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International Journal of Pharmaceutics 463 (2014) 146– 154

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journa l h o me pag e: www.elsev ier .com/ locate / i jpharm

harmaceutical nanotechnology

nionic polymers and 10 nm Fe3O4@UA wound dressings supportuman foetal stem cells normal development and exhibit greatntimicrobial properties

lexandru Mihai Grumezescua, Alina Maria Holbanb,∗, Ecaterina Andronescua,eorge Dan Mogos anuc, Bogdan Stefan Vasilea, Mariana Carmen Chifiriucb,eronica Lazarb, Eugen Andreid, Andrei Constantinescud, Horia Maniud

University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Science and Engineering of Oxide Materials andanomaterials, Polizu Street No. 1-7, 011061 Bucharest, RomaniaUniversity of Bucharest, Faculty of Biology, Microbiology Department, Aleea Portocalelor, No. 1-3, 060101 Bucharest, RomaniaDepartment of Pharmacognosy & Phytotherapy, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rares Street, 200349raiova, RomaniaFlow Cytometry and Cell Therapy Laboratory, Institute of Cellular Biology and Pathology “Nicolae Simionescu” (ICBP), Bucharest, Romania

r t i c l e i n f o

rticle history:eceived 19 July 2013ccepted 21 August 2013vailable online 29 August 2013

eywords:agnetite nanoparticlesound dressing

egenerative medicineodium alginate

a b s t r a c t

The aims of this study were the development, characterization and bioevaluation of a novel biocompati-ble, resorbable and bio-active wound dressing prototype, based on anionic polymers (sodium alginate –AlgNa, carboximethylcellulose – CMC) and magnetic nanoparticles loaded with usnic acid (Fe3O4@UA).The antimicrobial activity was tested against Staphylococcus aureus grown in biofilms. The biocompatibil-ity testing model included an endothelial cell line from human umbilical vein and human foetal progenitorcells derived from the amniotic fluid, that express a wide spectrum of surface molecules involved in dif-ferent vascular functions and inflammatory response, and may be used as skin regenerative support. Theobtained results demonstrated that CMC/Fe3O4@UA and AlgNa/Fe3O4@UA are exhibiting structural andfunctional properties that recommend them for further applications in the biomedical field. They could

arboximethylcelluloseuman foetal progenitor cells

be used alone or coated with different bio-active compounds, such as Fe3O4@UA, for the development ofnovel, multifunctional porous materials used in tissues regeneration, as antimicrobial substances releas-ing devices, providing also a mechanical support for the eukaryotic cells adhesion, and exhibiting theadvantage of low cytotoxicity on human progenitor cells. The great antimicrobial properties exhibitedby the newly synthesized nano-bioactive coatings are recommending them as successful candidates for

devic

improving the implanted

. Introduction

When skin and mucosa become compromised, environmen-al and skin associated microorganisms are able to gain access tonderlying tissues, where the physical conditions are optimal forolonization and growth. Staphylococcus aureus is one of the mostommon bacterium isolated from both acute and chronic woundsf various etiologies. Their prevalence has been highly demon-trated in surgical site infections as well penetrating trauma andurn wounds (Keen et al., 2010; Pastar et al., 2013). Frequency

f S. aureus drug resistant strains in wound infections has beenhown to be exceedingly high (Fazli et al., 2009). S. aureus, in itsethicillin susceptible and methicillin resistant form (MRSA), is

∗ Corresponding author. Tel.: +40 721600737.E-mail address: alina m h@yahoo.com (A.M. Holban).

378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2013.08.026

es surfaces used in regenerative medicine.© 2013 Elsevier B.V. All rights reserved.

a common opportunistic pathogen, responsible for the majorityof all superficial skin infections, resulting in increased morbidity,mortality, and tremendous health-care costs (Pastar et al., 2013).Another wound associated medical complication is represented bybiofilm embedded S. aureus infections or multispecific staphylococ-cal biofilms.

Appropriate wound care represents one of the key aspects fora successful wound healing strategy. Using novel wound dressingswith improved anti-adherent surface has proved to determine goodresults against microbial colonization (Anghel et al., 2012a). Fur-thermore, a very efficient approach seems to include the usageof certain natural and synthetic compounds with proved anti-microbial activity. This strategy seems to improve significantly the

efficiency of novel developed anti-microbial nanosystems (Saviucet al., 2011a,b; Chifiriuc et al., 2012), being considered useful toolsfor fighting against infections without taking the risk of developingantibiotic resistance.

