, all mediators contributed together toa significant indirect and large effect, which reduced toinsignificance the direct effect of previous victimizing. The

42fact that mediation between victimizing and re-victimizing
occurred through a group modified by behaviours andstates suggests that the impact of victimizing is larger for
women than for men. The effects of victimizing in changing
behaviours and increasing risks (which differ with gender)
produce effects on both mental health care and law
enforcement [12].

CONCLUSIONSIn multivariate models, all the three researched
consumption models (binge drinking, drugs consumption,
mixed alcohol and drugs consumption), masculine gender,
clashing or fighting experiences, money loss, low
performance in school or at work, experience of being a
victim of robbery or theft were identified as predictors forproblems with the police in young people aged 18-20 years
with driver’s license in Timis County Romania.

REFERENCES
1. Viner RM, Ozer EM, Denny S, Marmot M, Resnick M, Fatusi
A, Currie C. Adolescence and the social determinants ofhealth. The Lancet. 2012; 379(9826): 1641-52.
2. http://www.who.int/mediacentre/factsheets/fs345/en/, accesat
la 02.02.2016
3. http://www.who.int/features/factfiles/youth_roadsafety/en/
accesat la 02.02.2016
4. Toroyan T, Peden M (eds), Youth and Road Safety, Geneva,
World Health Organization, 2007
5. Shope JT, Bingham CR. Teen driving: motor-vehicle crashes
and factors that contribute. Am J Prev Med. 2008; 35(3Suppl): S261-71.
6. Vlaicu B. Comportamente cu risc la studenții din județul Timiș.
Ed. Eurobit, Timișoara. 2009
7. Zuckerman M. Sensation seeking and risky behavior, 2007.
Washington, DC, US: American Psychological Association,
xix, 309 pp. http://dx.doi.org/10.1037/11555-002
8. Steinberg L, Albert D, Cauffman E, et al. Age differences insensation seeking and impulsivity as indexed by behavior
and self-report: evidence for a dual systems model.
Developmental Psychology. 2008; 44(6): 1764.
9. Romer D. Adolescent risk taking, impulsivity, and brain
development: Implications for prevention. Dev. Psychobiol.
2010; 52:263-276.
10. Finkelhor D, Ormrod RK, Turner HA. Poly-victimization: A
neglected component in child victimization Child Abuse &Neglect 2007; 31:7-26.
11. Julian D. Ford JD, Elhai JD, Connor DF, Frueh
C.Poly-Victimization and Risk of Posttraumatic, Depressive,
and Substance Use Disorders and Involvement inDelinquency in a National Sample of Adolescents. Journal of
Adolescent Health 2010; 46 (6): 545-552.
12. Testa M, Hoffman JH, Livingston JA. Alcohol and sexual risk
behaviorsasmediatorsofthesexualvictimization–revictimization relationship. Journal of
consulting and clinical psychology. 2010; 78(2): 249.

Fiziologia - Physiology • 2016.26.2(90)PREDICTORI AI EXPERIMENTĂRII DE NECAZURICU POLIȚIA LA TINERI DE 18-20 ANI CU PERMISDE CONDUCERE, CONSUMATORI DE ALCOOLȘI DROGURI DIN JUDEȚUL TIMIȘ, ROMÂNIAREZUMAT
Cercetarea și-a propus evaluarea unor predictori ai experimentării de necazuri cu poliția la tineri care posedă
permis de conducere și sunt consumatori de alcool de tip binge drinking, de droguri și consum mixt alcool-droguri,
din județul Timiș, România. Eșantionului de 1605 tineri cu vârste între 18-20 de ani, 51,4% elevi și 48,6%
studenți, cu fete semnificativ mai bine reprezentate, i s-a aplicat un studiu populațional transversal. Dintre tinerii
care conduc, 29,5%, au raportat necazuri cu poliția 15,8% dintre băieți și 4,6% dintre fete. După modelul deconsum al băuturilor alcoolice și/sau al consumului de droguri în ultimele 30 de zile, raportarea incidentului este
semnificativ mai crescută la oricare din modelele de consum la băieți și la consumatoarele de droguri și
consumul mixt la fete, comparativ cu non-consumatorii. Predictori pentru problemele cu poliția sunt toatemodelele de consum, sexul masculin, experiențe de încăierare sau bătaie, pierderea de bani, performanțele
reduse la școală și muncă, experiența de victimă a unui jaf sau furt.
Cuvinte cheie: tineri, necazuri cu poliția, consum de alcool și droguri

