Perilipin

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Research ArticlePerilipin Expression Reveals Adipogenic Potential of hADSCsinside Superporous Polymeric Cellular Delivery Systems

Sorina Dinescu,1 Bianca Galateanu,1 Adriana Lungu,2 Eugen Radu,3 Sorin Nae,4

Horia Iovu,2 and Marieta Costache1

Department o Biochemistry and Molecular Biology, University o Bucharest, - SplaiulIndependentei, Bucharest,Romania Advanced Polymer Materials Group, Department o Bioresources and Polymer Science, University Politehnica o Bucharest,

Calea Victoriei, Bucharest, Romania Molecular Biology and Pathology Research Lab “Molimagex”, University Hospital Bucharest, Splaiul Independentei,

Bucharest, Romania Faculty o Medicine, Ovidius University o Constanta, Aleea Universitatii, Constanta, Romania

Correspondence should be addressed to Marieta Costache; [email protected]

Received February ; Accepted April ; Published May

Academic Editor: Kibret Mequanint

Copyright © Sorina Dinescu et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Recent progress in tissue engineering and regenerative medicine envisages the use o cell-scaffold bioconstructs to best mimic the

natural in vivo microenvironment. Our aim was not only to develop novel D porous scaffolds or regenerative applications by the association o gelatin (G), alginate (A), and polyacrylamide (PAA) major assets but also to evaluate their in vitro potential tosupport human adipose-derived stem cells (hADSCs) adipogenesis. G-A-PAA biomatrix investigated in this work is an interestingsubstratecombining the advantages o the threeindividual constituents, namely, biodegradability o G, hydrophilicity o A and PAA,superior elasticity at compression with respect to the G-A andPAAcontrols, andthe capacity to generate porous scaffolds. hADSCsinside these novel interpenetrating polymer networks (IPNs) were able to populate the entire scaffold structure and to display theircharacteristic spindle-like shape as a consequence o a good interaction with G component o the matrices. Additionally, hADSCsproved to display the capacity to differentiate towards mature adipocytes, to accumulate lipids inside their cytoplasm, and to expressperilipin late adipogenic marker inside novel IPNs described in this study. On long term, this newly designed biomatrix aims torepresent a stem cell delivery system product dedicated or modern regenerative strategies.

1. Introduction

Modern tissue engineering (E) applications require thecorrelation between the composition, structure, and charac-teristics o the material and the biological component. Teinteraction o the scaffold with cells, uids, and tissues isstrongly dependent on the chemistry o the material, sincethe physicochemical eatures o the material can decisively inuence cell adherence.

Polymers are versatile natural and synthetic compoundsdisplaying a large panel o properties that make themsuitable or a wide range o E applications. Despite theirspecic biodegradability and biocompatibility, some mate-rials do not possess appropriate mechanical properties orbiodegradation rate. In this context, we recently developed

and investigated various multicomponent scaffolds basedon semi- and interpenetrating polymer networks (IPNs) by

combining natural, synthetic, biodegradable, and/or non-biodegradable macromolecular components such as gelatin-alginate [, ], gelatin-alginate-polyacrylamide (PAA) [],broin-PAA [], gelatin-poly(−hydroxyethyl methacrylate)(PHEMA) [], and collagen-sericin [, ]. Te underlyingprinciple was that the natural polymers (i.e., collagen, gelatin,and alginate) would impair biodegradability to the resultingbi- or tricomponent scaffolds, while displaying improvedoverall properties. Furthermore, the presence o collagenor gelatin in a scaffold’s ormulation coners cell adhesionproperties, while also ensuring enzymatic biodegradation. Inaddition, macromolecular elements with high water affinity,such as PAA and alginate, enhance the degradation rate

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 830791, 9 pageshttp://dx.doi.org/10.1155/2014/830791

http://dx.doi.org/10.1155/2014/830791http://dx.doi.org/10.1155/2014/830791

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BioMed Research International

o multicomponent scaffolds due to improved accessibility o the substrate to hydrolytic attack [, ], thus improvingthe overall water affinity o such multicomponent scaffolds.aking together all these eatures, we recently synthesized andcharacterized a tricomponent gelatin-alginate-PAA system asappealing substrates or sof tissue regeneration [].