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A.M. Grumezescu et al. / International Jo

Usnic acid (UA) has proved its high anti-microbial activitygainst Gram-positive bacteria species (Segatore et al., 2012;asero et al., 2013).

We have previously reported that a non-water dispersibleanofluid based Fe3O4@fatty acid and UA, is able to potentate theicrobicidal and anti-biofilm activity of UA on S. aureus strains

Grumezescu et al., 2011a). However, there are some limitationsf the obtained nanofluid, due to its apolar nature and waternsolubility and highly aggregative properties. Another recentlyublished paper reports improved and extended microbiologicalpplications of UA, by the successful fabrication of novel waterispersible nanostructures based on magnetite and usnic acidFe3O4@UA). This nanosystem exhibited an efficient antimicrobialctivity against planktonic and adherent cells, especially on Gram-ositive strains (Grumezescu et al., 2013).

Stem and progenitor cells hold the potential to open up newmportant perspectives to treat many degenerative diseases, as wells for cell therapy and regenerative medicine. The term ‘regener-tive medicine’ refers to a new and expanding field in biomedicalesearch that focuses on the development of innovative therapiesllowing the body to replace, restore and regenerate damaged oriseased cells, tissues and organs. It combines several technologi-al approaches including the use of soluble molecules, biomaterials,issue engineering, gene therapy, stem cell transplantation and theeprogramming of cell and tissue types. Because of its easy accessi-ility, skin is becoming an attractive model organ for regenerativeedicine (Dieckmann et al., 2010). The engraftment of engineered

issue substitutes fabricated by seeding stem cells on biodegrad-ble, biocompatible scaffolds has been proposed as a more suitableolution to repair injured tissues (Cima et al., 1991; Cohen et al.,993). This approach is based on the ability of bone marrow-derivedesenchymal stem cells (MSC) to generate virtually all cell lineages

Bianco et al., 2001) in vitro as well as to engraft and home in vivoFerrari et al., 1998; Duan et al., 2003; Forte et al., 2009).

Biocompatible and biodegradable polymers can provide a con-enient support for SC differentiation leading to tissue formation.caffolds used for tissue engineering should have the followingharacteristics: biocompatibility, biodegradability, reproducibility,igh porosity with interconnection pores, and no potential of seri-us immunological or foreign body reaction (Forte et al., 2009).urthermore, biological studies and clinical practice have high-ighted that, in addition to compositional requirements, a threeimensional interconnected porous polymer structure improvesissue development and provides temporary mechanical supportCancedda et al., 2007; Yoshikawa et al., 2009). Preliminary in vitroC differentiation studies investigated the performance of com-ercially available porous collagen sponges (William et al., 2012).

xpected results with biodegradable polymers allow implantedells to synthesize their own extracellular matrix in situ, and afteregeneration of the foreign polymeric material to attempt a tem-orally match creation of the new innate one. One of the majorroblems are the bacterial infections occurred at the site of an

njured tissue could elicit a pro-inflammatory response that canmpair the success of lesion repair and closure (Ahmed et al., 2011).n vitro studies have shown that soluble products of both S. aureuslanktonic cells and biofilms inhibit wound healing and signifi-antly reduced human keratinocytes viability and increased theirpoptosis, especially when grown in biofilms (Kirker et al., 2009).

The pores size of porous materials used as scaffolds for tis-ue regeneration varies depending on a particular application. Forkin growth and wound healing the optimum pore size is in theange of 20–120 �m (Yannas, 1992). By contrast, in biomaterials

oaded with therapeutic agents, pores with size less than 10 �m iniameter are needed to administer release of the agent by a slow,

ocal, continuous and controlled flux (Elsner and Zilberman, 2009).herefore, in order to obtain a multifunctional devices acting as

of Pharmaceutics 463 (2014) 146– 154 147

scaffold with controlled release, there may come a need to com-bine different pore sizes within the same structure (Elsner et al.,2012).

The aim of this study was to develop a novel biocompatibleand resorbable Fe3O4@UA-based bio-active wound dressing proto-type, with increases resistance to S. aureus colonization. Moreover,we enhanced the relevance of the biocompatibility testing modelincluding two cell lines: EA.hy926 (endothelial cell line from humanumbilical vein) and AFSC (human foetal progenitor cells derivedfrom the amniotic fluid), that express a wide spectrum of surfacemolecules involved in different vascular functions and inflam-matory response, and may be used as skin regenerative supportand thus interacting with the proposed bio-active wound dressingmodel.