Fiziologia - Physiology • 2016.26.2(90)43ACUTE POSTERIOR MULTIFOCAL PLACOID PIGMENT
EPITHELIOPATHY - 18 MONTHS FOLLOW-UP
Preda M.A 1, Ciobotea D.1, Vlaicu B.2, Boruga O.1, Munteanu M.1
1Department of Ophthalmology, „Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
2Departemnt of Hygiene, „Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania

Abstract Acute posterior multifocal placoid pigment epitheliopathy (APMPPE), represents an acquired inflammatory
disorder affecting the retinal pigment epithelium, and choroid of young adults. The disease is characterized by multiple
yellow-white placoid subretinal lesions of the posterior pole. We present the case of a young woman, 25 years old, who
attended our department complaining of sudden vision loss in both eyes, without any history of ocular or generaldiseases. After a complex ocular and general examination, a set of laboratory exams and ocular investigations, weestablished the diagnosis of acute posterior multifocal placoid pigment epitheliopathy. Local and general treatment was
initiated. The patient was followed for 18 months to note the disease’s evolution, considering the risk of recurrence.
Keywords Epitheliopathy, placoid subretinal lesions, fluorescein angiography

INTRODUCTION
The acute posterior multifocal placoid pigment
epithelium (APMPPE) was first described by Gass in 1968.
The study presented clinical and fluorescein angiography
findings in three young women with loss of central vision inboth eyes (1). The disease is self-limited and consists ofmultiple yellow-white placoid subretinal lesions of theposterior pole. The lesions coexist in different stages of
evolution, frequently bilateral and normally resolving inweeks to months, leaving outlined areas of retinal pigment
epithelial scars. In the beginning, a rapid onset of blurredvision associated with central and periferic scotomas isnoted. Some studies have reported that the patients
experienced photopsias prior to vision loss (2,3). Symptoms
are usually unsymmetrical, bilateral and can appear several
days apart. Visual acuity can range from 20/25 to handmotion depending on the degree of foveal involvement. The
APMPPE’s etiology is not completely elucidated, it isthought to be a delayed-type hypersensitivity-induced
occlusive vasculitis. Its immune nature is by cause of its
association with HLA-B7 and HLA-DR2 genetic haplotypes(4, 5). The physiopathology is based on an inflammatory
process in the choriocapillaris, which leads to hypoperfusion
and ischemia of the retinal pigmented epithelium causing intime its atrophy and hyperpigmentation.

MATERIAL AND METHODS
We present the case of a 25 years old woman, who
attended our department accusing sudden vision loss inboth eyes, without any history of ocular or generaldiseases. A general and ophthalmological examination was
carried out, blood samples were taken. The
ophthalmological examination consisted of: testing visualacuity, intraocular pressure measurement, anterior andposterior pole examination, ocular echography, fundus
photography, optical coherence tomography(OCT),
fluorescein angiography.
Due to the disease’s association with immune
responses and inflammatory process the generalexamination implied: general consult, sacral-iliac
radiography, blood pressure measurement, blood testscomplete blood count, erythrocyte sedimentation rate
(ESR), fibrinogen, C reactive protein and specificdeterminations : HLA-B27, HLA-A29, HLA-DR2, HLA-B7.
At presentation, visual acuity was 20/25 in right eye and20/100 in the left eye, the anterior segment was
unremarkable, intraocular pressure (IOP) was normal, andthe vitreous cavity was clear in both eyes. Multiple
yellowish-white placoid lesions in the posterior pole of botheyes were observed on fundus examination. Fig 1. OCT
showed hyper-reflectivity and alterations at the level of thephotoreceptors and RPE, especially in the left eye where it
showed a retinal pigment epithelium detachment. Fig. 2.
Fluorescein angiography showed early hyperfluorescentlesions at the posterior pole with late staining, without
macular edema or papillitis. Fig 3. Ocular echography was
normal. Fig 4. The blood tests revealed an inflammatory
reaction ESR= 40 mm/h, CRP=15 mg/l and HLA-B27
positive. Sacral-iliac Rx disclosed the pinching of thesacroiliac joint space.