In a dynamic view, adipose tissue (A) through itscellular component, the adipocytes, generates a wide rangeo signal molecules such as growth actors, proteins relatedto the immune system, and adipokines []. Particularly,subcutaneous adipose depots are accessible and abundant,in contrast with the bone marrow (BM), the traditionalmesenchymal stem cells (MSCs) harvesting source. In thisperspective, A has become an attractive option or adipose-derived stem cells isolation (ADSCs). ADSCs ound in thestromal-vascular raction (SVF) o the A have the ability todifferentiate into cells o several lineages such as adipocytes,osteoblasts, chondrocytes, myocytes, endothelial cells,hematopoietic cells, hepatocytes, and neuronal cells [–].

Te main promoters o adipogenic differentiation, PPAR and C/EBP, act synergistically to activate transcriptiono genes that produce the adipocyte phenotype, althoughhormones are required or terminal differentiation [, ].Mature adipocytes synthesize A-specic products, suchas adipocyte atty acid-binding protein (aP) and perilipin[]. Lipid droplet-associated protein, perilipin, coats lipiddroplets in mature adipocytes and acts as a protective layeragainst the physiological lipases.

In this context, our aim was not only to develop acombinatory approach o the gelatin, alginate, and PAA majorassets to design novel D porous scaffolds or sof tissueregenerative applications, but also to evaluate their in vitropotential to support hADSCs differentiation towards matureandunctional adipocytes. On long term, thisnewly designedbiomatrix aims to represent a stem cell delivery system prod-uct dedicated or modern regenerative strategies. Tereore,essential unctional properties such as the water affinity, themechanical properties, and the enzymatic degradation o the porous tricomponent gelatin-alginate-PAA scaffolds wereevaluated. In addition, cell behavior and distribution, as wellas the potential to accumulate lipid droplets and to expresslate adipogenic markers such as perilipin during in vitroadipogenesis, were also assessed.

2. Materials and Methods

.. Materials. Gelatin B (urther named Gel) rom bovineskin (Sigma) was used as % (w/v) aqueous solution.Sodium alginate (SA) was used as % (w/v) aqueous solution.Acrylamide (AAm) or electrophoresis >% (HPLC), N,N-methylenebis(acrylamide) (MBA) %, triethanolamine(EA), ammonium persulate (APS), glutaric aldehyde (GA)as aqueous solution %, and calcium chloride anhydrous(CaCl2) were purchased rom Sigma and used withouturther purication. Ethylene diamine tetra-acetic acid(tetrasodium salt tetrahydrate) (EDA) rom Sigma-Aldrichwas used as received. Sodium azide (%) was purchasedrom Avocado Research Chemicals Ltd. Collagenase type I o

Clostridium histolyticum with a collagen activity ≥ unitsper mg (collagen digestion units) was rom Sigma. All thesalts necessary to prepare phosphate buffer saline (PBS) weresupplied by Sigma-Aldrich.

Human subcutaneous adipose tissue which served asstem cells source or this study was harvested rom adult

patients undergoing elective abdominoplasty. All the sub- jects offered their written inormed consent to participatein this study and none o them had diabetes or severesystemic illness or was taking medication known to affectadipose tissue metabolism. All the medical procedures wereperormed in compliance with the Helsinki Declaration,with the approval o the Emergency Hospital or PlasticSurgery and Burns Ethical Committee (Reerence number/..). hADSCs were manipulated using sterileTermo Scientic Nunc labware disposables. MesenPRO RSculture medium and StemPro Adipogenesis DifferentiationKit (Gibco, Lie echnologies, Foster City, CA) were used topropagate and differentiate hADSCs. Glutaraldehyde, bovineserum albumin (BSA), riton X-, and Oil Red O dye werepurchased rom Sigma-Aldrich Co. RNA extraction was per-ormed using RIzol Reagent provided by Invitrogen, FosterCity, CA, USA, and the qPCR LightCycler FastStart DNAMaster SYBR Green I Kit was provided by Roche, Mannheim,Germany. All the primary and secondary antibodies used inthis study were purchased rom Santa Cruz Biotechnology,Inc. (Heidelberg, Germany).