2. Materials and methods

2.1. Materials

All chemicals were used as received. FeCl3, FeSO4·7H2O,NH4OH (25%), usnic acid (UA), and CH3OH were purchased fromSigma–Aldrich ChemieGmbh (Munich, Germany).

2.2. Fabrication of 10 nm structures

Fe3O4 nanoparticles are usually prepared by wet chemical pre-cipitation from aqueous iron salt solutions by means of alkalinemedia (Grumezescu et al., 2011b; Saviuc et al., 2011c). Briefly,500 mg of UA and 8 mL of NH4OH (25%) were added in 200 mLdeionized water under vigorous stirring. Then, 1 g of FeCl3 and 1.6 gof FeSO4·7H2O were dissolved in 200 mL of deionized water andFe+3/Fe2+ solution was dropped into the basic solution of UA accord-ing to our recently published paper (Grumezescu et al., 2013).After precipitation, magnetite-usnic acid crystals (Fe3O4@UA) wererepeatedly washed with methanol, separated with a strong NdFeBpermanent magnet. Subsequently, the Fe3O4@UA was added intothe 100 mL solution of acetic acid 0.1 N and stirred for 10 min. Afterthis, the Fe3O4@UA was separated with a strong NdFeB perma-nent magnet, repeatedly washed with deionized water and finallydispersed in deionized water.

2.3. Fabrication of wound dressing based anionic polymers and10 nm structures

Wound dressing based anionic (sodium alginate – AlgNa, car-boximethylcellulose – CMC) polymers and 10 nm structures wasprepared as follow: solutions containing 5% (w/v) AlgNa or 5% (w/v)CMC and 1% (w/v) Fe3O4@UA were prepared in deionized water andmixed for 10 min. Prepared gels were casted into glass Petri dishes(12.5 cm in diameter; 20 mL) to be lyophilized.

2.4. Characterization

2.4.1. TEMThe transmission electron microscopy images were obtained on

finely powdered samples using a TecnaiTM G2 F30 S-TWIN high res-olution transmission electron microscope (HRTEM) from FEI. Themicroscope was operated in transmission mode at 300 kV withTEM point resolution of 2 A and line resolution of 1 A. The finely

Fe3O4@UA powder was dispersed into pure ethanol and ultrason-icated for 15 min. After that diluted sample was put onto a holeycarbon coated copper grid and left to dry before it was analyzedthrough TEM.

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37 C of the inoculated plates. After the addition of wound dress-ings plates were incubated for 24 h at 37 ◦C. After the incubationperiod inhibition zones around wound dressings were analyzed.

48 A.M. Grumezescu et al. / International Jo

.4.2. XRDX-ray diffraction analysis was performed on a Shimadzu XRD

000 diffractometer at room temperature. In all the cases, Cu K�adiation (� = 15,406 A at 15 mA and 30 kV) was used. The samplesere scanned in the Bragg angle 2� range of 10–80◦.

.4.3. FT-IRA Nicolet 6700 FT-IR spectrometer (Thermo Nicolet, Madison,

I) connected to software of the OMNIC operating system (Version.0 Thermo Nicolet) was used to obtain FT-IR spectra of preparedound dressings. The samples were placed in contact with attenu-

ted total reflectance (ATR) on a multibounce plate of ZnSe crystalt controlled ambient temperature (25 ◦C). FT-IR spectra were col-ected in the frequency range of 4000–650 cm−1 by co-adding 32cans and at a resolution of 4 cm−1 with strong apodization. Allpectra were ratioed against a background of an air spectrum.

.4.4. SEMSEM analysis was performed on a HITACHI S2600N electron

icroscope, at 25 keV, in secondary electrons fascicle, on samplesovered with a thin silver layer.