Address for correspondence: Prof. Dr. Vlaicu Brigitha, Microbiology Department, UMF ’’Victor Babes” Timisoara, B-dul Victor
Babes, Nr. 16, Timişoara, phone: 0256/490412, 0256/492101; e-mail: vlaicu@umft.ro44

Fiziologia - Physiology • 2016.26.2(90)Fig.1 Fundus photography at presentation, multiple
yellowish-white placoid lesions in the posterior pole.

Fig. 2 OCT findings at presentation, macular oedema in right eye,hyper-reflectivity in left eye

Fiziologia - Physiology • 2016.26.2(90)Fig.3 Fluorescein angiography at presentation, both eyes - early
hyperfluorescent lesions at the posterior polewith late staining, without macular edema or papillitis

Fig 4. Ocular echography at presentation, normal aspect

45

After analyzing the results and establishing thediagnosis of acute posterior multifocal placoid pigment
epithelium in both eyes and sacroiliitis, we opted forgeneral treatment with Azathioprine 50 mg/day andMethilprednisolone 200 mg/day and intravitreal treatment
with triamcinolone 0.06ml/eye.
Considering the risk of recurrence we followed thepatient’s evolution for 18 months, evaluating the possible
changes in the optical coherence tomography and fundus
photography (5).

RESULTS
One month after treatment the visual acuity improved
significantly, 20/20 in both eyes. Fundus photographyhighlighted the grey-white retinal lesions, which indicates
there is no inflammatory process, and the lesions areclinically mute.Fig 5 OCT findings showed normal reflectivity
and organization. Fig 5.

Six months after treatment the results are conserved.
Fig 6 Eighteen months after treatment was initiated theresultsfindings
are stable,
no otherscar
activeinflammatory flares were
Fig 5. One month after treatment, fundus photography– grey-whitish
lesions
reported.
Fig
6
OCT findings- normal reflectivity, no macular oedema

46

Fiziologia - Physiology • 2016.26.2(90)Fig. 6 OCT and Fundus photography findings after 6 and 18 months- grey-whitish scar lesions, normal reflectivity

DISCUSSIONSCONCLUSIONSAll patients with a new diagnosis of APMPPE should
receive a full neurologic and systemic work-up to evaluate
for vasculitis and other associated systemic conditions.
It is known that approximately 33% of patients report a
preceding viral or flu-like episode prior to APMPPE
symptom onset. APMPPE has been described in cases
associated with A streptococcus and adenovirus infections,
hepatitis B, Lyme disease, tuberculosis, thyroiditis,
polyarteritis nodosa, nephritis, sarcoidosis, scleritis,
ulcerative colitis and post-vaccination. Other associations
have been reported including HLA-B7 and HLA-DR2
genetic haplotypes, vein occlusion, retinal vasculitis, optic
disc edema, subhyaloid hemorrhage and rare choroidal
neovascular membrane formation (6, 7, 8).
In the majority of the cases the prognosis is positive,
most of the patient achieving visual acuity of 20/40 or more.
The visual recovery period can range from 4 weeks to 6
months. An interesting review by Fiore T el al. assessed 183
articles related to APMPPE. The conclusion was that 25% of
patients had a visual acuity of 20/50 or worse. The visual
prognosis depends on the foveal involvement. Regarding
the treatment, in the literature steroids have been reported
to bring benefits in cases with foveal involvement andassociated with vasculitis. More studies are needed toestablish dosage, duration, and effects of steroid
treatment.(8, 9, 10)