.. Methods

... Scaffold Synthesis. IPNs based on G, SA, and PAA wereprepared using a previously described three-step procedure[] which was adapted or this particular study. Briey, semi-IPNs were initially generated by the ree-radical copolymer-ization o AA and MBA in water in the presence o G andSA. A weight ratio o : : between G, SA, and AA wasused, with a total solid content (%) o %. A redox initiatingsystem based on APS (% molar with respect to AA andMBA) and EA (/ molar with respect to APS) was usedto perorm the polymerization reaction at room temperature(R). Te molar ratio between MBA and AA was . : . Acopolymerization stock solution was prepared through thedissolution o AA, MBA, and the corresponding amount o APS in distilled water, under stirring, at R. mL o thissolution was urther mixed with mL o G solution andwith mL o SA solution, at ∘C. Te resulting mixture was

degassed using an ultrasound bath (Elma S H, Elmasonic)or minutes at ∘C and nally EA was added under

vigorous stirring. Te copolymerization reaction o AA andMBA was allowed, or hours, at R and, consequently,semi-IPNs consisting in crosslinked PAA and uncrosslinkedG and SA were obtained. Furthermore, the materials werecooled or hours at ∘C, to allow physical gelation o G. Ina second step, the crosslinking o G was perormed throughtheir immersion in GA .% or hours, at R. Te thirdphase o the synthesis consisted in the crosslinking o SA by immersing the samples or hours in a % CaCl2 aqueoussolution. As a result, calcium alginate (A) was ormed. TeG-A-PAA hydrogel was urther extracted in distilled water

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at ∘C or our days. Gravimetric measurement was usedto conrm the success o the IPNs ormation. Te synthesiso porous scaffolds was perormed through a reeze-dryingtreatment as previously reported [].

In order to investigate the potential advantages o thetricomponent IPN, control G-A and PAA hydrogels with

the same % (%) were synthesized ollowing similar pro-cedures and submitted to lyophilization to generate porousmaterials. While PAA was obtained through the redoxinitiated ree-radical polymerization o the correspondingmonomer and crosslinker (molar ratio MBA/AA o ./, = 21%), G-A hydrogels were obtained using a three-stepcrosslinking. Briey, the preparation o the bicomponent G-SA solution (Gel/SA o / and = 21%) was ollowed bythephysical gelation o G ( hours at ∘C). Ten, the crosslinkingo G was perormed through the immersion o the specimensin GA .% or hours, at R. Te third phase consistedin the crosslinking o SA by immersion o the samples or hours in a % CaCl2 aqueous solution to generate A. Allthe control samples were puried as described or G-A-PAA,ollowed by reeze-drying.

... Determination o Water Affinity. Te swelling behavioro the G-A-PAA and control reeze-dried hydrogels wasinvestigated in ddw, at ∘C, using the conventional gravi-metric method. Te swelling ratio (SR) was calculated atpredened time intervals, using the ollowing equation:

SR = − 0

0∗ 100, ()

where is the weight o swollen hydrogel at time and 0is the initial weight o the dry hydrogel beore incubation

in ddw. Te samples were weighed afer the excess o waterwas removed with lter paper. Te maximum swelling degree(MSD) represents the maximum value obtained afer reach-ing equilibrium. Afer the swelling experiment, the sampleswere dried and the dry mass was measured in order to allow comparison with the initial dry mass.

... Mechanical Properties. Te mechanical behaviour o the investigated hydrogels swollen at equilibrium (in ddw at ∘C) was studied using Brookeld C texture analyzerat room temperature. Cylinder samples with the diametero mm and a thickness o mm were xed on a baseplate and uniaxially compressed by the upper plate connected

to a -gram cell. Te test speed was set at . mm/s.A stress versus strain graph was plotted. Te compressionmodulus was calculated rom the slope o the linear part o the compression curve at % strain.