.5. Biological assays

.5.1. Biocompatibility testingStem cells culture: To analyze the biological effects of anionic

olymers (CMC/Fe3O4@UA and AlgNa/Fe3O4@UA) on human cells,e used a human endothelial cell line EA.hy926 (human endothe-

ial umbilical vein cell line, which was kindly donated by Dr. Coraean Edgell (Department of Pathology, University of North Car-lina, Chapel Hill). The amniotic fluid (AF) samples (n = 4), kindlyonated by GeneticLab SRL, were collected during the amniocente-is (5 mL), isolated, processed, characterized, and stored in the ICBPtem cell bank. Human foetal AF samples were obtained upon writ-en informed consent and complied with European Union (EU) andational legislation regarding human samples collection, manip-lation and personal data protection. All samples were tested forhe absence of HIV1/2, HBV, HCV and HTLV and processed within

h from collection, during which the samples were suspendedn sterile saline phosphate buffer containing 100 U/mL penicillin,00 mg/mL streptomycin and 100 mg/mL neomycin.

Both human cell cultures, derived from the amniotic fluid stemells (AFSC) and the EA.hy926 line have been used for biocompat-bility testing of CMC and AlgNa samples alone and as resorbableA coated polymer samples. Human AFSC and EA.hy926 were cul-

ured in AmnioMax (Gibco, Invitrogen GmbH, Karlsruhe, Germany),MEM and RPMI 1640 (Sigma–Aldrich, USA) media, containing0% foetal calf serum (FCS), and, penicillin (100 U/mL), strepto-ycin and neomycin (100 mg/mL), 1% (PSN) antibiotics (Gibco,

nvitrogen GmbH, Karlsruhe, Germany), using 6, 12, and 24-wellell culture plates (Falcon, Becton Dickinson GmbH, Heidelberg,ermany). Adherent cells were harvested and washed with phos-hate buffered saline solution (PBS, GIBCO, Invitrogen GmbH)pH 7.4), and detached from the culture flasks by addition of.2–2 mL/cm2, of 0.25% trypsin/0.1% ethylendiamine tetraaceticcid (Sigma–Aldrich, Taufkirchen, Germany) for 3 min at 37 ◦C, andentrifuged at 1050 rpm/10 min at 4 ◦C. Subsequently, the EA.hy926nd AFSC were collected and washed twice with RPMI1640 with0% FCS. Tridimensional wound dressings based CMC or AlgNa ande3O4@UA were incubated with EA.hy926 and AFSC cultures, main-ained for 5 days at 37 ◦C in a humidified atmosphere of 5% CO2, and

nalyzed at 24-48-72-96-120 h intervals. For cell detachment andhe wound dressings digestion, 10 �L from 100 mg/mL collagenaseV (Sigma–Aldrich, USA) and 10 �L from 1 mg/mL hyaluronidaseSigma–Aldrich, USA) in DMEM growth media (Sigma–Aldrich,

of Pharmaceutics 463 (2014) 146– 154

USA) were used. As cell culture controls, not treated EA.hy926 andAFSC lines were used.

2.5.2. Morphology, viability, cytotoxicity, and cell cycle assayThe cell morphology was analyzed with a Nikon Eclipse TS100

(Nikon, Japan) fluorescence microscope. Viability assessmentswere performed using (0.4%) Trypan blue (Invitrogen, USA) stainingusing an automated cell counter Countess (Invitrogen, USA) accord-ing to the manufacturer protocols. Cells were filtered using 100 �mfilters in order to remove tissue debris. Propidium Iodide (PI) stain-ing was performed for cytotoxicity tests. The percentage of livingcells was calculated by determining the percentage of total eventsexcluding the PI stained cells for flow cytometry analysis. The cellswere incubated with 50 �L from a stock solution of 400 �g/mL PI,for 30 min at 37 ◦C, in dark conditions, followed by a PBS wash andfiltered prior to flow cytometry acquisition. The flow cytometryanalysis was performed using a GALLIOS (Beckman Coulter, USA).

For cytotoxicity samples testing the logarithmic FL2/FL3 dot plotsetting was carried out, and for cell cycle analysis, the linear FL2histogram was performed. The data was analyzed using Summitsoftware version 4.3 (Dako, USA) and expressed as the percentageof cell population in different cell cycle phases.

2.5.3. Antimicrobial assayStrains and growth conditions: S. aureus ATCC 25923 was pur-

chased from American Type Culture Collection (ATCC). A singlecolony obtained on LB agar (Oxoid) was stab inoculated in 5 mLLuria Broth and incubated overnight at 37 ◦C, 200 rpm shaking.Overnight cultures were diluted to a standard optical density of0.5 McFarland (∼107–108 colony forming units) and this inoculumwas used for the following experiments.