In the case we presented, the treatment option to combine
local treatment with general one brought positive results. We
administrated intravitreal injection with triamcinolone 0,06 ml
per eye. After only one injection per eye the visual acuity
improved to 20/20 in both eyes. The OCT outcome suggests
significant change in reflectivity and the absence of any
macular oedema, this status was maintained throughout theentire evaluation period. Fundus photography revealed a stable
ocular aspect after treatment and during the 18 months offollow-up, the existing lesions became scars with no sign ofinflammatory flares. It is a fact that combining general treatment
with local one potentiate the result. The general treatment with
Azathioprine 50 mg/day and Methilprednisolone 200 mg/day
was continued for six months.
In conclusion, one month after the treatment beginning
the visual acuity reached 20/20 in both eyes and the result isstill conserved clinically and morphologically. The patient’s
well-being is regained. Ophthalmological reviews are madeevery six months

Fiziologia - Physiology • 2016.26.2(90)References
1. Gass JD. Acute posterior multifocal placoid pigment
epitheliopathy. Arch Ophthalmol. 1968 Aug;80(2):177-85
2. O’Halloran HS et al. Acute multifocal placoid pigment
epitheliopathy and central nervous system involvement: nine47

new cases and a review of the literature. Ophthalmology. 2001
May;108(5):861-8
3. Burés-Jelstrup A, Adán A, Casaroli-Marano R. Acute posterior
multifocal placoid pigment epitheliopathy. A study of 16 cases.
Arch Soc Esp Oftalmol. 2007 May;82(5):291-7
4. Jones NP. Acute posterior multifocal placoid pigment
epithliopathy. Br J Ophthalmol. 1995 Apr;79(4):384-9 \5. Hsu CT, Harlan JB, Goldberg MF, Dunn JP. Acute posterior
multifocal placoid pigment epitheliopathy associated with asystemic necrotizing vasculitis. Retina. 2003 Feb;23(1):64-8.
6. Quillen DA et al. The white dot syndromes. Am J Ophthalmol.
2004 Mar;137(3):538-50

7 Comu S, Verstraeten T, Rinkoff JS, Busis NA. Neurologicalmanifestation of acute posterior multifocal placoid pigment
epitheliopathy. Stroke. 1996 May;27(5):996-1001
8.Fiore T el al. Acute posterior multifocal placoid pigment
epitheliopathy: outcome and visual prognosis. Retina. 2009
Jul-Aug;29(7):994-1001
9. Steiner S, Goldstein D. Imaging in the Diagnosis andManagement of APMPPE. International Ophthalmology
Clinics. 2012;52(4):211-219.
10.Mirza G, Jampol L. Acute posterior multifocal placoid pigment
epitheliopathy. In Ryan SJ. Retina, 5th Edition. Elsevier, 2013:
1341-1346.

EPITELIOPATIE PIGMENTARĂ PLACOIDĂ ACUTĂ
MULTIFOCALĂ – 18 LUNI DE URMĂRIRE CLINICĂ
Rezumat Epiteliopatia pigmentară placoidă acută multifocală (EPPAM) este o boală inflamatorie dobândită careafectează epiteliul pigmentar retinian şi coroida tinerilor. Boala este caracterizată de multiple leziuni placoide
subretiniene galbene-albicioase dispuse la nivelul polului posterior. Prezentăm cazul unei femei de 25 de ani, cares-a prezentat în clinica noastră acuzând scăderea bruscă a acuității vizuale la ambii ochi, fără existența vreunuiistoric de boli oculare sau generale.După ce am efectuat o examinare generală şi oculară complexă, investigații
oftalmologice specifice, analize de laborator am stabilit diagnosticul de epiteliopatie pigmentară placoidă acută
multifocală. Am inițiat tratament local şi general. Datorită riscului mare de recurențe, am urmărit evoluția şistabilitatea rezultatului timp de 18 luni.
Cuvinte cheie Coriocapilară, leziuni subretiniene, vasculită

48

Fiziologia - Physiology • 2016.26.2(90)