... In Vitro Degradation by Collagenase. In vitro degrada-tion o the hydrogel G-A-PAA and o the control G-A hydro-gel was investigated by incubation o cylindrical reeze-driedsamples (Φ = × mm) in collagenase solution, ollowinga procedure reported elsewhere [, ]. Briey, the sampleswere initially immersed in . mL ris-HCl buffer (. M,pH .) in the presence o NaN3 (.% w/v) and CaCl2( mM) at ∘C. Afer one hour, . mL collagenase solution

( U/mL), dissolved in ris-HCl buffer, was added. Tedegradation o G was stopped at predened time intervalsby adding . mL EDA solution (.M) and subsequentcooling on ice. Ten, the samples were washed three times or minutes with ice-cooled ris-HCl buffer and three timeswith double-distilled water. Te remaining materials were

dried or the determination o the gel raction (the insolublepolymer raction remaining afer degradation)

Degradation ⋅ extentddw , % =0 −

0× 100, ()

where 0 is the initial mass o the sample, while is theweight o the sample afer degradation treatment.

... In Vitro Cell Culture Model and Cell-Scaffold Biohybrid Achievement. hADSCs were isolated as previously described[] and seeded on top o G-A and G-A-PAA biomatricesat an initial density o 6 × 105 cells/cm2 afer propagation in

MesenPRO RS Medium. In our experiments, the porous Dconstructs resulted afer hADSCs populated G-A and G-A-PAA biomatrices were dened as biohybrids. Consequently,they are urther addressed as hADSCs/G-A and hADSCs/G-A-PAA, respectively.

Regarding the adipogenic differentiation protocol, thebioconstructs were exposed to proadipogenic conditionsor days using StemPro Adipogenesis Differentiation Kit(Gibco, Lie echnologies, Foster City, CA). Bioconstructsinduction towards the adipogenic lineage was started only afer hours afer cell seeding into scaffolds. hADSCspotential o differentiation towards the adipogenic lineagewas assessed at , , , , and days afer induction.

Te time point when the systems were rst exposed to thechondrogenic cocktail was considered .

... Scanning Electron Microscopy Evaluation o Biohybridsduring Adipogenesis. Te resulting biohybrids were subjectedto SEM analysis at days afer hADSCs seeding and at , , ,, and days afer adipogenic induction. All the sampleswere xed with .% glutaraldehyde (Sigma-Aldrich Co.) or hours at ∘C and then subjected to reeze-drying. Tecross-sections were gold-coated and then analyzed using aQuanta Inspect F SEM device equipped with a eld emissiongun (FEG) with . nm resolution and with an X-ray energy dispersive spectrometer (EDS).

... Intracellular Lipid Accumulation inside Biohybrids Sub- jected to Adipogenesis. Te presence o neutral lipid dropletsin the cytoplasm o the differentiating cells inside G-A-PAAand G-A scaffolds was investigated by Oil Red O staining at ,, , , and days o induction. For this purpose, the bio-hybrids were xed overnight in % paraormaldehyde. Aferpermeabilization in % bovine serum albumin (BSA)/.%riton X- solution or hours, both hADSCs/G-A-PAAand hADSCs/G-A bioconstructs were exposed to Oil RedO staining solution or hours at ∘C. Te resultingbioconstructs were analyzed by bright-eldmicroscopy usingan Olympus IX inverted microscope and images were

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: Primer sequences used or adipogenic marker identica-tion by qPCR.

arget Nucleotide sequence

Perilipin F -AGCCCAGAAGACCACA-

Perilipin R -CAGCCAGAAGCAAC-

BP F

-AGGCACGCGCACAC-

BP R -GGGCAGCCAGGCCAAA-

RPLA F -CCGGAGGAGAAGAGGAAAGAGA-

RPLA R -GAGGACCCGGAGCAA-

BP: AAA-box binding protein; RPLA: ribosomal protein LA.

captured with Cell F Imaging Sofware (Olympus, Hamburg,Germany, ).