Antimicrobial assay: In order to test the antimicrobial activity ofnewly fabricated resorbable wound dressings S. aureus diluted cul-tures were used to swab inoculate Petri dishes containing MullerHinton agar (Oxoid). After inoculation the plates were allowed todry for 10 min at room temperature and resorbable wound dress-ings were added at three time points. First time point (T0): wounddressings were added immediately after inoculation; second timepoint (T1): wound dressings were added after 6 h; and third timepoint (T2): wound dressings were added after 12 h of incubation at

◦

Fig. 1. XRD pattern of Fe3O4 (a) and Fe3O4@UA (b).

A.M. Grumezescu et al. / International Journal of Pharmaceutics 463 (2014) 146– 154 149

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. Results and discussion

Magnetite is a fascinating material, which has proved its appli-ability in many biomedical fields, such us magnetic resonancemaging (Ling et al., 2012), cancer treatment (Voicu et al., 2013;rumezescu et al., 2012a), drug targeting (Park et al., 2012;rumezescu et al., 2011c), stabilization of essential oils (Anghelt al., 2012b) or inhibition of microbial biofilm developmentAnghel et al., 2012c).

By combining the biodegradability properties of AlgNa and CMCith the antimicrobial and antibiofilm properties of usnic acid,otentiated by the magnetite nanoparticles carriers (Grumezescut al., 2012b,d,e), we aimed to prepare novel wound dressing withmproved antimicrobial properties.

The structure of prepared Fe3O4@UA was analyzed by XRD andhe results are plotted in Fig. 1. Six characteristic peaks observedt 2� region of 20–70◦ were marked by their corresponding indices2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0), respectively, which

atch well with the XRD data of the Fe3O4 nanocrystals. The results

evealed that chemical modification of the Fe3O4with UA did notnduce any significant changes in the phase property of Fe3O4 andre in good agreement with previously published literature (Pannd shen, 2013; Jiang et al., 2012).

Fig. 3. FT-IR spectra of AlgNa (a) AlgNa/Fe3O4@

) images of Fe3O4@UA.

The nanoparticles were successfully prepared as shown in Fig. 2.Typical TEM analysis for Fe3O4@UA was performed in order toget direct information on particle size and morphology. The pre-pared water dispersible nanoparticles are monodispersed and havea mean diameter of 10 nm.

FT-IR spectroscopy was used to examine the integrity of func-tional groups after interaction with water dispersible nanoparticlesand freeze-drying process, as shown in Fig. 3. Characteristic peaksassignment of CMC and CMC/Fe3O4@UA are 3200–3600 cm−1 forthe hydroxyl group and for C O C bonds at 1022 and 1051 cm−1;the peaks at 1589 cm−1 1412 cm−1 is due to the COO− asym-metric and symmetric stretching vibrations of COO groups onthe CMC (Grumezescu et al., 2012f). Characteristic peaks assign-ment of AlgNa and AlgNa/Fe3O4@UA, characteristic bands are at1030 and 1081 cm−1 of the C O C stretching vibration, the band at2920 cm−1 of C H stretching, and a broad band due to the hydrogenbound OH group appeared between 3200 and 3500 cm−1 attributedto the complex vibrational stretching, associated with free, interand intra molecular bound hydroxyl groups. In the alginate spec-

−1

trum there are two specific strong absorption bands at 1598 cmand 1408 cm−1 attributed to asymmetric and symmetric stretch-ing vibrations of COO groups on the AlgNa (Pascalau et al., 2012;Balaure et al., 2013).

UA (b), CMC/Fe3O4@UA (c) and CMC (d).

150 A.M. Grumezescu et al. / International Journal of Pharmaceutics 463 (2014) 146– 154

0×, a2 1000×) and CMC/Fe3O4@AU (b1 – 200×, b2 1000×).

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Fig. 4. SEM micrographs of AlgNa/Fe3O4@AU (a1 – 15

SEM micrographs are plotted in Fig. 4. In the case oflgNa/Fe3O4@UA the homogeneously-dispersed Fe3O4@UA in theound dressing matrix can be observed in Fig. 4(a1,2). Fig. 4(b1,2)resents the morphology of the CMC/Fe3O4@UA. There can bebserved the Fe3O4@UA aggregates on the surface of this type ofound dressings (Fig. 4(b2)). Also, the SEM images of fabricatedound dressings show an open, interlaced and highly porous net-ork, in the range of 20–120 �m.