... qPCR Evaluation o Perilipin Late Adipogenic Marker Expression. Afer hADSCs/G-A and hADSCs/G-A-PAA bio-

constructs ragments were exposed to RIzol Reagent inaccordance with the manuacturer’s instructions, total RNAwas isolated and assessed or concentration, purity (Nan-oDrop spectrophotometer, Shimadzu, Duisburg, Germany),and integrity on the BioAnalyzer (Agilent echnologies,Waldbronn, Germany). Late adipogenic marker perilipinexpression was evaluated by qPCR, perormed on a LightCy-cler . carrousel-based system using LightCycler FastStartDNA Master SYBR Green I Kit. Te genes coding orribosomal protein LA (RPLA) and AAA-box bindingprotein (BP) were used as reerence genes in order tonormalize the levels o perilipin adipogenic marker duringdata processing. Primer sequences used or gene expression

assessment are presented in able .

... Conocal Fluorescence Microscopy Assessment o Per-ilipin Protein Expression in D Culture Conditions. Perilipinprotein expression was assessed at , , , , and days aferadipogenic induction using a Carl Zeiss LSM conocalmicroscope. Briey, both hADSCs/G-A and hADSCs/G-A-PAA biohybrids were xed with % PFA, permeabilized with% BSA/.% riton X- solution, incubated overnight at∘C with rabbit polyclonal anti-perilipin antibody solution,andnally exposed to RIC conjugated goat anti-rabbit sec-ondary antibody. Cell nuclei were stained using DAPI dye or min and the resulting labeled constructs were visualized in

conocal uorescence microscopy using a conocal aperturethat corresponded to a back-projected size o airy unit.Te and nm laser lines were used or excitation anduorescence emission was detected at – nm or DAPIand – nm or RIC. Carl Zeiss Zen sofware

version . was used or image acquisition and analysis.

... Statistical Analysis. Te spectrophotometric and geneexpression data were statistically analyzed using GraphPadPrism . Sofware, one-way ANOVA, and Bonerroni test.Te experiments were perormed with = 3 biologicalreplicatesand each data set is presented as the average o threereplicates (mean ± standard deviation).

100200

300

400

500

600

700800

900

0

S w

e l l i n g d e g r e e ( % )

G-A

PAA

G-A-PAA

0 200 400 600 800 1000 1 200 1 400 1 600 1800

Swelling time (min)

(a)

C o m p r e s s i v e m o

d u l u s ( P a )

80000

70000

60000

50000

40000

30000

20000

10000

0G-A PAA G-A-PAA

(b)

F: (a) Evolution othe swelling degreeo thestudiedhydrogeland o the control samples, in distilled water, as obtained afer minutes. (b) Effect o the composition on the compressive modulusobtained at room temperature and compressive strain o %.

3. Results and Discussions

.. Scaffold Synthesis and Physicochemical Characterization

... Swelling Behavior. Te affinityagainst water is extremely important since it directly impacts various key propertiessuch as () the mechanical behavior, () the capacity o thehydrogels to generate porous scaffolds through reeze-drying,and () the ability to be loaded with or to deliver water-soluble species. Te results o the swelling test are graphically depicted in Figure (a). Te tricomponent hydrogel G-A-

PAA had the highest MSD value, namely, % ± , whencompared to the control hydrogels. Te lowest swelling,with a MSD value o % ± , was noticed or G-A,while the control PAA had a MSD value o % ± . Teswelling is directly determined by the crosslinking density o each network. Interestingly, the combination o the threepolymers in a complex IPN with the same % as the controlhydrogels increased the affinity or water. Tis is normal sinceeach additional component, intercalated at molecular level,plays the role o a diluent or the resulting tricomponentIPN. Accordingly, the maximum swelling degree increases.Furthermore, with respect to the time needed to reach theswelling equilibrium, the tricomponent IPN is again the