Biocompatibility assay revealed no cytotoxic reactions of anyMC/Fe3O4@UA or AlgNa/Fe3O4@UA anionic polymers againstested stem cells.

Even if EA.hy926 and AFSC cells were added directly uponhe CMC or AlgNa polymers during the loading of cell culturelates or after 24 hours of culture, similar results were obtainedn CMC/Fe3O4@UA and AlgNa/Fe3O4@UA samples. The biodegra-ation observed as dissolution of the polymers, as well as the partialgglomeration tendency of anionic polymers in cell culture media,ay increase with high electrolyte content, in the used media with-

ut FCS. However, the addition of 10% FCS in cell culture mediatabilized the CMC or AlgNa against agglomeration. These dataonfirm previous biocompatibility studies and the interaction ofSC with silver nanoparticles (Greulich et al., 2009; Engler et al.,

013). In this regard, our results proved that CMC/Fe3O4@UA andlgNa/Fe3O4@UA did not significantly influence the adherence androliferative potential of EA.hy926 and AFSC cells to their surfacep to 6 days of incubation.

The examination of the cell monolayer morphology assessedy automated cell counter statistics and conformed by opti-al microscopy, demonstrate that the CMC/Fe3O4@UA andlgNa/Fe3O4@UA have no cytotoxic effect on these human foetaltem cells morphology (average viable cell size = 8.15 �m ± 0.91

tandard deviation) (Fig. 6). Moreover, the polymer matrix adher-nce pattern as revealed by cell counting in the viability analysis ofuman foetal MSC was not significantly affected by the presence ofMC/Fe3O4@UA and AlgNa/Fe3O4@UA.

Fig. 5. The percentage of EA.hy926 and AFSC viability and the expression of theproliferation rate on CMC, CMC/Fe3O4@UA; AlgNa, AlgNa/Fe3O4@UA samples.

The tested CMC/Fe3O4@UA and AlgNa/Fe3O4@UA did not signif-icantly interfere in vitro with the cellular cycle phases, as revealedby the flow cytometry analysis. As it could be noticed in Fig. 5, thepercentages of EA.hy926/AFSC cells in different phases of the cellu-lar cycle were similar to the control cells. These results, correlatedwith the absence of the cytotoxicity revealed by Trypan blue andPI staining assay are pleading for the possibility of the in vivo useof these wound dressings for regenerative medicine, with no risksfor the occurrence of cytotoxic and proliferative side effects.

The cell cycle histograms revealed some differences in the cel-lular cycle progression. Even if the cell cycle moves in the normalsequence of the cellular phases, we can observe a delay of pro-gression from G1 → S → G2 on AlgNa (4) and AlgNa/Fe O @UA (6)

3 4samples compared with the viability and cell number (Fig. 7).

One of the main reasons that human foetal cells are not mov-ing into the S phase may include insufficient cell growth, injured

A.M. Grumezescu et al. / International Journal of Pharmaceutics 463 (2014) 146– 154 151

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ig. 6. Microscopy images of foetal mesenchymal stem cells monolayer. (1–2) ClgNa/Fe3O4@UA.

NA, or different cell culture conditions (growth factors, cell cul-ure media composition etc.). In our study, the cell proliferation andhe cell cycle increase significantly for CMC and CMC/Fe3O4@UAamples after 72–96 h. However, in the presence of AlgNa andlgNa/Fe3O4@UA a minor delay between G1/Go and S phase coulde noticed, in which no cellular division or cell growth may occur,ut without supporting cell injury due to the increase in cell num-er. Further details regarding the progression of the cell cyclehrough the S phase remain to be investigated.

The complex relationship between stem cells and polymericcaffolds has been analyzed in few studies, so far (Forte et al., 2013;oneno et al., 2005; Li et al., 2005; Turner et al., 2004). It was alreadyeported that scaffold and polymer chemistry, geometry, and man-facturing process play a pivotal role in directing cardiac residenttem cell commitment towards the cardiac phenotype (Forte et al.,009). Freshly isolated MSC could be used as a suitable model totudy adult stem cell culture onto a series of scaffolds, which havelready proven effective in vitro (Forte et al., 2013) and are undervaluation for clinical applications. On this purpose, polymer films,icrobeads, and scaffolds have been challenged for their capacity

o sustain MSC adhesion, growth, and multilineage differentiation.In this study, cell culture analysis indicated that foetal human

Cs adhere well on both CMC/Fe3O4@UA and AlgNa/Fe3O4@UA,ithout any evidence of toxic effects or cell cluster formation, as

emonstrated by the absence of floating cells or cell aggregatesithin the Petri dishes during the culture. As expected, cell distri-

ution onto CMC/Fe3O4@UA and AlgNa/Fe3O4@UA polymers wasniform, with cells retaining their spindle-shaped morphology

ls of EA.hy926 and AFSC; (3–4) +CMC and +AlgNa; (5–6) +CMC/Fe3O4@UA and

(Fig. 6). Notably, the cell cultures were able to migrate throughthe unpressed anionic polymer and colonize the inner layer, dataobserved through the difference of detached viable counted cells.This phenomenon, already observed when using endothelial cells,has been attributed to the larger pores and the open structuresupporting cell migration within the scaffold [50].

Simulating the in vitro human foetal progenitor cells turnoveron anionic polymers is an important step to evaluate the possibleuse of the respective biomaterials in a clinical context to regener-ate a complex and dynamic tissue. Regarding this, further studiesare ongoing to test adhesion and proliferation rate on both anionicpolymers with differentiated chondrocytes and osteocytes derivedfrom human foetal SC lines. Furthermore, gene expression stud-ies will be necessary to verify the effect of anionic polymers onSC differentiation (adipo-, osteo-, and chondro-differentiation) andtheir possible use in bone tissue engineering. Likewise, in vivoimplants of seeded polymers in mice might provide relevant infor-mation on their potential use in the field of regenerative medicine.Precautions need to be considered for using actively proliferatingfoetal human cells in vivo, so that implanted cells remain controlledby the body’s molecular signals and avoid development of malig-nancy.

Microbiology results revealed that the fabricated wounddressings exhbited a great antimicrobial potential, significantly

inhibiting S. aureus development. AlgNa/Fe3O4@UA has proved toinduce the most effective bacterial growth inhibition, being activeat all-time points used, the inhibition diameter zones decreasing,as expected, in the following order: T0 � T1 � T2 (Fig. 8).

152 A.M. Grumezescu et al. / International Journal of Pharmaceutics 463 (2014) 146– 154

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ig. 7. Cell cycle histograms. R7 = Go–G1. R8 = S. R9 = G2/M. (1–2) Controls of EA.hy

These results demonstrate that the newly fabricated AlgNaased nano-bioactive resorbable wound dressing is inducing a

icrobicidal effect on microbial cultures of different densities,ainly due to the bioactive Fe3O4@UA activity. Furthermore, due to

ts absorbent ability, this nano-bioactive material can be efficiently

ig. 8. The aspect of bacterial growth inhibition zones after the removal of the fabricated were added immediately after microbial inoculation, (b) T1, wound dressings were added

fter 12 h incubation of the inoculated plates.

d AFSC; (3–4) +CMC and +AlgNa; (5–6) +CMC/Fe3O4@UA and AlgNa/Fe3O4@UA.

used also in the treatment of infected wounds, since its additionon the surface of an inoculated plate, incubated for 12 h induced

a drastic decrease of the microbial growth. CMC/Fe3O4@UA mate-rials also exhibited a significant anti-staphylococcal activity, butlower comparing with AlgNa-based nano-bioactive materials. This

ound dressings added on the plate at different time points. (a) T0, wound dressings after 6 h incubation of the inoculated plates, (c) T2, wound dressings were added

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bservation may be explained by the fact that AlgNa and CMC pro-ide different controlled release kinetics for the active compoundFe3O4@UA).

. Conclusions

The obtained results demonstrated that CMC and AlgNa anionicolymers are exhibiting structural and functional properties thatecommend them for further applications in the biomedical field.hey could be used alone or coated with different bio-active com-ounds as Fe3O4@UA for the development of novel, multifunctionalorous materials used in tissues regeneration, as antimicro-ial substances releasing devices, providing also a mechanicalupport for the eukaryotic cells adhesion, and exhibiting thedvantage of low cytotoxicity on human progenitor cells. Thereat antimicrobial properties exhibited by the newly synthesizedano-bioactive coatings, are recommending them as successfulandidates for improving implanted devices surfaces used in regen-rative medicine.

cknowledgements

This paper is supported by the Sectorial Operational Pro-ramme for Human Resources Development, financed by theuropean Funding Programme, under project number POSDRU07/1.5/S/80765.

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