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1009080706050403020

100 D e

g r a d a t i o n e x t e n t ( % )

0 100 200 300 400 500

Degradation time (min)

G-A

G-A-PAA

∗ ∗ ∗ ∗

F : In vitro degradation with collagenase I, at ∘C: degra-dation extents versus degradation time. ∗ Tese degradation extent values do not indicate a total enzymatic degradation but thedisintegration o the scaffolds which become too ragile due to thedegradation o G.

leader, swelling aster than the control materials (PAA needs minutes to reach equilibrium andG-Aneeds minutes,while G-A-PAA swells in only minutes maximum). Inorder to estimate i uncrosslinked products are releasedrom the hydrogels during swelling, the nal dry mass aferthe maximum swelling was compared with the initial dry mass o each sample. No signicant differences were noticed,conrming the insolubility o the analyzed materials.

... Mechanical Behavior. Te compression moduli werecalculated using the linear part o the stress-strain curvesobtained at % strain. Figure (b) is representative in thisrespect and it shows that the compressive moduli decrease

in the order G-A > PAA > G-A-PAA. Te maximum value(65 ± 5 kPa) was obtained or the IPN G-A. Te ollowing

value, 52 ± 4 kPa, was obtained or the synthetic hydrogelPAA. Te lowest value, 15 ± 2 kPa, was obtained or G-A-PAA. First, these values indicate that the tricomponentIPN is the most elastic hydrogel when compared to the twocontrol materials prepared using the same %. Furthermore,the elasticity perectly correlates the swelling behavior o the hydrogels increasing with the amount o water rom thehydrogel swollen at the maximum: G-A

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hADSCs/G-A

2

d a y s a f e r s e e

d i n g

(a)

hADSCs/G-A-PAA

(b)

4

w e e k s a f e r

a d i p o g e n i c i n d u c t i o n

(c) (d)

F : SEM micrographs showing hADSCs morphology and distribution inside hADSCs/G-A-PAA (b, d) and hADSCs/G-A (a, c)bioconstructs at days afer cell seeding (a, b) and afer weeks o induced in vitro adipogenesis (c, d).

phenotype is initiated. Late adipogenic marker perilipin,responsible or lipid storage, was evaluated in terms o geneexpression pattern in the conditions o hADSCs/G-A andhADSCs/G-A-PAA D cultures during days o inducedadipogenic differentiation (Figure ).

In our conditions, perilipin was rst statistically detected( < 0.001) at days afer adipogenic induction inboth hADSCs/G-A and hADSCs/G-A-PAA biohybrids, ascompared to days o adipogenesis. Perilipin levels o gene

expression highly increased ( < 0.001) between and days o induced adipogenesis in both constructs, suggestinga continuous differentiation process supported by the G-Aand G-A-PAA materials and the gradual accumulation o lipids inside differentiating cells. Interestingly, a statistically signicant difference ( < 0.05) was ound between perilipintranscript levels in hADSCs/G-A and hADSCs/G-A-PAAbioconstructs, whereas this difference was not detected at days. Fourweeks aferadipogenic induction,perilipin mRNAlevels signicantly increased ( < 0.001) in both systemsas compared to days, with no statistic difference betweenhADSCs/G-A-PAA and control biohybrids. According to ourresults, the increasing prole o perilipin gene expression

suggests that an adipogenic differentiation process was activeinside the D bioconstructs, while similar patterns o geneexpression between the hADSCs/G-A and hADSCs/G-A-

PAA suggest the nontoxic effect o PAA in the compositiono the tested scaffolds.

Perilipin Protein Expression. Late adipogenic marker perilipinprotein expression was evaluated by conocal uorescencemicroscopy at days afer seeding and at , , , , and

days afer adipogenic induction o hADSCs/G-A andhADSCs/G-A-PAA biohybrids. Images displaying the earliestpositive expression o perilipin at days afer induction andimages o -week differentiating hADSCs were capturedat ∼ m in the scaffolds’ depth and are presented inFigure .

Afer one week o adipogenic commitment, RIClabeled perilipin molecules were observed attached to small

vesicles surrounding the blue DAPI stained nucleus in ew cells (Figures (a) and (b)) conrming the Oil Red Ostaining observations. In addition, days afer adipogenicinduction, hADSCs inside G-A-PAA’s pores highly expressedperilipin as all the nuclei were proved to be surrounded

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hADSCs/G-A

1

w e e k

100m

(a)

hADSCs/G-A-PAA

100m

(b)

4 w

e e k s

100m

(c)

100m

(d)

F : Lipid droplet accumulation inside hADSCs/G-A-PAA (b, d) and hADSCs/G-A (a, c) bioconstructs afer week (a, b) and weekso adipogenic differentiation (c, d), as revealed by Oil Red O staining.

hADSCs/G-AhADSCs/G-A-PAA

0.03

0.02

0.01

0.00

F o l d i n c r e a s e

T0 3 d 7 d 14 d 28 d

∗

###

Perilipin

∙ ∙ ∙

F : Perilipin gene expression pattern in hADSCs/G-A and hADSCs/G-A-PAA bioconstructs during days o induced adipogenicdifferentiation, as revealed by qPCR. Statistical meaning (

∗ < 0.05 (hADSCs/G-A-PAA versus hADSCs/G-A bioconstruct: days);

###

<0.001 (hADSCs/G-A-PAA: days versus days);

∙∙∙ < 0.001 (hADSCs/G-A: days versus days)).

by bright red vesicles (Figures (c) and (d)). Perilipinexpression inside hADSCs/G-A biohybrid was ound to besimilar.

Our experiments show that, as a result o the adi-pogenic differentiation progress, adipocytes inside G-A and

G-A-PAA biomatrices start to gradually accumulate lipids.Consequently, this increase o intracellular lipids determinesthe overexpression o perilipin, since perilipin unctions as alipid droplet gatekeeper that controls lipases access to lipids,in a phosphorylation-dependent manner.

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hADSCs/G-A

1

w e e k

(a)

hADSCs/G-A-PAA

(b)

4

w e e k s

(c) (d)

F : Conocal microscopy micrographs displaying perilipin protein expression in hADSCs/G-A and hADSCs/G-A-PAA biohybridsduring adipogenesis.

4. Conclusions

Te tricomponent material G-A-PAA investigated in thiswork is an interesting substrate combining the advantageso the three individual constituents, namely, biodegradability o G, hydrophilicity o A and PAA, superior elasticity atcompression with respect to the G-A and PAA controls, and,

nevertheless, the capacity to generate porous scaffolds.hADSCs inside these novel IPNs were able to populate

the entire scaffold structure and to display their characteristicspindle-like shape as a consequence o a good interactionwith G component o the matrices. Additionally, hADSCsproved to display the capacity to differentiate towards matureadipocytes, to accumulate lipids inside their cytoplasm, andto express perilipin late adipogenic marker inside novelIPNs described in this study. Consequently, the presenceo the synthetic PAA component in the ormulation o the scaffold did not inuence either hADSCs behavior incontact with the material or the evolution o the adipogenicdifferentiation process, but it ensured a more appropriate D

microenvironment or cells, resembling in vivo conditions.Furthermore, as a perspective or tissue engineering advance,hADSCs/G-A-PAA could be an eligible candidate as cellulardelivery system at the injury sites or regenerative purposes.

Conflict of Interests

Te authors declare that there is no conict o interestsregarding the publication o this paper.

Authors’ Contribution

Sorina Dinescu and Bianca Galateanu have contributedequally to this work.

Acknowledgments

Tis research was supported by the European Social Fundingthrough the Sectorial Operational Programme or Human

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Resources Development POSDRU//./S/ and by the Romanian CNCS-UEFISCDI, Complex Exploratory Research Project (Grant no. PCCE/). Te authorsthank Dr. Eugeniu Vasile or the help he provided with theScanning Electron Microscopy assays.

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